A possible explanation of the results of this study couldbe that midbrain hyperechogenicity is a TCS marker ofnigral vulnerability or susceptibility to neurodegeneration.This has been suggested by one study of healthy elderlysubjects, which found reduced 18F-Dopa uptake measuredby PET in a subgroup with hyperechogenic SN in TCScompared to subjects with normal midbrain echogenicity.8

Reports of midbrain hyperechogenicity in asymptomaticcarriers of the Parkin gene mutation also suggest that mid-brain echogenicity could be related to PD risk.9 Increasedprevalence of hyperechogenicity in the area of the SN inpatients with ET as compared to healthy controls wouldthen appear consistent with the increased risk for PD thathas been established in ET patient cohorts.3–5 TCS mightprove useful as a screening tool to identify those ET patientsat particular risk to develop PD and prospective studies inlarger ET samples are needed to determine if those withhyperechogenic SN will go on to develop PD while thesewithout will not.

REFERENCES

1. Wenning GK, Kiechl S, Seppi K, et al. Prevalence of movementdisorders in men and women aged 50–89 years (Bruneck Studycohort): a population-based study. Lancet Neurol 2005;4:815-820.

2. Dogu O, Sevim S, Camdeviren H, et al. Prevalence of essentialtremor: door-to-door neurologic exams in Mersin Province, Tur-key. Neurology 2003;61:1804-1806.

3. Jankovic J. Essential tremor: clinical characteristics. Neurology2000;54:S21–S25.

4. Koller WC, Busenbark K, Miner K. The relationship of essentialtremor to other movement disorders: report on 678 patients. Es-sential Tremor Study Group. Ann Neurol 1994;35:717-723.

5. Zorzon M, Capus L, Pellegrino A, Cazzato G, Zivadinov R.Familial and environmental risk factors in Parkinson’s disease: acase-control study in north-east Italy. Acta Neurol Scand 2002;105:77-82;654-658.

6. Schmidauer C, Sojer M, Seppi K, et al. Transcranial ultrasoundshows nigral hypoechogenicity in restless legs syndrome. AnnNeurol 2005;58:630-634.

7. Berg D, Becker G, Zeiler B, et al. Vulnerability of the nigrostriatalsystem as detected by transcranial ultrasound. Neurology 1999;53:1026-1031.

8. Berg D, Siefker C, Ruprecht-Dorfler P, Becker G. Relationship ofsubstantia nigra echogenicity and motor function in elderly sub-jects. Neurology 2001;56:13-17.

9. Walter U, Klein C, Hilker R, Benecke R, Pramstaller PP, DresslerD. Brain parenchyma sonography detects preclinical parkinsonism.Mov Disord 2004;19:1445-1449.

10. Deuschl G, Bain P, Brin M. Consensus statement of the MovementDisorder Society on Tremor. Ad Hoc Scientific Committee. MovDisord 1998;13 (Suppl 3):2-23.

11. Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinicaldiagnosis of idiopathic Parkinson’s disease: a clinico-pathologicalstudy of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181-184.

12. Bain PG, Findley LJ, Atchison P, et al. Assessing tremor severity.J Neurol Neurosurg Psychiatry 1993;56:868-873.

13. Cohen O, Pullman S, Jurewicz E, Watner D, Louis ED. Resttremor in patients with essential tremor: prevalence, clinical cor-relates, and electrophysiologic characteristics. Arch Neurol 2003;60:405-410.

14. Lee MS, Kim YD, Im JH, Kim HJ, Rinne JO, Bhatia KP. 123I-IPTbrain SPECT study in essential tremor and Parkinson’s disease.Neurology 1999;52:1422-1426.

15. Berg D, Siefker C, Becker G. Echogenicity of the substantia nigrain Parkinson’s disease and its relation to clinical findings. J Neurol2001;248:684-689.

