Research ArticleAssociation between AKT1 Gene Polymorphism rs2498794 andSmoking-Related Traits with reference to Cancer Susceptibility

Daisuke Nishizawa1 Shinya Kasai1 Junko Hasegawa1 Naomi Sato23 Fumihiko Tanioka4

Haruhiko Sugimura3 Kazutaka Ikeda1 and Yoh Dobashi5

1Addictive Substance Project Tokyo Metropolitan Institute of Medical Science Tokyo 156-8506 Japan2Department of Clinical Nursing Hamamatsu University School of Medicine Hamamatsu 431-3192 Japan3Department of Tumor Pathology Hamamatsu University School of Medicine Hamamatsu 431-3192 Japan4Department of Pathology Iwata City Hospital Iwata 438-8550 Japan5Department of Pathology Saitama Medical Center Jichi Medical University Saitama 330-8503 Japan

Correspondence should be addressed to Daisuke Nishizawa nishizawa-dsigakukenorjp

Received 3 March 2015 Revised 14 May 2015 Accepted 21 May 2015

Academic Editor Diego Forero

Copyright copy 2015 Daisuke Nishizawa et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

To clarify the potential role of variability within and around theAKT1 gene in smoking behaviors we performed a single-nucleotidepolymorphism (SNP) analysis of the AKT1 gene in an elderly Japanese cohort Genotypes of the rs2498794 SNP which is locatedin the fifth intron region of the AKT1 gene were marginally but significantly associated with smoking duration in the total 999samples of former and current smokers Interestingly this SNP had a marginally significant association with individual cancerhistory (past and current) especially in groups with a shorter smoking duration (lt44 years) and fewer cigarettes per day (le20)These data suggest that the rs2498794 polymorphism of the AKT1 gene is associated with a long smoking duration and may beinvolved in the predisposition to cancer when the smoking duration is short or the cigarettes per day is rate low

1 Introduction

Akt (also called protein kinase B) is a serine-threonine kinasethat was first identified in mice as the cellular homologueof the murine thymoma oncogene v-Akt [1 2] Three mam-malian isoforms of its gene products have been identifiedmdashAkt1 (PKB120572) Akt2 (PKB120573) and Akt3 (PKB120574)mdashshowing abroad tissue distribution [1ndash3] Akt1 is the most ubiquitouslyexpressed isoform Although Akt2 and Akt3 are also ubiqui-tously expressed Akt2 is expressed predominantly in insulin-responsive tissues and Akt3 is expressed predominantly inthe testes and brain [4] Each Akt isoform is a downstreameffector of the growth factor signaling pathway and func-tions as a mediator of the phosphoinositide-3 kinase (PI3K-Akt) pathway [5] The stimuli that emanate from activatedgrowth factor receptors activate this kinase cascade throughPI3K and a second messenger phosphatidylinositol (345)-trisphosphate which then binds to Akt Consequently Akt is

phosphorylated and activated by PI3K-dependent kinases 1and 2 and activates various substrates including mammaliantarget of rapamycin Bad Bax Mdm2 and Foxo [2 5 6]Akts phosphorylate and regulate various substrates that areinvolved in diverse cellular functions including cell growthsurvival apoptosis and metabolism through the activationof translation [1ndash3 7 8] Protein overexpression and acti-vation and somatic aberrations of PI3K-Akt pathway geneshave been commonly observed in a variety of malignanciesand this pathway has been extensively investigated as one ofthe critical mechanisms in tumorigenesis and as a target forcancer therapy [7 9]

With regard to genetic aberrations AKT1 amplificationhas been reported in carcinomas of the lungs stomach breastand prostate [2 10 11] AKT2 gene amplification has beenobserved in carcinomas of the breast ovaries and pancreasand associated with a poor prognosis in several of thesecancers [2 11 12]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 316829 12 pageshttpdxdoiorg1011552015316829

2 BioMed Research International

Additionally genetic variations of AKTs such as single-nucleotide polymorphisms (SNPs) have also beenwell recog-nized to modulate gene function These variations are asso-ciated with a predisposition to and determinant of clinicaloutcomes of endometrial and lung cancers [13ndash15]

Akt1 is also centrally involved in neuronal survival andplasticity [16] AKT1 variations have been reported to beassociated with Parkinsonrsquos disease schizophrenia metham-phetamine use disorder and bipolar disorder [16ndash21] In lightof the critical role of Akt in maintaining proper cellularfunction and tumorigenesis andor tumor progression thescreening ofAKT SNPs is important Among theAKT genesthe present study focused on the AKT1 gene which is themost ubiquitously expressed and assumed to play centralroles in various functions and pathologies

With the goal of identifying allelic variants that signifi-cantly contribute to pathogenesis and smoking-related traitsglobal tests of associationswere performed between each SNPof the AKT1 gene and cancer predisposition and smokingbehaviors

2 Methods

21 Subjects The participants in the initial analysis thatexplored possible associations between AKT1 gene poly-morphisms and the susceptibility to common cancers andsmoking behavior included a total of 999 patients whopresented at or were admitted to Iwata City Hospital in JapanThe inclusion criteria for this study were being Japaneseambulatory able to communicate orally and 60 years of ageor older Numerous participants in this study had varioussmoking habits and completed a questionnaire that con-sisted of various questions about lifestyle including alcoholconsumption smoking diet and cancer history [22 23]Peripheral blood samples were collected from these subjectsfor the gene analysis The detailed demographic and clinicalcharacteristics of the subjects with a focus on cancer andsmoking behaviors are provided in Table 1 These data wereused in the statistical analyses Smoking duration (years)cigarettes smoked per day (CPD) and the product of thesetwo (ie the Brinkman (smoking) index) were incorporatedin the analysis for smoking behaviors

The study protocol was approved by the InstitutionalReview Boards at Hamamatsu University School of Medicine(Hamamatsu Japan) and the Tokyo Metropolitan Institute ofMedical Science (Tokyo Japan) All of the subjects providedinformed written consent for the genetics studies

22 Genotyping Genomic DNA was extracted from whole-blood samples using a QIAamp DNA BloodMaxi kit accord-ing to the manufacturerrsquos instructions (Qiagen HamburgGermany) The extracted DNA was dissolved in TE buffer(10mM Tris-HCl and 1mM ethylenediaminetetraacetic acidpH 80) before use The DNA concentration was adjusted to100 ng120583L for whole-genome genotyping and approximately5ndash50 ng120583L for genotyping the specific rs2498794 SNP usinga NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech-nologies Wilmington DE USA)

Briefly whole-genome genotyping was performed usingthe Infinium assay II with an iScan system (Illumina SanDiego CA USA) according to the manufacturerrsquos instruc-tions HumanCytoSNP-12 v20 (total markers 301232) Bead-Chips were used to genotype the 300 samples from thepatientswith clinical data for cancer and smoking historyTheBeadChips included a number of probes that are specific tocopy number variation markers but most of the BeadChipswere for SNP markers on the human autosome or sexchromosome In the data-cleaning process the samples witha genotype individual level call rate lt095 were intended tobe excluded from the analyses Additionally markers with agenotype call frequency lt095 or ldquoCluster seprdquo (ie an indexof genotype cluster separation) lt01 were excluded from thesubsequent association study Markers were not excludedbased on the heterozygosity rates and results of Hardy-Weinberg equilibrium (HWE) tests for the whole-genomegenotyping data but the HWE tests were conducted for theselected individual SNPs for association analyses Tests forpopulation substructure and relatedness were not conductedbecause it was assumed that all of the subjects were unrelatedand genetically homogeneous Japanese mostly living in theKanto orTokai areaAs a result a total of 291523 SNPmarkerssurvived the filtration process and were used for the datasetof the association analyses

To genotype the rs2498794 SNP using a total of 700DNA samples in the subsequent association study afteran initial exploratory association study the TaqMan allelicdiscrimination assay (Life Technologies Carlsbad CA USA)was basically adopted To perform the TaqMan allelic dis-crimination assaywith a LightCycler 480 (RocheDiagnosticsBasel Switzerland) TaqMan SNP Genotyping Assays (LifeTechnologies) were used that contained sequence-specificforward and reverse primers to amplify the polymorphicsequence and two probes labeled with VIC and FAM dye todetect both alleles of the candidate Tag SNP rs2498794 (assayID C 193159 10) Real-time polymerase chain reactionwas performed in a final volume of 10 120583L that contained2times LightCycler 480 Probes Master (Roche Diagnostics) 40timesTaqMan Genotyping Assays 5ndash50 ng genomic DNA as thetemplate and up to 10120583LH

2O (Roche Diagnostics) The

thermal conditions were the following 95∘C for 10minfollowed by 45 cycles of 95∘C for 10 s and 60∘C for 60 swith final cooling at 50∘C for 30 s Afterward endpointfluorescence was measured for each sample well and eachgenotype was determined based on the presence or absenceof each type of fluorescence

23 Linkage Disequilibrium Analysis To initially analyzeSNPswithin and around theAKT1 gene region genotype datafor approximately 300000 SNP markers that resulted fromwhole-genome genotyping with the patient samples withclinical data for cancer and smoking history were basicallyused and the genotype data for all of the SNPs with AKT1gene annotation were extracted for a total of 300 samplesThe minor allele threshold for the SNP selection was set at0001 which indicates the inclusion of at least oneminor allelecarrier in the 300 samples As a result the rs28546406 SNPwas dropped based on the minor allele frequency criterion

BioMed Research International 3

Table 1 Demographic data of patients

119899 Minimum Maximum Mean SD MedianAll subjects 999

Male 612Female 387

Age (years) 999 60 94 7358 586 7300Height (cm) 996 130 180 15686 869 15800Weight (kg) 996 30 101 5475 998 5400Smoking status 999

Current smokers 130Exsmokers 392Never-smokers 477

Smoking behaviors 522Smoking period (year) 520 100 6800 4050 1440 4400CPDa 520 100 10000 2144 1342 2000Brinkman (smoking) index 519 900 348000 85332 58864 70000

Cancer status (present history) 999Present 89Absent 874Unknown 36

Cancer status (past history) 999Present 128Absent 848Unknown 23

Cancer sites (presentpast history) 105136Oral mucosatongue 22Upper digestive tract 1536Lower digestive tract 823Liver 64Biliary tract (gallbladderbile duct) 21Pancreas 22Breast 610Lung 1112Hematolymphoid system 62Ovary 11Uterus 35Kidney 18Bladder 27Prostate 3520Thyroid 11Skin 11Bone 10Others 21

aCigarettes smoked per day

Of the seven available SNPs with minor allele frequenciesabove 0001 that were located within the exon and intronregions and approximately within the 10 kbp 51015840- and 31015840-flanking regions of the AKT1 SNPs for the associationstudies were selected based on standard tagging strategiesregardless of the functionality of the SNPs [24ndash26] Toidentify relationships between the SNPs that were used inthe study and reduce the burden of tests because some

tests were not independent a linkage disequilibrium (LD)analysis was performed for 300 samples using Haploview v41 [27] To estimate the LD strength between the SNPs thecommonly used 1198631015840 and 1199032 values were pairwise-calculatedusing the genotype dataset of each of the seven SNPs Linkagedisequilibrium blocks were defined among the SNPs withminor allele frequencies above 005 that showed ldquostrong LDrdquobased on the default algorithm of Gabriel et al [28] in which

4 BioMed Research International

the upper and lower 95 confidence limits on 1198631015840 for strongLD were set at 098 and 07 respectively Tag SNPs in theLD block were consequently determined using the Taggersoftware package with default settings which is incorporatedin Haploview and has been detailed in a previous report [26]The Tag SNPs in the LD block and common SNPs outside theblock with minor allele frequencies above 005 were selectedfor the association analyses

24 Statistical Analysis A total of 300 subjects were used forthe initial LD and association analyses For all of the genotypedata that were used in these analyses the distributions werechecked in the entire cohort using the1205942 test and the absenceof significant deviation from the theoretical distributionthat was expected from Hardy-Weinberg equilibrium wasconfirmed Prior to the analyses the subjects were dividedinto two subgroups based on the presence and absence ofcommon cancers (present and past illness) in addition todividing the subjects into three smoking subgroups currentsmokers exsmokers and never-smokers (Table 1) To explorethe associations between the clinical characteristics of thetotal of 999 subjects the 1205942 test or Mann-Whitney testwas performed overall and statistical significance was setto 119875 lt 005 To explore the associations between theSNPs and phenotypes related to smoking and cancer inthe initial 300 subjects the 1205942 test was performed overalland SNPs that showed 119875 lt 005 in the analysis wereconsidered nominally significant and selected for furtheranalysis In the following confirmatory stage of the analysis inthe remaining 699 subjects the 1205942 test was again performedoverall to corroborate the association that was observed inthe exploratory stage of the analysis Analyses of interactionsbetween genotypes of the candidate SNP and smoking-related phenotypes such as smoking history smoking periodCPD and the Brinkman index were conducted with thestatistical significance set to 119875 lt 005 after dividing the entiresample of subjects with available smoking-related phenotypedata into two groups based on categorical phenotypes orhigherlower values of quantitative phenotypes comparedwith each median value considering the classifications ofprevious reports and expected correlations among the pheno-typic values [29 30] Statistical corrections for multiple testssuch as Bonferroni adjustments on the multiple parametersanalyzed were not performed in the present exploratorystudy as a whole because it would be too conservative forgenetic association studies [31] meaning that the likelihoodof type II errors is increased by Bonferroni adjustments andtruly important differences could be deemed nonsignificant[32] All of the statistical analyses were performed using SPSS180J software (International BusinessMachinesCorporationArmonk NY USA)

Statistical power analyses were performed using GPower313 [33] Power analyses for the 1205942 tests revealed that theexpected power (1 minus type II error probability) was 999and 100 for Cohenrsquos conventional ldquomediumrdquo effect size of030 [34] when the degrees of freedom and type I errorprobability were set at 1 and 005 respectively and the samplesizes were 300 and 699 respectively corresponding to the

sample sizes of the exploratory analyses and subsequentconfirmatory analyses in the present study However forthe same type I error probability and sample sizes of 300and 699 the expected power decreased to 410 and 753respectively when Cohenrsquos conventional ldquosmallrdquo effect sizewas 010 Conversely the estimated effect sizeswere 01617 and01060 for the same type I error probability and sample sizes of300 and 699 respectively to achieve 80 power Therefore asingle analysis in the present studywas expected to detect trueassociations with the phenotype with 80 statistical powerfor effect sizes from large to moderately small but not verysmall

3 Results

We explored the contribution of the SNPs in and aroundAKT1 SNPs to various smoking traits and individual cancerhistory in the initial 300 subjects followed by confirmatoryanalyses in the remaining 699 subjects In the analyses of theclinical data prior to association analyses concerning SNPsin a total of 999 subjects significant differences were foundin sex (1205942 = 9876 119875 = 00017) height (119880 = 71176500119875 = 00104) CPD (119880 = 20187000 119875 = 00304) and theBrinkman index (119880 = 19746500 119875 = 00170) betweenindividuals with a current and past cancer history and thosewithout any cancer history Subjects who were male weretaller had a greater CPD and had a higher Brinkman indexwere more susceptible to any cancer compared with controls(ie higher in risk of cancer Table 2) In the list of cancerhistory (Table 1) 95 of 105 subjects with a current history ofcancer and 127 of 136 subjects with a past history of cancerwere smoking-related cases according to Surgeon Generalrsquosreport [35] and the list of cancers that are associated withsmoking is becoming longer [36] In the analysis that focusedonly on smoking-related cancers in which cancers of theuterus thyroid biliary tract (gallbladderbile duct) ovariesbones and other organs (Table 1) were excluded from theanalysis and significant differences were found in smokinghistory (1205942 = 7244 119875 = 00071) sex (1205942 = 17325119875 lt 00001) height (U = 64641500 119875 = 00006) CPD (U= 20086000 119875 = 00316) and the Brinkman index (U =19732500 119875 = 00209) between individuals with a currentand past history of cancer and those without any historyof cancer A similar analysis was conducted after stratifyingthe data by sex which revealed significant differences insmoking history (male 1205942 = 0146 119875 = 07020 female1205942= 5603 119875 = 00179) and age (male U = 28378000119875 = 00256 female U = 9833000 119875 = 09731) between thecancer and control subjects In the analysis that focused onlyon the smoking-related cancers mentioned above significantdifferences were again found in smoking history (male 1205942 =0284 119875 = 05942 female 1205942 = 4483 119875 = 00342) and age(male U = 28071000 119875 = 00244 female U = 8682000119875 = 06950) between the cancer and control subjects Femalesubjects without a smoking history and male subjects whowere older were more susceptible to cancer compared withcontrols (ie they had a higher risk of cancer details notshown)

BioMed Research International 5

Table2Com

paris

onso

fclin

icaldatabetweenpresenceabsence

ofcancer

histo

ry

119899Minim

umMaxim

umMean

SDMedian

Statistic

a119875

Dire

ctionof

association

Sex

962

988

000167lowast

Higherc

ancerrisk

inmales

Cancer

(malefemale)

14863

Con

trol(malefemale)

437314

Age

(years)

970

7422850

006514

Cancer

214

6488

7412

573

7400

Con

trol

756

6094

7343

589

7300

Height(cm

)968

7117650

001035lowast

Higherh

eightincancer

subjects

Cancer

213

140

175

15820

784

16000

Con

trol

755

130

180

15645

892

1570

0Weight(kg)

967

8238600

056215

Cancer

213

30101

5453

1019

5400

Con

trol

754

3090

5477

990

5400

Smok

inghisto

ry971

310

007831

Cancer

(ever-sm

okersnever-sm

okers)

9212

2Con

trol(ever-smokersnever-sm

okers)

377380

Smok

ingperio

d(year)

501

2062450

007276

Cancer

122

1000

6400

4245

1363

4600

Con

trol

379

100

6800

3971

1464

4400

CPD

b501

201870

0003041lowast

HigherC

PDin

cancer

subjects

Cancer

122

250

8000

2375

1510

2000

Con

trol

379

100

10000

2077

1290

2000

Brinkm

an(smoking)

index(BI)

500

1974650

001700lowast

HigherB

Iincancer

subjects

Cancer

122

10000

336000

100164

70825

86000

Con

trol

378

900

348000

8093

85474

768000

a Uvaluefor

Mann-Whitney

testand1205942valuefor1205942test

b Cigarettessmoked

perd

ay

lowastPlt005

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

Additionally genetic variations of AKTs such as single-nucleotide polymorphisms (SNPs) have also beenwell recog-nized to modulate gene function These variations are asso-ciated with a predisposition to and determinant of clinicaloutcomes of endometrial and lung cancers [13ndash15]