Apolipoprotein E ε4 Allele isAssociated with ParkinsonDisease Risk in a Mexican

Mestizo Population

Marisol Lopez, PhD1, Jorge Guerrero, BSc,2

Petra Yescas, MSc,2 Marie-Catherine Boll, MD, PhD,3

Itziar Familiar, MD,2 Adriana Ochoa, MSc,2

Astrid Rasmussen, MD, PhD,2 andMarıa Elisa Alonso, MD2*

1Department of Biological Systems, Universidad AutonomaMetropolitana Xochimilco, Mexico City, Mexico;

2Department of Neurogenetics and Molecular Biology,Instituto Nacional de Neurologıa y Neurocirugıa Manuel

Velasco Suarez, Mexico City, Mexico; 3Division ofNeurology, Instituto Nacional de Neurologıa y Neurocirugıa

Manuel Velasco Suarez, Mexico City, Mexico

Abstract: We investigated the association between apoli-poprotein E (APOE) alleles and genotypes and Parkinsondisease (PD) in 229 unrelated Mexican Mestizo PD patientsand 229 controls. Results showed that both APOE-�4 alleleand APOE �4/�3 genotype are associated with PD (OR �1.736, P � 0.011; OR � 1.688, P � 0.019, respectively).Mean age at onset of PD was not associated to any APOEallele or genotype, but was significantly earlier in familialPD when compared to sporadic cases (P � 0.025). © 2007Movement Disorder Society

Key words: Parkinson disease; apolipoprotein E; age atonset.

Parkinson disease (PD) is the second most commonneurodegenerative disorder, with an estimated preva-lence in industrialized countries of about 1% of individ-uals older than 60 years.

The three cardinal features of PD are rest tremor,rigidity, and bradykinesia.1 Familial aggregation studies

*Correspondence to: Marıa Elisa Alonso, MD, Instituto Nacional deNeurologıa y Neurocirugıa Manuel Velasco Suarez, Departamento deNeurogenetica, Insurgentes Sur 3877, Colonia La Fama, Tlalpan,14269 Mexico D.F., Mexico. E-mail: elisaav@servidor.unam.mx

Received 5 October 2006; Accepted 25 October 2006Published online 17 January 2007 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/mds.21340

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Movement Disorders, Vol. 22, No. 3, 2007

have suggested that genetic factors play a role in theetiology of PD.2 There are a number of families in whichPD segregates in a Mendelian fashion and five geneshave been identified that, when mutated, result in eitherautosomal recessive or dominant PD.3 However, the ma-jority of PD cases are sporadic and the etiology stemsfrom a combination of genetic and environmental causes.

Several association studies have focused on the iden-tification of genes that confer increased susceptibility toPD, among them is apolipoprotein E (APOE).4,5

APOE is a polymorphic protein with three isoforms,ε2, ε3, and ε4, present in elevated concentrations in thebrain. It has been speculated that APOE may play a partin the etiology of neurodegenerative diseases, such as PDand Alzheimer disease (AD).4,6

The APOE-ε4 allele has been shown to influence bothrisk and age at onset of AD.6 In contrast, the role ofAPOE in determining risk of PD has yielded conflictingresults. Some investigations have not found a significantdifference in APOE allele frequencies between PD pa-tients and control individuals,7 and others have reportedcontradictory results in relation to the association ofalleles/genotypes with the risk for developing PD or itsage at onset (AAO).5,6-10 To clarify this issue we studieda large cohort of PD patients from the Mexican Mestizopopulation. Given the genetic admixture profile of ourpopulation we believe that our results have significantimplications for the etiology of PD among Latin Amer-ican populations.

PATIENTS AND METHODS

We studied 229 unrelated Parkinson disease (PD) pa-tients recruited consecutively from January 2003 untilJanuary 2006 in the outpatient Movement Disorder andNeurogenetic Clinics at the National Institute of Neurol-ogy and Neurosurgery (Mexico City). PD was diagnosedthrough manifestations of two or more cardinal featuresof PD: initially unilateral resting tremor, bradykinesia orrigidity, levodopa-responsiveness and/or absence of clin-ical features atypical of PD.1 The reported AAO wasdefined as the age at which an affected individual firstnoticed one of the cardinal signs of PD. Pedigrees wereobtained by direct interviews with the patients and inonly 20 cases the family history was unknown. Weconsidered a family history positive if at least one firstdegree relative was affected. The 229 controls werepair-matched for age and gender, and without diagnosisof a neurodegenerative disorder. They were the spousesof patients with PD or of patients with other diseases thataccompanied them to the institution. To assess an ethnichomogeneity, only cases and controls with both parentsand four grandparents of Mexican Mestizo origin were

included. This study received approval from the NationalInstitute of Neurology and Neurosurgery Internal Re-view Board and informed consent was obtained from allsubjects.