Akt1 is also centrally involved in neuronal survival andplasticity [16] AKT1 variations have been reported to beassociated with Parkinsonrsquos disease schizophrenia metham-phetamine use disorder and bipolar disorder [16ndash21] In lightof the critical role of Akt in maintaining proper cellularfunction and tumorigenesis andor tumor progression thescreening ofAKT SNPs is important Among theAKT genesthe present study focused on the AKT1 gene which is themost ubiquitously expressed and assumed to play centralroles in various functions and pathologies

With the goal of identifying allelic variants that signifi-cantly contribute to pathogenesis and smoking-related traitsglobal tests of associationswere performed between each SNPof the AKT1 gene and cancer predisposition and smokingbehaviors

2 Methods

21 Subjects The participants in the initial analysis thatexplored possible associations between AKT1 gene poly-morphisms and the susceptibility to common cancers andsmoking behavior included a total of 999 patients whopresented at or were admitted to Iwata City Hospital in JapanThe inclusion criteria for this study were being Japaneseambulatory able to communicate orally and 60 years of ageor older Numerous participants in this study had varioussmoking habits and completed a questionnaire that con-sisted of various questions about lifestyle including alcoholconsumption smoking diet and cancer history [22 23]Peripheral blood samples were collected from these subjectsfor the gene analysis The detailed demographic and clinicalcharacteristics of the subjects with a focus on cancer andsmoking behaviors are provided in Table 1 These data wereused in the statistical analyses Smoking duration (years)cigarettes smoked per day (CPD) and the product of thesetwo (ie the Brinkman (smoking) index) were incorporatedin the analysis for smoking behaviors

The study protocol was approved by the InstitutionalReview Boards at Hamamatsu University School of Medicine(Hamamatsu Japan) and the Tokyo Metropolitan Institute ofMedical Science (Tokyo Japan) All of the subjects providedinformed written consent for the genetics studies

22 Genotyping Genomic DNA was extracted from whole-blood samples using a QIAamp DNA BloodMaxi kit accord-ing to the manufacturerrsquos instructions (Qiagen HamburgGermany) The extracted DNA was dissolved in TE buffer(10mM Tris-HCl and 1mM ethylenediaminetetraacetic acidpH 80) before use The DNA concentration was adjusted to100 ng120583L for whole-genome genotyping and approximately5ndash50 ng120583L for genotyping the specific rs2498794 SNP usinga NanoDrop ND-1000 Spectrophotometer (NanoDrop Tech-nologies Wilmington DE USA)

Briefly whole-genome genotyping was performed usingthe Infinium assay II with an iScan system (Illumina SanDiego CA USA) according to the manufacturerrsquos instruc-tions HumanCytoSNP-12 v20 (total markers 301232) Bead-Chips were used to genotype the 300 samples from thepatientswith clinical data for cancer and smoking historyTheBeadChips included a number of probes that are specific tocopy number variation markers but most of the BeadChipswere for SNP markers on the human autosome or sexchromosome In the data-cleaning process the samples witha genotype individual level call rate lt095 were intended tobe excluded from the analyses Additionally markers with agenotype call frequency lt095 or ldquoCluster seprdquo (ie an indexof genotype cluster separation) lt01 were excluded from thesubsequent association study Markers were not excludedbased on the heterozygosity rates and results of Hardy-Weinberg equilibrium (HWE) tests for the whole-genomegenotyping data but the HWE tests were conducted for theselected individual SNPs for association analyses Tests forpopulation substructure and relatedness were not conductedbecause it was assumed that all of the subjects were unrelatedand genetically homogeneous Japanese mostly living in theKanto orTokai areaAs a result a total of 291523 SNPmarkerssurvived the filtration process and were used for the datasetof the association analyses

To genotype the rs2498794 SNP using a total of 700DNA samples in the subsequent association study afteran initial exploratory association study the TaqMan allelicdiscrimination assay (Life Technologies Carlsbad CA USA)was basically adopted To perform the TaqMan allelic dis-crimination assaywith a LightCycler 480 (RocheDiagnosticsBasel Switzerland) TaqMan SNP Genotyping Assays (LifeTechnologies) were used that contained sequence-specificforward and reverse primers to amplify the polymorphicsequence and two probes labeled with VIC and FAM dye todetect both alleles of the candidate Tag SNP rs2498794 (assayID C 193159 10) Real-time polymerase chain reactionwas performed in a final volume of 10 120583L that contained2times LightCycler 480 Probes Master (Roche Diagnostics) 40timesTaqMan Genotyping Assays 5ndash50 ng genomic DNA as thetemplate and up to 10120583LH

2O (Roche Diagnostics) The

thermal conditions were the following 95∘C for 10minfollowed by 45 cycles of 95∘C for 10 s and 60∘C for 60 swith final cooling at 50∘C for 30 s Afterward endpointfluorescence was measured for each sample well and eachgenotype was determined based on the presence or absenceof each type of fluorescence

23 Linkage Disequilibrium Analysis To initially analyzeSNPswithin and around theAKT1 gene region genotype datafor approximately 300000 SNP markers that resulted fromwhole-genome genotyping with the patient samples withclinical data for cancer and smoking history were basicallyused and the genotype data for all of the SNPs with AKT1gene annotation were extracted for a total of 300 samplesThe minor allele threshold for the SNP selection was set at0001 which indicates the inclusion of at least oneminor allelecarrier in the 300 samples As a result the rs28546406 SNPwas dropped based on the minor allele frequency criterion

BioMed Research International 3

Table 1 Demographic data of patients

119899 Minimum Maximum Mean SD MedianAll subjects 999

Male 612Female 387

Age (years) 999 60 94 7358 586 7300Height (cm) 996 130 180 15686 869 15800Weight (kg) 996 30 101 5475 998 5400Smoking status 999

Current smokers 130Exsmokers 392Never-smokers 477

Smoking behaviors 522Smoking period (year) 520 100 6800 4050 1440 4400CPDa 520 100 10000 2144 1342 2000Brinkman (smoking) index 519 900 348000 85332 58864 70000

Cancer status (present history) 999Present 89Absent 874Unknown 36

Cancer status (past history) 999Present 128Absent 848Unknown 23

Cancer sites (presentpast history) 105136Oral mucosatongue 22Upper digestive tract 1536Lower digestive tract 823Liver 64Biliary tract (gallbladderbile duct) 21Pancreas 22Breast 610Lung 1112Hematolymphoid system 62Ovary 11Uterus 35Kidney 18Bladder 27Prostate 3520Thyroid 11Skin 11Bone 10Others 21

aCigarettes smoked per day

Of the seven available SNPs with minor allele frequenciesabove 0001 that were located within the exon and intronregions and approximately within the 10 kbp 51015840- and 31015840-flanking regions of the AKT1 SNPs for the associationstudies were selected based on standard tagging strategiesregardless of the functionality of the SNPs [24ndash26] Toidentify relationships between the SNPs that were used inthe study and reduce the burden of tests because some

tests were not independent a linkage disequilibrium (LD)analysis was performed for 300 samples using Haploview v41 [27] To estimate the LD strength between the SNPs thecommonly used 1198631015840 and 1199032 values were pairwise-calculatedusing the genotype dataset of each of the seven SNPs Linkagedisequilibrium blocks were defined among the SNPs withminor allele frequencies above 005 that showed ldquostrong LDrdquobased on the default algorithm of Gabriel et al [28] in which

4 BioMed Research International

the upper and lower 95 confidence limits on 1198631015840 for strongLD were set at 098 and 07 respectively Tag SNPs in theLD block were consequently determined using the Taggersoftware package with default settings which is incorporatedin Haploview and has been detailed in a previous report [26]The Tag SNPs in the LD block and common SNPs outside theblock with minor allele frequencies above 005 were selectedfor the association analyses

24 Statistical Analysis A total of 300 subjects were used forthe initial LD and association analyses For all of the genotypedata that were used in these analyses the distributions werechecked in the entire cohort using the1205942 test and the absenceof significant deviation from the theoretical distributionthat was expected from Hardy-Weinberg equilibrium wasconfirmed Prior to the analyses the subjects were dividedinto two subgroups based on the presence and absence ofcommon cancers (present and past illness) in addition todividing the subjects into three smoking subgroups currentsmokers exsmokers and never-smokers (Table 1) To explorethe associations between the clinical characteristics of thetotal of 999 subjects the 1205942 test or Mann-Whitney testwas performed overall and statistical significance was setto 119875 lt 005 To explore the associations between theSNPs and phenotypes related to smoking and cancer inthe initial 300 subjects the 1205942 test was performed overalland SNPs that showed 119875 lt 005 in the analysis wereconsidered nominally significant and selected for furtheranalysis In the following confirmatory stage of the analysis inthe remaining 699 subjects the 1205942 test was again performedoverall to corroborate the association that was observed inthe exploratory stage of the analysis Analyses of interactionsbetween genotypes of the candidate SNP and smoking-related phenotypes such as smoking history smoking periodCPD and the Brinkman index were conducted with thestatistical significance set to 119875 lt 005 after dividing the entiresample of subjects with available smoking-related phenotypedata into two groups based on categorical phenotypes orhigherlower values of quantitative phenotypes comparedwith each median value considering the classifications ofprevious reports and expected correlations among the pheno-typic values [29 30] Statistical corrections for multiple testssuch as Bonferroni adjustments on the multiple parametersanalyzed were not performed in the present exploratorystudy as a whole because it would be too conservative forgenetic association studies [31] meaning that the likelihoodof type II errors is increased by Bonferroni adjustments andtruly important differences could be deemed nonsignificant[32] All of the statistical analyses were performed using SPSS180J software (International BusinessMachinesCorporationArmonk NY USA)