Genomic DNA was genotyped for apolipoprotein E(APOE) polymorphisms by the method of Hixson andVernier with slight modifications.11 APOE genotype andallele frequencies between PD cases and controls werecompared by logistic regression using the status case/control as the dependent variable and APOE alleles andgenotypes as independent variables. The relative risk wasestimated through calculations of the odds ratio (OR)with 95% confidential interval (CI). Mean AAO of dis-ease and APOE alleles or familial/sporadic PD was eval-uated by Student’s t test. Hardy-Weinberg equilibriumwas evaluated based on the �2 test. Values of P � 0.05were considered statistically significant.

RESULTS

Demographic characteristics, clinical data, and APOEgenotype and allele frequencies of the PD patients andcontrol subjects studied are shown in the Table 1. Alleleand genotype frequencies were in accordance withHardy-Weinberg equilibrium. The most frequent geno-type both in PD cases and controls was ε3/ε3 genotype.Logistic regression analysis revealed that only APOE ε4allele and ε4/ε3 genotype were associated with PD risk(OR � 1.736, P � 0.011; OR � 1.688, P � 0.019,respectively).

Mean age at onset of PD was not associated to anyAPOE polymorphism, including APOE-ε4 allele (P �0.063). To further explore this issue, we performed

TABLE 1. Sample characteristics and distribution of APOEgenotypes and alleles in Parkinson disease patients and

controls

Characteristics PD cases Controls

n 229 229Men/women 138/91 138/91Mean age, yr, (SD) 62.28 � 12.85 63.97 � 11.23Mean age at onset 54.12 � 12.37 –Familial/sporadic 37/172 –Unknown family history 20 –APOE

Genotypes (%)ε4/ε4 1.3 0.4ε4/ε3 21.8 14.0ε4/ε2 0.4 0.9ε3/ε3 68.1 78.2ε3/ε2 8.3 6.6

Alleles (%)ε4 12.4 7.9ε3 83.2 88.4ε2 4.4 3.7

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Movement Disorders, Vol. 22, No. 3, 2007

Kaplan-Meier survival analysis to plot AAO distributioncurves for different genotype groups and to estimate thedifference between the curves using log-rank statistics.We did not find significant differences between the AAOsurvival curves in APOE-ε-4 allele carriers vs. noncarri-ers (P � 0.43; Fig. 1), or between the ε2/ε3, ε3/ε3, andε4/ε- genotype groups (P � 0.10; data not shown).

Of the 229 PD patients, 172 comprised sporadic cases(70 men and 102 women) and 37 were familial cases (21men, 16 women). Twenty patients (15 men and 5women) were eliminated due to unknown family history.When we compared the mean AAO of the disease be-tween sporadic (54.99 �11.65 years) and familial PDcases (50.03 �14.39 years) we found that it was signif-icantly earlier in those with a positive family history(P � 0.025).

All results were similar with and without gender ad-justment in PD patients and controls.

DISCUSSION

The results from numerous case-control studies ofAPOE and PD are inconsistent. Some authors report noassociation with any APOE allele,7 while others havefound a positive association with different APOE alleles.The French Parkinson’s Disease Genetics Study Group8

found no association between APOE-ε4 allele and PD,but ε2 allele was significantly more frequent in sporadicPD than in controls. A meta-analysis of 22 studies be-tween PD an APOE estimated a positive associationbetween the APOE-ε2 allele and sporadic PD.9 Unlikethese two reports we did not detect any association withPD risk for the APOE-ε2 allele. Our results support the

association of APOE ε4/ε3 genotype and APOE-ε4 allelewith PD risk in the total sample. However, no associationwas found between APOE-ε4 allele and age at onset ofthe disease. These findings are in agreement with aprevious report where APOE-ε4 allele was associated toPD risk,4 but in disagreement4,12 with respect to theinfluence of this APOE variant in decreasing AAO.

Disregarding APOE genotypes, when we comparedthe mean AAO of the disease between sporadic (54.99�11.65 years) and familial PD cases (50.03 �14.39years) we found that the familial cases were significantlyyounger at disease onset (P � 0.025), in agreement withZareparsi et al.12

The difference in results among several studies be-tween APOE and PD risk may be due to different pop-ulations analyzed in these studies (European Caucasian,Chinese and North American Caucasian) or differencesin statistical power due to sample size. Also, our inves-tigation shows the importance of analyzing PD patientsfamily history taking into account that Parkinson diseaseetiology is most likely a complex interaction of geneticand environmental factors, the controversies observedmay result from differences in critical environmentalexposure that act on risk PD alleles.

There is increasing evidence that APOE alleles havesignificant association with AD, but results have beencontradictory in multiple sclerosis, amyotrophic lateralsclerosis and PD.4 The impact of the APOE isoforms onPD has shown to be more heterogeneous among popu-lations than in AD. This probably results from differentmechanisms which become active in both disorders.

Mexican Mestizo populations consist of gene poolsderived from three primary sources: Amerindian, Euro-pean-Spanish, and African in different proportions de-pending on the geographic region and socio-economicstatus. In our study, PD cases and control individualswere of comparable ethnic origin (Mexican Mestizo)with a low socio-economic status and mainly from thecentral region of Mexico, which would indicate a majorAmerindian contribution, followed by European-Spanishand with almost no African component.13,14

The possible association of APOE and PD risk hasbeen widely investigated in different populations7-10,15

but is underrepresented in Latin American populations,including Mexican Mestizo. To the best of our knowl-edge this is the first investigation of APOE and PD inMexican Mestizo individuals. Our data will extend theliterature in regard to the knowledge of the genetic struc-ture of the Mexican Mestizo population.

Acknowledgments: This study was funded partly by a grantfrom Consejo Nacional de Ciencia y Tecnologıa de Mexico

FIG. 1. Kaplan-Meier curves comparing ages at onset of Parkinsondisease (PD) according to apolipoprotein E-ε4 allele status. The log-rank test was not significant (P � 0.43).

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Movement Disorders, Vol. 22, No. 3, 2007

(CONACYT 6836). We thank Sanjay I. Bidichandani for crit-ical revision of the manuscript and helpful comments.

REFERENCES

1. Samii A, Nutt JG, Ransom BR. Parkinson’s disease. Lancet 2004;363:1783–1793.

2. Alonso ME, Otero E, D’ Regules R, Figueroa HH. Parkinson’sdisease: a genetic study. Can J Neurol Sci 1986;13:248–251.

3. Kachergus J, Mata IF, Hulihan M, et al. Identification of a novelLRRK2 mutation linked to autosomal dominant parkinsonism:evidence of a common founder across European populations. Am JHum Genet 2005;76:672–680.

4. Li YJ, Hauser MA, Scott WK, et al. Apolipoprotein E controls therisk and age at onset of Parkinson disease. Neurology 2004;62:2005–2009.

5. Maraganore DM, Farrer MJ, Hardy JA, McDonnell SK, Schaid DJ,Rocca WA. Case-control study of debrisoquine 4-hydroxylase,N-acetyltransferase 2, and apoliprotein E gene polymorphisms inParkinson�s disease. Mov Disord 2000;15:714–719.

6. Saunders AM, Strittmatter WJ, Schemel D, et al. Association ofapolipoprotein E allele ε4 with late-onset familial and sporadicAlzheimer’s disease. Neurology 1993;43:1467–1472.