Statistical power analyses were performed using GPower313 [33] Power analyses for the 1205942 tests revealed that theexpected power (1 minus type II error probability) was 999and 100 for Cohenrsquos conventional ldquomediumrdquo effect size of030 [34] when the degrees of freedom and type I errorprobability were set at 1 and 005 respectively and the samplesizes were 300 and 699 respectively corresponding to the

sample sizes of the exploratory analyses and subsequentconfirmatory analyses in the present study However forthe same type I error probability and sample sizes of 300and 699 the expected power decreased to 410 and 753respectively when Cohenrsquos conventional ldquosmallrdquo effect sizewas 010 Conversely the estimated effect sizeswere 01617 and01060 for the same type I error probability and sample sizes of300 and 699 respectively to achieve 80 power Therefore asingle analysis in the present studywas expected to detect trueassociations with the phenotype with 80 statistical powerfor effect sizes from large to moderately small but not verysmall

3 Results

We explored the contribution of the SNPs in and aroundAKT1 SNPs to various smoking traits and individual cancerhistory in the initial 300 subjects followed by confirmatoryanalyses in the remaining 699 subjects In the analyses of theclinical data prior to association analyses concerning SNPsin a total of 999 subjects significant differences were foundin sex (1205942 = 9876 119875 = 00017) height (119880 = 71176500119875 = 00104) CPD (119880 = 20187000 119875 = 00304) and theBrinkman index (119880 = 19746500 119875 = 00170) betweenindividuals with a current and past cancer history and thosewithout any cancer history Subjects who were male weretaller had a greater CPD and had a higher Brinkman indexwere more susceptible to any cancer compared with controls(ie higher in risk of cancer Table 2) In the list of cancerhistory (Table 1) 95 of 105 subjects with a current history ofcancer and 127 of 136 subjects with a past history of cancerwere smoking-related cases according to Surgeon Generalrsquosreport [35] and the list of cancers that are associated withsmoking is becoming longer [36] In the analysis that focusedonly on smoking-related cancers in which cancers of theuterus thyroid biliary tract (gallbladderbile duct) ovariesbones and other organs (Table 1) were excluded from theanalysis and significant differences were found in smokinghistory (1205942 = 7244 119875 = 00071) sex (1205942 = 17325119875 lt 00001) height (U = 64641500 119875 = 00006) CPD (U= 20086000 119875 = 00316) and the Brinkman index (U =19732500 119875 = 00209) between individuals with a currentand past history of cancer and those without any historyof cancer A similar analysis was conducted after stratifyingthe data by sex which revealed significant differences insmoking history (male 1205942 = 0146 119875 = 07020 female1205942= 5603 119875 = 00179) and age (male U = 28378000119875 = 00256 female U = 9833000 119875 = 09731) between thecancer and control subjects In the analysis that focused onlyon the smoking-related cancers mentioned above significantdifferences were again found in smoking history (male 1205942 =0284 119875 = 05942 female 1205942 = 4483 119875 = 00342) and age(male U = 28071000 119875 = 00244 female U = 8682000119875 = 06950) between the cancer and control subjects Femalesubjects without a smoking history and male subjects whowere older were more susceptible to cancer compared withcontrols (ie they had a higher risk of cancer details notshown)

BioMed Research International 5

Table2Com

paris

onso

fclin

icaldatabetweenpresenceabsence

ofcancer

histo

ry

119899Minim

umMaxim

umMean

SDMedian

Statistic

a119875

Dire

ctionof

association

Sex

962

988

000167lowast

Higherc

ancerrisk

inmales

Cancer

(malefemale)

14863

Con

trol(malefemale)

437314

Age

(years)

970

7422850

006514

Cancer

214

6488

7412

573

7400

Con

trol

756

6094

7343

589

7300

Height(cm

)968

7117650

001035lowast

Higherh

eightincancer

subjects

Cancer

213

140

175

15820

784

16000

Con

trol

755

130

180

15645

892

1570

0Weight(kg)

967

8238600

056215

Cancer

213

30101

5453

1019

5400

Con

trol

754

3090

5477

990

5400

Smok

inghisto

ry971

310

007831

Cancer

(ever-sm

okersnever-sm

okers)

9212

2Con

trol(ever-smokersnever-sm

okers)

377380

Smok

ingperio

d(year)

501

2062450

007276

Cancer

122

1000

6400

4245

1363

4600

Con

trol

379

100

6800

3971

1464

4400

CPD

b501

201870

0003041lowast

HigherC

PDin

cancer

subjects

Cancer

122

250

8000

2375

1510

2000

Con

trol

379

100

10000

2077

1290

2000

Brinkm

an(smoking)

index(BI)

500

1974650

001700lowast

HigherB

Iincancer

subjects

Cancer

122

10000

336000

100164

70825

86000

Con

trol

378

900

348000

8093

85474

768000

a Uvaluefor

Mann-Whitney

testand1205942valuefor1205942test

b Cigarettessmoked

perd

ay

lowastPlt005

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

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International Journal of

Volume 2014

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Microbiology

BioMed Research International 3

Table 1 Demographic data of patients

119899 Minimum Maximum Mean SD MedianAll subjects 999

Male 612Female 387

Age (years) 999 60 94 7358 586 7300Height (cm) 996 130 180 15686 869 15800Weight (kg) 996 30 101 5475 998 5400Smoking status 999

Current smokers 130Exsmokers 392Never-smokers 477

Smoking behaviors 522Smoking period (year) 520 100 6800 4050 1440 4400CPDa 520 100 10000 2144 1342 2000Brinkman (smoking) index 519 900 348000 85332 58864 70000

Cancer status (present history) 999Present 89Absent 874Unknown 36

Cancer status (past history) 999Present 128Absent 848Unknown 23

Cancer sites (presentpast history) 105136Oral mucosatongue 22Upper digestive tract 1536Lower digestive tract 823Liver 64Biliary tract (gallbladderbile duct) 21Pancreas 22Breast 610Lung 1112Hematolymphoid system 62Ovary 11Uterus 35Kidney 18Bladder 27Prostate 3520Thyroid 11Skin 11Bone 10Others 21

aCigarettes smoked per day

Of the seven available SNPs with minor allele frequenciesabove 0001 that were located within the exon and intronregions and approximately within the 10 kbp 51015840- and 31015840-flanking regions of the AKT1 SNPs for the associationstudies were selected based on standard tagging strategiesregardless of the functionality of the SNPs [24ndash26] Toidentify relationships between the SNPs that were used inthe study and reduce the burden of tests because some

tests were not independent a linkage disequilibrium (LD)analysis was performed for 300 samples using Haploview v41 [27] To estimate the LD strength between the SNPs thecommonly used 1198631015840 and 1199032 values were pairwise-calculatedusing the genotype dataset of each of the seven SNPs Linkagedisequilibrium blocks were defined among the SNPs withminor allele frequencies above 005 that showed ldquostrong LDrdquobased on the default algorithm of Gabriel et al [28] in which

4 BioMed Research International

the upper and lower 95 confidence limits on 1198631015840 for strongLD were set at 098 and 07 respectively Tag SNPs in theLD block were consequently determined using the Taggersoftware package with default settings which is incorporatedin Haploview and has been detailed in a previous report [26]The Tag SNPs in the LD block and common SNPs outside theblock with minor allele frequencies above 005 were selectedfor the association analyses

24 Statistical Analysis A total of 300 subjects were used forthe initial LD and association analyses For all of the genotypedata that were used in these analyses the distributions werechecked in the entire cohort using the1205942 test and the absenceof significant deviation from the theoretical distributionthat was expected from Hardy-Weinberg equilibrium wasconfirmed Prior to the analyses the subjects were dividedinto two subgroups based on the presence and absence ofcommon cancers (present and past illness) in addition todividing the subjects into three smoking subgroups currentsmokers exsmokers and never-smokers (Table 1) To explorethe associations between the clinical characteristics of thetotal of 999 subjects the 1205942 test or Mann-Whitney testwas performed overall and statistical significance was setto 119875 lt 005 To explore the associations between theSNPs and phenotypes related to smoking and cancer inthe initial 300 subjects the 1205942 test was performed overalland SNPs that showed 119875 lt 005 in the analysis wereconsidered nominally significant and selected for furtheranalysis In the following confirmatory stage of the analysis inthe remaining 699 subjects the 1205942 test was again performedoverall to corroborate the association that was observed inthe exploratory stage of the analysis Analyses of interactionsbetween genotypes of the candidate SNP and smoking-related phenotypes such as smoking history smoking periodCPD and the Brinkman index were conducted with thestatistical significance set to 119875 lt 005 after dividing the entiresample of subjects with available smoking-related phenotypedata into two groups based on categorical phenotypes orhigherlower values of quantitative phenotypes comparedwith each median value considering the classifications ofprevious reports and expected correlations among the pheno-typic values [29 30] Statistical corrections for multiple testssuch as Bonferroni adjustments on the multiple parametersanalyzed were not performed in the present exploratorystudy as a whole because it would be too conservative forgenetic association studies [31] meaning that the likelihoodof type II errors is increased by Bonferroni adjustments andtruly important differences could be deemed nonsignificant[32] All of the statistical analyses were performed using SPSS180J software (International BusinessMachinesCorporationArmonk NY USA)

Statistical power analyses were performed using GPower313 [33] Power analyses for the 1205942 tests revealed that theexpected power (1 minus type II error probability) was 999and 100 for Cohenrsquos conventional ldquomediumrdquo effect size of030 [34] when the degrees of freedom and type I errorprobability were set at 1 and 005 respectively and the samplesizes were 300 and 699 respectively corresponding to the

sample sizes of the exploratory analyses and subsequentconfirmatory analyses in the present study However forthe same type I error probability and sample sizes of 300and 699 the expected power decreased to 410 and 753respectively when Cohenrsquos conventional ldquosmallrdquo effect sizewas 010 Conversely the estimated effect sizeswere 01617 and01060 for the same type I error probability and sample sizes of300 and 699 respectively to achieve 80 power Therefore asingle analysis in the present studywas expected to detect trueassociations with the phenotype with 80 statistical powerfor effect sizes from large to moderately small but not verysmall

3 Results

We explored the contribution of the SNPs in and aroundAKT1 SNPs to various smoking traits and individual cancerhistory in the initial 300 subjects followed by confirmatoryanalyses in the remaining 699 subjects In the analyses of theclinical data prior to association analyses concerning SNPsin a total of 999 subjects significant differences were foundin sex (1205942 = 9876 119875 = 00017) height (119880 = 71176500119875 = 00104) CPD (119880 = 20187000 119875 = 00304) and theBrinkman index (119880 = 19746500 119875 = 00170) betweenindividuals with a current and past cancer history and thosewithout any cancer history Subjects who were male weretaller had a greater CPD and had a higher Brinkman indexwere more susceptible to any cancer compared with controls(ie higher in risk of cancer Table 2) In the list of cancerhistory (Table 1) 95 of 105 subjects with a current history ofcancer and 127 of 136 subjects with a past history of cancerwere smoking-related cases according to Surgeon Generalrsquosreport [35] and the list of cancers that are associated withsmoking is becoming longer [36] In the analysis that focusedonly on smoking-related cancers in which cancers of theuterus thyroid biliary tract (gallbladderbile duct) ovariesbones and other organs (Table 1) were excluded from theanalysis and significant differences were found in smokinghistory (1205942 = 7244 119875 = 00071) sex (1205942 = 17325119875 lt 00001) height (U = 64641500 119875 = 00006) CPD (U= 20086000 119875 = 00316) and the Brinkman index (U =19732500 119875 = 00209) between individuals with a currentand past history of cancer and those without any historyof cancer A similar analysis was conducted after stratifyingthe data by sex which revealed significant differences insmoking history (male 1205942 = 0146 119875 = 07020 female1205942= 5603 119875 = 00179) and age (male U = 28378000119875 = 00256 female U = 9833000 119875 = 09731) between thecancer and control subjects In the analysis that focused onlyon the smoking-related cancers mentioned above significantdifferences were again found in smoking history (male 1205942 =0284 119875 = 05942 female 1205942 = 4483 119875 = 00342) and age(male U = 28071000 119875 = 00244 female U = 8682000119875 = 06950) between the cancer and control subjects Femalesubjects without a smoking history and male subjects whowere older were more susceptible to cancer compared withcontrols (ie they had a higher risk of cancer details notshown)

BioMed Research International 5

Table2Com

paris

onso

fclin

icaldatabetweenpresenceabsence

ofcancer

histo

ry

119899Minim

umMaxim

umMean

SDMedian

Statistic

a119875

Dire

ctionof

association

Sex

962

988

000167lowast

Higherc

ancerrisk

inmales

Cancer

(malefemale)

14863

Con

trol(malefemale)

437314

Age

(years)

970

7422850

006514

Cancer

214

6488

7412

573

7400

Con

trol

756

6094

7343

589

7300

Height(cm

)968

7117650

001035lowast

Higherh

eightincancer

subjects

Cancer

213

140

175

15820

784

16000

Con

trol

755

130

180

15645

892

1570

0Weight(kg)

967

8238600

056215

Cancer

213

30101

5453

1019

5400

Con

trol

754

3090

5477

990

5400

Smok

inghisto

ry971

310

007831

Cancer

(ever-sm

okersnever-sm

okers)

9212

2Con

trol(ever-smokersnever-sm

okers)

377380

Smok

ingperio

d(year)

501

2062450

007276

Cancer

122

1000

6400

4245

1363

4600

Con

trol

379

100

6800

3971

1464

4400

CPD

b501

201870

0003041lowast

HigherC

PDin

cancer

subjects

Cancer

122

250

8000

2375

1510

2000

Con

trol

379

100

10000

2077

1290

2000

Brinkm

an(smoking)

index(BI)

500

1974650

001700lowast

HigherB

Iincancer

subjects

Cancer

122

10000

336000

100164

70825

86000

Con

trol

378

900

348000

8093

85474

768000

a Uvaluefor

Mann-Whitney

testand1205942valuefor1205942test

b Cigarettessmoked

perd

ay

lowastPlt005

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

the upper and lower 95 confidence limits on 1198631015840 for strongLD were set at 098 and 07 respectively Tag SNPs in theLD block were consequently determined using the Taggersoftware package with default settings which is incorporatedin Haploview and has been detailed in a previous report [26]The Tag SNPs in the LD block and common SNPs outside theblock with minor allele frequencies above 005 were selectedfor the association analyses

24 Statistical Analysis A total of 300 subjects were used forthe initial LD and association analyses For all of the genotypedata that were used in these analyses the distributions werechecked in the entire cohort using the1205942 test and the absenceof significant deviation from the theoretical distributionthat was expected from Hardy-Weinberg equilibrium wasconfirmed Prior to the analyses the subjects were dividedinto two subgroups based on the presence and absence ofcommon cancers (present and past illness) in addition todividing the subjects into three smoking subgroups currentsmokers exsmokers and never-smokers (Table 1) To explorethe associations between the clinical characteristics of thetotal of 999 subjects the 1205942 test or Mann-Whitney testwas performed overall and statistical significance was setto 119875 lt 005 To explore the associations between theSNPs and phenotypes related to smoking and cancer inthe initial 300 subjects the 1205942 test was performed overalland SNPs that showed 119875 lt 005 in the analysis wereconsidered nominally significant and selected for furtheranalysis In the following confirmatory stage of the analysis inthe remaining 699 subjects the 1205942 test was again performedoverall to corroborate the association that was observed inthe exploratory stage of the analysis Analyses of interactionsbetween genotypes of the candidate SNP and smoking-related phenotypes such as smoking history smoking periodCPD and the Brinkman index were conducted with thestatistical significance set to 119875 lt 005 after dividing the entiresample of subjects with available smoking-related phenotypedata into two groups based on categorical phenotypes orhigherlower values of quantitative phenotypes comparedwith each median value considering the classifications ofprevious reports and expected correlations among the pheno-typic values [29 30] Statistical corrections for multiple testssuch as Bonferroni adjustments on the multiple parametersanalyzed were not performed in the present exploratorystudy as a whole because it would be too conservative forgenetic association studies [31] meaning that the likelihoodof type II errors is increased by Bonferroni adjustments andtruly important differences could be deemed nonsignificant[32] All of the statistical analyses were performed using SPSS180J software (International BusinessMachinesCorporationArmonk NY USA)

Statistical power analyses were performed using GPower313 [33] Power analyses for the 1205942 tests revealed that theexpected power (1 minus type II error probability) was 999and 100 for Cohenrsquos conventional ldquomediumrdquo effect size of030 [34] when the degrees of freedom and type I errorprobability were set at 1 and 005 respectively and the samplesizes were 300 and 699 respectively corresponding to the

sample sizes of the exploratory analyses and subsequentconfirmatory analyses in the present study However forthe same type I error probability and sample sizes of 300and 699 the expected power decreased to 410 and 753respectively when Cohenrsquos conventional ldquosmallrdquo effect sizewas 010 Conversely the estimated effect sizeswere 01617 and01060 for the same type I error probability and sample sizes of300 and 699 respectively to achieve 80 power Therefore asingle analysis in the present studywas expected to detect trueassociations with the phenotype with 80 statistical powerfor effect sizes from large to moderately small but not verysmall