7. Eerola J, Launes J, Hellstrom O, Tienari PJ. Apolipoprotein E(APOE), PARKIN and cathecol-o-methyltransferase (COMT)genes and susceptibility to sporadic Parkinson�s disease in Finland.Neurosci Lett 2002;330:296–298.

8. The French Parkinson’s Disease Genetics Study Group. Apoli-poprotein E genotype in familial Parkinson’s disease. J NeurolNeurosurg Psychiatry 1997;63:394–395.

9. Huang X, Chen PC, Poole C. ApoE-ε2 allele associated withhigher prevalence of sporadic Parkinson disease. Neurology 2004;62:2198–2202.

10. Pankratz N, Byder l, Halter C, et al. Presence of an APOE4 alleleresults in significantly earlier onset of Parkinson disease and ahigher risk with dementia. Mov Disord 2006;21:45–49.

11. Hixson JE, Vernier DT. Restriction isotyping of human apoli-poprotein E by gene amplification and cleavage with Hha I. J LipidRes 1990;31:545–548.

12. Zareparsi S, Kaye J, Camicioli R, et al. Modulation of the age atonset of Parkinson’s disease by apolipoprotein E genotypes. AnnNeurol 1997;42:655–658.

13. Lisker R, Ramırez V, Babinsky V. Genetic structure of autochtho-nous populations of Meso-America: Mexico. Hum Biol 1996;68:395–394.

14. Gomez M, Clark RM, Nath SK, et al. Genetic admixture ofEuropean FRDA genes is the cause of Friedreich ataxia in theMexican population. Genomics 2004;84:779–784.

15. Ibarreta D, Gomez-Isla T, Portera-Sanchez A, Parrilla R, AyusoMS. Apolipoprotein E genotype in Spanish patients of Alzheimer�sor Parkinson�s disease. J Neurol Sci 1995;134:146–149.

Pattern of Subclinical PulmonaryDysfunctions in Parkinson’s

Disease and the Effect ofLevodopa

Pramod Kumar Pal, MBBS, MD, DM,1*Talakad N. Sathyaprabha, MBBS, MD,2

Prasad Tuhina, MSc,2 and Kandavel Thennarasu, PhD3

1Department of Neurology, National Institute of MentalHealth and Neurosciences, Bangalore, Karnataka, India;

2Department of Neurophysiology, National Institute ofMental Health and Neurosciences, Bangalore, Karnataka,India; 3Department of Biostatistics, National Institute ofMental Health & Neurosciences, Bangalore, Karnataka,

India

Abstract: We performed a detailed evaluation of pulmonaryfunction in 53 patients with idiopathic Parkinson’s disease(PD) who did not have symptoms of pulmonary or cardiacdysfunction. There was a significant pulmonary dysfunc-tion of restrictive type which partially responded to levo-dopa. Compared to men, women were more severely af-fected. Pulmonary function assessment is recommended inPD, irrespective of severity of disease. © 2007 MovementDisorder Society

Key words: Parkinson’s disease; pulmonary function tests

Pulmonary dysfunctions of obstructive, restrictive,and mixed types have been described in patients withadvanced Parkinson’s disease (PD),1–3 but its prevalencein early PD is not known. The pulmonary dysfunctionsprobably result from poorly coordinated activity of therespiratory muscles, but it is not known if its severity hasgender predilection or correlates with any motor sign ofPD. Finally the role of levodopa in improving pulmonaryfunctions in PD is debatable.2,4,5 Therefore we undertookthis study to determine the severity and pattern of sub-clinical pulmonary dysfunction in PD and to study theeffect of levodopa.

PATIENTS AND METHODS

Subjects

Fifty-three non-demented patients [15 women and 38men; age, 53.3 � 9.5 years (mean � SD)] with idio-

*Correspondence to: P.K. Pal, National Institute of Mental Healthand Neurosciences (NIMHANS), Bangalore 560 029, Karnataka, India.E-mail: pal.pramod@rediffmail.com

Received 27 September 2006; Accepted 25 October 2006Published online 17 January 2007 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/mds.21330

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