3 Results

We explored the contribution of the SNPs in and aroundAKT1 SNPs to various smoking traits and individual cancerhistory in the initial 300 subjects followed by confirmatoryanalyses in the remaining 699 subjects In the analyses of theclinical data prior to association analyses concerning SNPsin a total of 999 subjects significant differences were foundin sex (1205942 = 9876 119875 = 00017) height (119880 = 71176500119875 = 00104) CPD (119880 = 20187000 119875 = 00304) and theBrinkman index (119880 = 19746500 119875 = 00170) betweenindividuals with a current and past cancer history and thosewithout any cancer history Subjects who were male weretaller had a greater CPD and had a higher Brinkman indexwere more susceptible to any cancer compared with controls(ie higher in risk of cancer Table 2) In the list of cancerhistory (Table 1) 95 of 105 subjects with a current history ofcancer and 127 of 136 subjects with a past history of cancerwere smoking-related cases according to Surgeon Generalrsquosreport [35] and the list of cancers that are associated withsmoking is becoming longer [36] In the analysis that focusedonly on smoking-related cancers in which cancers of theuterus thyroid biliary tract (gallbladderbile duct) ovariesbones and other organs (Table 1) were excluded from theanalysis and significant differences were found in smokinghistory (1205942 = 7244 119875 = 00071) sex (1205942 = 17325119875 lt 00001) height (U = 64641500 119875 = 00006) CPD (U= 20086000 119875 = 00316) and the Brinkman index (U =19732500 119875 = 00209) between individuals with a currentand past history of cancer and those without any historyof cancer A similar analysis was conducted after stratifyingthe data by sex which revealed significant differences insmoking history (male 1205942 = 0146 119875 = 07020 female1205942= 5603 119875 = 00179) and age (male U = 28378000119875 = 00256 female U = 9833000 119875 = 09731) between thecancer and control subjects In the analysis that focused onlyon the smoking-related cancers mentioned above significantdifferences were again found in smoking history (male 1205942 =0284 119875 = 05942 female 1205942 = 4483 119875 = 00342) and age(male U = 28071000 119875 = 00244 female U = 8682000119875 = 06950) between the cancer and control subjects Femalesubjects without a smoking history and male subjects whowere older were more susceptible to cancer compared withcontrols (ie they had a higher risk of cancer details notshown)

BioMed Research International 5

Table2Com

paris

onso

fclin

icaldatabetweenpresenceabsence

ofcancer

histo

ry

119899Minim

umMaxim

umMean

SDMedian

Statistic

a119875

Dire

ctionof

association

Sex

962

988

000167lowast

Higherc

ancerrisk

inmales

Cancer

(malefemale)

14863

Con

trol(malefemale)

437314

Age

(years)

970

7422850

006514

Cancer

214

6488

7412

573

7400

Con

trol

756

6094

7343

589

7300

Height(cm

)968

7117650

001035lowast

Higherh

eightincancer

subjects

Cancer

213

140

175

15820

784

16000

Con

trol

755

130

180

15645

892

1570

0Weight(kg)

967

8238600

056215

Cancer

213

30101

5453

1019

5400

Con

trol

754

3090

5477

990

5400

Smok

inghisto

ry971

310

007831

Cancer

(ever-sm

okersnever-sm

okers)

9212

2Con

trol(ever-smokersnever-sm

okers)

377380

Smok

ingperio

d(year)

501

2062450

007276

Cancer

122

1000

6400

4245

1363

4600

Con

trol

379

100

6800

3971

1464

4400

CPD

b501

201870

0003041lowast

HigherC

PDin

cancer

subjects

Cancer

122

250

8000

2375

1510

2000

Con

trol

379

100

10000

2077

1290

2000

Brinkm

an(smoking)

index(BI)

500

1974650

001700lowast

HigherB

Iincancer

subjects

Cancer

122

10000

336000

100164

70825

86000

Con

trol

378

900

348000

8093

85474

768000

a Uvaluefor

Mann-Whitney

testand1205942valuefor1205942test

b Cigarettessmoked

perd

ay

lowastPlt005

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table2Com

paris

onso

fclin

icaldatabetweenpresenceabsence

ofcancer

histo

ry

119899Minim

umMaxim

umMean

SDMedian

Statistic

a119875

Dire

ctionof

association

Sex

962

988

000167lowast

Higherc

ancerrisk

inmales

Cancer

(malefemale)

14863

Con

trol(malefemale)

437314

Age

(years)

970

7422850

006514

Cancer

214

6488

7412

573

7400

Con

trol

756

6094

7343

589

7300

Height(cm

)968

7117650

001035lowast

Higherh

eightincancer

subjects

Cancer

213

140

175

15820

784

16000

Con

trol

755

130

180

15645

892

1570

0Weight(kg)

967

8238600

056215

Cancer

213

30101

5453

1019

5400

Con

trol

754

3090

5477

990

5400

Smok

inghisto

ry971

310

007831

Cancer

(ever-sm

okersnever-sm

okers)

9212

2Con

trol(ever-smokersnever-sm

okers)

377380

Smok

ingperio

d(year)

501

2062450

007276

Cancer

122

1000

6400

4245

1363

4600

Con

trol

379

100

6800

3971

1464

4400

CPD

b501

201870

0003041lowast

HigherC

PDin

cancer

subjects

Cancer

122

250

8000

2375

1510

2000

Con

trol

379

100

10000

2077

1290

2000

Brinkm

an(smoking)

index(BI)

500

1974650

001700lowast

HigherB

Iincancer

subjects

Cancer

122

10000

336000

100164

70825

86000

Con

trol

378

900

348000

8093

85474

768000

a Uvaluefor

Mann-Whitney

testand1205942valuefor1205942test

b Cigarettessmoked

perd

ay

lowastPlt005

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

rs4983556

rs17846832

rs2498794

rs2494743

rs2498787

rs4983387

rs45478396

6

67 66

57

33

50

96

99

49

49

Block 1 (16kb)

D998400

1 2 3 4 5 7 8

(a)

rs4983556

rs17846832

rs2498794

rs45478396

1

1

1 22

18

20

82

81 90

1

1

1

44

0

0

0

0

0

0

0

0

0

2 3 4 5 7 8

Block 1 (16kb)

r2

rs2494743

rs2498787

rs4983387

(b)

Figure 1 State of linkage disequilibrium (LD) between the SNPs within the exon and intron regions and approximately within the 10 kbp 51015840-and 31015840-flanking regions of the AKT1 gene on chromosome 14 based on the genotype data of the 300 samples used in the initial exploratoryassociation analysis Numbers in squares in which two SNPs face represent the percentage of 1198631015840 and 1199032 values calculated from the genotypedata of the SNPs in (a) and (b) respectively Blank squares represent1198631015840 = 1 or 1199032 = 1

rs4983556

rs2494743

rs17846832

rs2498794

rs45478396

rs2498787

rs4983387

NP_0010144311AKT1

NM_0010144311

1kbp

Figure 2 Schematic structure of the human AKT1 gene (gene accession number NM 0010144311) and the location of the seven SNPs thatwere used in the association analyses based on the NCBI database (httpwwwncbinlmnihgovgene accessed May 13 2015) The geneis illustrated between the 31015840- and 51015840-flanking regions corresponding to left and right sides respectively Green boxes and a line connectingthem represent the exon and intron respectively

After whole-genome genotyping an LD analysis wasinitially conducted using the genotype data from 300 sam-ples among a total of 999 samples (Table 1) An LD blockwas observed among the seven SNPs with minor allelefrequencies above 0001 that were located within and aroundthe AKT1 gene region and all three SNPs in the blockwere selected as Tag SNPs in the LD block (Figure 1) Theschematic structure of the gene and location of the SNPsare illustrated in Figure 2 Only one SNP rs2498794 waslocated outside the block withminor allele frequencies above005 Therefore a total of four common SNPs (rs2498794rs2494743 rs2498787 and rs4983387) were selected for theassociation analyses Of these SNPs only one SNP rs2498794was found to be nominally significant (119875 lt 005) in the initialexploratory association analysis between the SNPs and smok-ing or cancer-related phenotypes (Table 3) This SNP was

significantly associated with cancer status (present illness)in the recessive model for the minor T allele in which theTT genotype of this SNP was associated with an increasedrisk of cancer A further analysis of the remaining 699samples to confirm the association that was observed in theexploratory analysis was conducted for this SNP Howeverno significant association was found for this sample set andthe association between the rs2498794 SNP and cancer status(present illness) was not significant even in the combined999-sample set (Table 4) Although a significant associationwas found between this SNP and cancer status (either presentor past history combined) in the confirmatory analysis withdecreased cancer risk in the TT genotype of this SNP theassociation was not significant in the combined sample set(Table 4) suggesting that the influence of this SNP on thesusceptibility to lifetime cancer risk may not be substantial

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

Table3Re

sults

ofexploratoryassociationanalysisbetweenthec

ommon

AKT1

SNPs

andsm

okingor

cancer-related

phenotypes

SNP

CHRa

Positionb

Locatio

nMAFc

Majorm

inor

alleles

119875values

fore

xploratory

associationanalysisin

geno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

dBrinkm

an(smok

ing)

index

rs4983556

14104302924

31015840-flanking

0015

TG

mdashmdash

mdashmdash

mdashmdash

rs17846832

14104309681

intro

n00233

CT

mdashmdash

mdashmdash

mdashmdash

rs2498794

14104316296

intro

n04167

CT

012008510

043lowast

049502930412

046

408090217

042006940265

047903440622

046

8044

50462

rs2494743

14104322765

intro

n04381

CT

058603510

472

050908860255

063203400822

099709800947

099309620906

098308600994

rs2498787

14104327626

intro

n04897

AG

090307360692

06130334060

0098409290906

08710

9310645

096308960787

07510

4550720

rs285464

0614

104333890

51015840-flanking

0GG

mdashmdash

mdashmdash

mdashmdash

rs4983387

14104339273

51015840-flanking

047

GA

073208900490

076004590824

05610

780037

3096308260926

08350659060

4070804880520

rs45478396

14104343869

51015840-flanking

000

67CT

mdashmdash

mdashmdash

mdashmdash

a Chrom

osom

enum

ber

b Chrom

osom

alpo

sition(bp)

c Minor

allelefrequ

ency

d Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandcancer

status

(present

illness)inther

ecessiv

emod

el(119875lt005)

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

Table4Re

sults

ofconfi

rmatoryassociationanalysisbetweenther

s2498794

SNPandsm

okingor

cancer-related

phenotypes

SNP

Stage

Pvaluesfor

confi

rmatoryandcombinedassociationanalyses

ingeno

typicdo

minantrecessiv

emod

elsfor

each

minor

allele

Cancer

status

(present

illness)

Cancer

status

(present

orpast

illnesses)

Smok

inghisto

rySm

okingperio

d(year)

CPD

aBrinkm

an(smok

ing)

index

rs2498794

confi

rmatory

020804530077

007509030038lowast

055208920336

014307430092

091208900744

01410

7330092

rs2498794

combined

091006650890

020504820190

09010

824076

3006

8064

10048dagger

061005290610

007304860070

a Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandlonger

perio

dsof

smok

inghisto

ryin

ther

ecessiv

emod

el(119875lt005)

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

Among the other phenotypic traits a significant associationwas found between the rs2498794 SNP and smoking duration(years) in the combined sample set (Table 4) Homozygouscarriers of the minor T allele in this SNP had longer smokinghistories comparedwith noncarriers suggesting that this SNPmay affect smoking behavior leading to a prolonged periodof smoking in TT carriers of this SNP

The observed association between the rs2498794 SNPand smoking duration in the total of 999 samples couldbe of interest but we could not draw definitive conclusionsabout such a relationship because an association was notfound in either the initial exploratory analyses or subsequentconfirmatory analyses We further examined interactiveeffects between this SNP and phenotypes in the overallsubjects with available smoking-related phenotype data suchas smoking history smoking period CPD and the Brinkmanindex We compared genotype data between the presenceand absence of cancer history after dividing the subjectsinto two groups based on longshort smoking historiesand higherlower values in quantitative variables comparedwith each median value This analysis resulted in significantassociations between the rs2498794 SNP and cancer status(present or past history combined) only in groups with ashorter smoking duration (lt44 years) and lower CPD (le20)Homozygous carriers of the minor T allele in this SNPwere fewer in cancer subjects than in controls when thesmoking duration was short and CPD rate was low (Table 5)suggesting that the TT genotype of this SNP may be relatedto lower susceptibility to cancer (ie this genotype could beassociated with a lower risk of cancer only when the smokingduration is short or CPD rate is low)

4 Discussion

The PI3KAkt pathway fulfills an important role in cellmetabolism proliferation apoptosis and metastasis [2 5]As one of the key components of this pathway somaticmutations of AKTs have also been reported An activatingmutation inAKT1 (E17K) which results in the growth factor-independent membrane translocation of Akt and increasedphosphorylation levels [37] was identified in various typesof cancers including melanoma and breast esophageal col-orectal endometrial ovarian and nonsmall cell lung cancers[38] Moreover an identical mutation in AKT2 was found tocause its membrane localization and the insulin-independentmembrane localization of the GLUT4 glucose transporterand subsequent hypoglycemia [39] AKT1 gene variationsincluding haplotypic variations have been reported to beassociated with various cellular pathological and biologicalphenotypes such as resistance to apoptosis in Epstein-Barrvirus-transformed lymphocyte cells [40] and the cellularresponse to DNA damage [40] Furthermore associationswith human behavior including psychiatric diseases havebeen reported [18 20] There is one report of an associationwith endometrial cancer susceptibility [15]The present studyexamined seven AKT1 gene variations in humans (to theextent that they were on the HumanCytoSNP-12 v20 Bead-Chip) and explored associations between these variationsand outcomes in cancer and smoking behavior that could

be related to each other The rs2498794 SNP was potentlyassociated with smoking duration Homozygous carriers ofthe T allele of this SNP had prolonged smoking durationscompared with noncarriersThe subsequent interactive asso-ciation study indicated that this SNP was also associated withthe susceptibility to cancer only in subgroups with a shortersmoking duration or lower CPD in which homozygouscarriers of the T allele of this SNP had a lower probabilityof predisposition to cancer (ie a decreased risk of cancer)The reason for this may lie in the fact that smoking forlonger periods of time and a higher CPD are well knowngeneral risk factors for cancer particularly lung cancerregardless of polymorphisms [41] Although future studiesshould attempt to replicate the present results the presentstudy demonstrated the possibility that the rs2498794 SNPmay be marginally associated with both smoking durationand cancer risk when the smoking duration is short and CPDrate is low

The candidate SNP that was selected in the present studyrs2498794 is located in the fifth intron region of the AKT1gene To date associations between variations in this sitewith pathological states clinical features or overt diseaseshave not been reported This SNP did not show strong LD(eg 1199032 ge 08) with other SNPs within and around theAKT1 gene region Additionally this SNP may not affectsplicing of the AKT1 gene or regulatory potential scoresaccording to SNP Function Prediction (FuncPred) in theSNPinfo Web Server which compiles information on SNPfunction predictions and ethnicity-specific allele frequencies(httpsnpinfoniehsnihgovsnpinfosnpfunchtm accessedMay 13 2015) Although these results appear to reflect arelatively low possibility that phenotypic alterations that arerelated to the rs2498794 SNP are attributable to alterationsin the function or expression of Akt1 that are caused by thisSNP or other SNPs that are in strong LD with this SNPfuture studies will clarify possible alterations in functionor expression that are related to such AKT1 SNPs Thers2494731 SNP which is located in the intron region (similarto the rs2498794 SNP) is annotated as an SNP that showsmoderately strong LD (eg 1199032 ge 07) with the rs2498794 SNPbased on the SNPinfoWeb Server and is reportedly associatedwith the risk of suicidal behavior in bipolar patients [42] Inaddition to the present results that showed a marginally sig-nificant association between the rs2498794 SNP and smokingduration in the combined 999 samples the rs2498794 SNPmay also be associated with neurobiological mechanisms thatunderlie psychiatric disorders or related phenotypes Futurestudies are needed to clarify the underlying mechanisms bywhich cancer or smoking-related phenotypes are modulatedby this SNP Our findings may provide novel insights forfuture investigations

Although the observed associations in the present studymight be restricted to the Japanese population and theunderlying mechanisms remain to be fully elucidated thepresent results suggest that the rs2498794 SNP may be amarker that predicts prolonged smoking duration and acornerstone for future association and functional studies thatfocus on this SNP

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

Table5Com

paris

onso

fgenotyped

atab

etweenpresenceabsence

ofcancer

histo

rystr

atified

bysm

oking-related

phenotypes

Genotype

Genotypicmod

elDom

inantm

odel

Recessivem

odel

TTTC

CC1205942

119875OR(95

CI)a

1205942

119875OR(95

CI)a

1205942

119875

Smokingh

istory

Ever-smokers

Cancer

1765

403281788

019381

1017

(0659ndash1570)

0005743

093959

0617(0349ndash1090)

2805891

009392

Con

trol

79175

126

Never-smokers

Cancer

1650

2512

34473

053943

1299

(0782ndash215

9)10

22436

031194

0966(053

0ndash17

61)

0012551

091080

Con

trol

68184

124

Smokingp

eriod(year)

Long

(ge44

)Ca

ncer

1331

240392140

082195

1226

(0635ndash2365)

0181513

067008

1226

(0635ndash2365)

0341154

055916

Con

trol

4386

62Short(lt44

)Ca

ncer

434

165917681

005188

1226

(0635ndash2365)

0369160

054346

0338(0115ndash0996)

4191842

004

062lowast

Con

trol

3688

64CP

Db

High(gt20)

Cancer

919

1215

2660

9046

612

1642

(074

3ndash3630)

1514011

021853

1194

(0484ndash2946

)0147578

070086

Con

trol

1836

38Lo

w(le20)

Cancer

846

285512995

006351

0849(0504ndash1428)

0382380

053633

0402(018

4ndash0880)

5491155

001911dagger

Con

trol

61139

88Brinkm

an(sm

oking)index

High(gt700)

Cancer

1233

2413

22869

051611

0961(0536ndash

1723)

0017720

089410

0671(0330ndash

1365)

1223659

026864

Con

trol

4376

61Lo

w(le700)

Cancer

532

163103819

021184

1130

(0586ndash218

1)0133278

071506

0469(0174ndash

1261)

2341073

01260

0Con

trol

3697

65a O

ddsratio

(95

confi

denceinterval)

b Cigarettessmoked

perd

ay

lowastSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

sesm

okingdu

ratio

nisshort

daggerSign

ificant

associationbetweenthem

inor

Talleleof

theS

NPandno

ncancerstatus(presento

rpastilln

ess)in

ther

ecessiv

emod

el(119875lt005)insubjectswho

seCP

Drateislow

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

Disclaimer

The funding agencies had no role in the study design datacollection and analysis decision to publish or preparation ofthe paper

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Daisuke Nishizawa and Yoh Dobashi contributed equally tothis study

Acknowledgments

The authors thank Mr Michael Arends for his assistancewith editing the paper We are grateful to the patients fortheir participation in this study and clinicians at IwataCity Hospital for collecting the clinical data This workwas supported by grants from the Ministry of EducationCulture Sports Science and Technology (MEXT) of Japan(Tokyo Japan nos 22790518 23390377 24659549 2479054425116532 26293347 26860360 and 26460438) Ministry ofHealth Labour andWelfare (MHLW) of Japan (Tokyo Japannos H21-3jigan-ippan-011 H22-Iyaku-015 H25-Iyaku-020H26-Kakushintekigan-ippan-060 and 14524680) SmokingResearch Foundation (Tokyo Japan) and Astellas Founda-tion for Research on Metabolic Disorders (Tokyo Japan)

References

[1] K M Nicholson and N G Anderson ldquoThe protein kinaseBAkt signalling pathway in human malignancyrdquo Cellular Sig-nalling vol 14 no 5 pp 381ndash395 2002

[2] D A Altomare and J R Testa ldquoPerturbations of the AKTsignaling pathway in human cancerrdquo Oncogene vol 24 no 50pp 7455ndash7464 2005

[3] E Gonzalez and T E McGraw ldquoThe Akt kinases isoformspecificity in metabolism and cancerrdquo Cell Cycle vol 8 no 16pp 2502ndash2508 2009

[4] B Vanhaesebroeck and D R Alessi ldquoThe PI3K-PDK1 connec-tion more than just a road to PKBrdquo Biochemical Journal vol346 part 3 pp 561ndash576 2000

[5] M Chen A Cassidy J Gu et al ldquoGenetic variations in PI3K-AKT-mTOR pathway and bladder cancer riskrdquo Carcinogenesisvol 30 no 12 pp 2047ndash2052 2009

[6] K D Courtney R B Corcoran and J A Engelman ldquoThe PI3Kpathway as drug target in human cancerrdquo Journal of ClinicalOncology vol 28 no 6 pp 1075ndash1083 2010

[7] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[8] S Koseoglu Z Lu C Kumar P Kirschmeier and J ZouldquoAKT1 AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively inducesapoptosis in 20 human tumor cell linesrdquo Cancer Biology andTherapy vol 6 no 5 pp 755ndash762 2007

[9] A Bellacosa C C Kumar A D Cristofano and J R TestaldquoActivation of AKT kinases in cancer implications for thera-peutic targetingrdquoAdvances in Cancer Research vol 94 no 1 pp29ndash86 2005

[10] Y Dobashi M Kimura H Matsubara S Endo J Inazawa andA Ooi ldquoMolecular alterations in AKT and its protein activationin human lung carcinomasrdquo Human Pathology vol 43 no 12pp 2229ndash2240 2012

[11] T Kirkegaard C J Witton J Edwards et al ldquoMolecularalterations in AKT1 AKT2 and AKT3 detected in breast andprostatic cancer by FISHrdquoHistopathology vol 56 no 2 pp 203ndash211 2010

[12] K Nakayama N Nakayama N Jinawath et al ldquoAmpliconprofiles in ovarian serous carcinomasrdquo International Journal ofCancer vol 120 no 12 pp 2613ndash2617 2007

[13] M A T Hildebrandt H YangM-C Hung et al ldquoGenetic vari-ations in the PI3KPTENAKTmTOR pathway are associatedwith clinical outcomes in esophageal cancer patients treatedwith chemoradiotherapyrdquo Journal of Clinical Oncology vol 27no 6 pp 857ndash871 2009

[14] X Pu M A T Hildebrandt C Lu et al ldquoPI3KPTENAKTmTOR pathway genetic variation predicts toxicity and distantprogression in lung cancer patients receiving platinum-basedchemotherapyrdquo Lung Cancer vol 71 no 1 pp 82ndash88 2011

[15] L-EWang HMa K S Hale et al ldquoRoles of genetic variants inthe PI3K and RASRAF pathways in susceptibility to endome-trial cancer and clinical outcomesrdquo Journal of Cancer Researchand Clinical Oncology vol 138 no 3 pp 377ndash385 2012

[16] M Hashimoto P Bar-On G Ho et al ldquo120573-synuclein regu-lates Akt activity in neuronal cells a possible mechanism forneuroprotection in Parkinsonrsquos diseaserdquo Journal of BiologicalChemistry vol 279 no 22 pp 23622ndash23629 2004

[17] T Toyota K Yamada S D Detera-Wadleigh and T YoshikawaldquoAnalysis of a cluster of polymorphisms inAKT1 gene in bipolarpedigrees a family-based association studyrdquo Neuroscience Let-ters vol 339 no 1 pp 5ndash8 2003

[18] E S Emamian D Hall M J Birnbaum M Karayiorgou andJ A Gogos ldquoConvergent evidence for impaired AKT1-GSK3120573signaling in schizophreniardquo Nature Genetics vol 36 no 2 pp131ndash137 2004

[19] M Ikeda N Iwata T Suzuki et al ldquoAssociation of AKT1 withschizophrenia confirmed in a Japanese populationrdquo BiologicalPsychiatry vol 56 no 9 pp 698ndash700 2004

[20] M Ikeda N Iwata T Suzuki et al ldquoPositive association ofAKT1 haplotype to Japanese methamphetamine use disorderrdquoInternational Journal of Neuropsychopharmacology vol 9 no 1pp 77ndash81 2006

[21] G Xiromerisiou G M Hadjigeorgiou A Papadimitriou EKatsarogiannis V Gourbali and A B Singleton ldquoAssociationbetweenAKT1 gene and Parkinsonrsquos disease a protective haplo-typerdquo Neuroscience Letters vol 436 no 2 pp 232ndash234 2008

[22] N Sato S Kageyama R Chen et al ldquoAssociation betweenneuropeptide Y receptor 2 polymorphism and the smokingbehavior of elderly Japaneserdquo Journal of Human Genetics vol55 no 11 pp 755ndash760 2010

[23] E Ella N Sato D Nishizawa et al ldquoAssociation betweendopamine beta hydroxylase rs5320 polymorphism and smokingbehaviour in elderly Japaneserdquo Journal of Human Genetics vol57 no 6 pp 385ndash390 2012

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 BioMed Research International

[24] C S Carlson M A Eberle M J Rieder J D Smith LKruglyak and D A Nickerson ldquoAdditional SNPs and linkage-disequilibrium analyses are necessary for whole-genome asso-ciation studies in humansrdquo Nature Genetics vol 33 no 4 pp518ndash521 2003

[25] C S Carlson M A Eberle M J Rieder Q Yi L Kruglyakand D A Nickerson ldquoSelecting a maximally informative setof single-nucleotide polymorphisms for association analysesusing linkage disequilibriumrdquoThe American Journal of HumanGenetics vol 74 no 1 pp 106ndash120 2004

[26] P I W de Bakker R Yelensky I Persquoer S B Gabriel M J Dalyand D Altshuler ldquoEfficiency and power in genetic associationstudiesrdquo Nature Genetics vol 37 no 11 pp 1217ndash1223 2005

[27] J C Barrett B Fry J Maller andM J Daly ldquoHaploview analy-sis and visualization of LD and haplotypemapsrdquo Bioinformaticsvol 21 no 2 pp 263ndash265 2005

[28] S B Gabriel S F Schaffner H Nguyen et al ldquoThe structure ofhaplotype blocks in the human genomerdquo Science vol 296 no5576 pp 2225ndash2229 2002

[29] S M Hartz S E Short N L Saccone et al ldquoIncreased geneticvulnerability to smoking at CHRNA5 in early-onset smokersrdquoArchives of General Psychiatry vol 69 no 8 pp 854ndash860 2012

[30] EH Lips VGaborieau J DMcKay et al ldquoAssociation betweena 15q25 gene variant smoking quantity and tobacco-relatedcancers among 17 000 individualsrdquo International Journal ofEpidemiology vol 39 no 2 pp 563ndash577 2010

[31] D R Nyholt ldquoGenetic case-control association studiesmdashcorrecting for multiple testingrdquo Human Genetics vol 109 no5 pp 564ndash565 2001

[32] T V Perneger ldquoWhatrsquos wrong with Bonferroni adjustmentsrdquoBritish Medical Journal vol 316 no 7139 pp 1236ndash1238 1998

[33] F Faul E Erdfelder A-G Lang and A Buchner ldquoGPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[34] J Cohen Statistical Power Analysis for the Behavioral SciencesAcademic Press New York NY USA 1977

[35] D S Greenberg ldquoTobacco after publicity surge surgeongeneralrsquos report seems to have little enduring effectrdquo Science vol145 no 3636 pp 1021ndash1022 1964

[36] B D Carter C C Abnet D Feskanich et al ldquoSmokingand mortalitymdashbeyond established causesrdquo The New EnglandJournal of Medicine vol 372 no 7 pp 631ndash640 2015

[37] J D Carpten A L Faber C Horn et al ldquoA transformingmutation in the pleckstrin homology domain of AKT1 incancerrdquo Nature vol 448 no 7152 pp 439ndash444 2007

[38] K Shoji K Oda S Nakagawa et al ldquoThe oncogenic mutationin the pleckstrin homology domain of AKT1 in endometrialcarcinomasrdquo British Journal of Cancer vol 101 no 1 pp 145ndash148 2009

[39] K Hussain B Challis N Rocha et al ldquoAn activating mutationof AKT2 and human hypoglycemiardquo Science vol 334 no 6055p 474 2011

[40] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[41] K Wakai M Inoue T Mizoue et al ldquoTobacco smoking andlung cancer risk an evaluation based on a systematic reviewof epidemiological evidence among the Japanese populationrdquoJapanese Journal of Clinical Oncology vol 36 no 5 pp 309ndash3242006

[42] L A V Magno D M Miranda F S Neves et al ldquoAssociationbetween AKT1 but not AKTIP genetic variants and increasedrisk for suicidal behavior in bipolar patientsrdquo Genes Brain andBehavior vol 9 no 4 pp 411ndash418 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology