Journal Club

埼玉医科大学　総合医療センター　内分泌・糖尿病内科Department of Endocrinology and Diabetes,

Saitama Medical Center, Saitama Medical University

松田　昌文Matsuda, Masafumi

2011 年 2 月 10 日　 8:30-8:55８階　医局

Rio Frio T, Bahubeshi A, Kanellopoulou C, Hamel N, Niedziela M, Sabbaghian N, Pouchet C, Gilbert L, O'Brien PK, Serfas K, Broderick P, Houlston RS, Lesueur F, Bonora E, Muljo S, Schimke RN, Bouron-Dal Soglio D, Arseneau J, Schultz KA, Priest JR, Nguyen VH, Harach HR, Livingston DM, Foulkes WD, Tischkowitz M.DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors.JAMA. 2011 Jan 5;305(1):68-77.

Khera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French BC, Phillips JA, Mucksavage ML, Wilensky RL, Mohler ER, Rothblat GH, Rader DJ.Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N Engl J Med. 2011 Jan 13;364(2):127-35.

http://library.med.utah.edu/WebPath/COW/COW084.html

Multinodular goiter の扱いは？　腺腫様甲状腺腫（ Adenomatous goiter ）として「真の腫瘤ではない」という扱いをされて来た。本来adenomatous goiter とう用語は適切ではないらしい。大きな結節は要細胞診

Sertoli-Leydig cell tumour

Sertoli-Leydig cell tumor is a rare cancer of the ovaries. The cancer cells produce and release a male sex hormone.The Sertoli cells are normally located in the male reproductive glands (the testes). They feed sperm cells. The Leydig cells, also located in the testes, release a male sex hormone called testosterone.

Sertoli cell: It is activated by follicle-stimulating hormone and has FSH-receptor on its membranes. Its main function is to nurture the developing sperm cells through the stages of spermatogenesis

Leydig cell: They produce testosterone in the presence of luteinizing hormone (LH).

Histological section through testicular parenchyma of a boar.1 Lumen of Tubulus seminiferus contortus2 spermatids3 spermatocytes4 spermatogonia5 Sertoli cell6 Myofibroblasts7 Leydig cells8 capillaries

JAMA. 2011;305(1):68-77

Program in Cancer Genetics, Departments of Oncology and Human Genetics (Drs Rio Frio, Foulkes, and Tischkowitz; Mr Bahubeshi; and Mss Hamel, Sabbaghian, and Pouchet), and Departments of Obstetrics and Gynecology (Dr Gilbert) and Pathology (Drs Arseneau and Nguyen), Department of Medical Genetics and Research Institute, McGill University Health Centre (Drs Rio Frio and Foulkes and Ms Hamel); Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital (Mr Bahubeshi, Mss Sabbaghian and Pouchet, and Drs Foulkes and Tischkowitz), McGill University, Montreal, Quebec, Canada; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts (Drs Kanellopoulou and Livingston); Department of Pediatric Endocrinology and Rheumatology, Poznan University of Medical Sciences, Poznan, Poland (Dr Niedziela); Department of Pathology, Etobicoke General Hospital, Toronto, Ontario, Canada (Dr O’Brien); Hereditary Breast Health Clinic, Health Sciences Centre Winnipeg, Winnipeg, Manitoba, Canada (Ms Serfas); Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, United Kingdom (Drs Broderick and Houlston); Genetic Cancer Susceptibility Group, International Agency for Research in Cancer, Lyon, France (Dr Lesueur); Medical Genetics Unit, Department of Gynaecology, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy (Dr Bonora); Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (Dr Muljo); Division of Endocrinology, Metabolism and Genetics, Internal Medicine Department, University of Kansas Medical Center, Kansas City (Dr Schimke); Department of Pathology, CHU Sainte-Justine, Montreal (Dr Bouron-Dal Soglio); Children’s Hospital and Clinics of Minnesota, St Paul (Dr Schultz); The International Pleuropulmonary Blas- toma Registry, St Paul (Dr Priest); Sector Patologı´a, Hospital Dr A. On˜ ativia, Salta, Argentina (Dr Harach). Dr Kanellopoulou is currently with the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda.

Context Nontoxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported, and published reports describe 5 families that also contain at least 1 individual with a Sertoli-Leydig cell tumor of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have been identified in families affected by pleuropulmonary blastoma (PPB: 胸膜肺芽腫 ), some of whom include cases of MNG and gonadal tumors such as SLCTs. Objective To determine whether familial MNG with or without SLCT in the absence of PPB was associated with mutations in DICER1.

Design, Setting, and Patients From September 2009 to September 2010, we screened 53 individuals from 2 MNG and 3 MNG/SLCT families at McGill University for mutations in DICER1. We investigated blood lymphocytes and MNG and SLCT tissue from family members for loss of the wild-type DICER1 allele (loss of heterozygosity), DICER1 expression, and microRNA (miRNA) dysregulation.

Main Outcome Measure Detection of germline DICER1 gene mutations in familial MNG with and without SLCT.

Dicer1, Dcr-1 homolog (Drosophila), also known as DICER1, is a protein which in humans is encoded by the DICER1 gene. It is present in many organisms as Dicer.

Function: This gene encodes a protein possessing an RNA helicase motif containing a DEXH box in its amino terminus and an RNA motif in the carboxy terminus. The encoded protein functions as a ribonuclease and is required by the RNA interference and microRNA (a.k.a. small temporal RNA) pathways to produce the active small RNA component that represses gene expression. Two transcript variants encoding the same protein have been identified for this gene.

http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=retrieve&dopt=default&rn=1&list_uids=23405

END

eFigure 4. Schematic diagram of the partial structure of DICER1 protein. The effect of the splice mutation found in family E is shown. Direct sequencing of the 390 bp and 318 bp PCR fragments revealed an in-frame excision of exon 18. The schematic diagram of DICER1 highlights the functional domains of the protein. The amino acid sequence shown represents the entire PAZ domain, with red amino acids indicating those lost as a result of the splice mutation. Underlined amino acids represent those in the PAZ domain which are part of the DICER1-specific loop.

eFigure 6. Schematic diagram of the position of reported germ-line DICER1 mutations. The exons are represented by vertical bars and the introns are represented by the intervening horizontal thin lines. Mutations are shown by their position along the gene, with exons shown approximately to scale. Mutations from this study are shown above the DICER1 gene structure in green type and previously reported mutations are shown below it in black type15,16 (* indicates mutation from Reference 16; other mutations from Reference 15). Notations were adjusted to reflect the NM_001195573 coding sequence (Ensembl no.: ENST00000393063). SLCT = Sertoli-Leydig cell tumor of the ovary; FMNG = familial multinodular goitre; PPB = pleuropulmonary blastoma; LC = lung cysts; CN = cystic nephroma; FCN = familial cystic nephroma; ERMS = embyronic rhabdomyosarcoma; RMS = rhabdomyosarcoma.

DICER1 mutations were also found in both MNG families previously linked to chromosome 14q (families D and E), but not in germline DNA from 71 individuals with differentiated thyroid cancer, 31 of whom also had MNG, and a further 9 individuals with MNG, all of whom had a family history of MNG and differentiated thyroid cancer .

DICER1 staining (reddish brown)

DICER1 staining (reddish brown)

None of the DICER1-related SLCTs showed evidence of LOH, and even though the numbers are small, these findings are consistent with the notion from animal models that DICER1 does not function as a classic tumor suppressor gene but that instead tumors develop as a result of miRNA dysregulation through a possible haploinsufficiency effect. The lack of correlation, however, between DICER1 mutation status and both mRNA and protein levels of DICER1 we observed here suggests that mechanism of tumorigenesis in human DICER1 mutation carriers may be complex and may differ between tissues.

This might indicate the presence of a close relationship between PAZ domain function and the regulation of thyroid development. miRNAs have a crucial role in human development and recent data have shown that germline DICER1 mutations are associated with early-onset malignancy. Perturbations of miRNAs in cancer are common, but constitutive defects in miRNAs have not previously been reported in humans. In light of the central role of DICER1 in miRNA processing, we looked for downstream evidence of miRNA dysregulation in tissues from heterozygotes in familiesDand E with nontruncating mutations (Figure 6), and found that 5 miRNAs were consistently decreased.

Results We identified and characterized germline DICER1 mutations in 37 individuals from 5 families. Two mutations were predicted to be protein truncating, 2 resulted in inframe deletions, and 1 was a missense mutation. Molecular analysis of the 3 SLCTs showed no loss of heterozygosity of DICER1, and immunohistochemical analysis in 2 samples showed strong expression of DICER1 in Sertoli cells but weak staining of Leydig cells. miRNA profiling of RNA from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers.

Conclusions DICER1 mutations are associated with both familial MNG and MNG with SLCT, independent of PPB. These germline DICER1 mutations are associated with dysregulation of miRNA expression patterns.

Message/Comments

多結節性甲状腺腫（ MNG ） 2 家系およびMNG ／卵巣 Sertoli-Leydig 細胞腫 3 家系の 53 人を対象に、 RNase III エンドリボヌクレアーゼ遺伝子 DICER1 の変異の有無を調査。 5 家系 37 人の生殖細胞にDICER1 変異が認められ、 MNG 家系のDICER1 変異保有者ではリンパ芽球様細胞系の miRNA 発現が不安定だった。miRNA が病態にどう関わるかは今後の課題Adenomatous goiter という言い方はMNG で

糖尿病患者における平均 IMT 値の分布糖尿病患者の約 70% が IMT 1.1mm 以上（異常値）を示す

糖尿病患者数

（

人

）

0

40

80

120

0 1 2 3正常

（ 332/1,105 ） 平均 IMT （ mm ）

n=1,105

〔小杉圭右 , 他 : 医薬ジャーナル , 36 （ 6 ） : 154-159, 2000 〕

糖尿病患者 1,105 例の頸動脈 IMT を評価し、平均 IMT 値の分布を検討

健常人、 IGT 、 2 型糖尿病患者の年代別頸動脈IMT

1.5

1.0

0

（ mm ）

0.5

**

**

***

*

30-39 40-49 50-59 60≦（年齢）*p ＜ 0.05 ： vs 健常人 〔平均 ±SE 〕

健常人（ n=55 ）

2 型糖尿病（ n=211 ）IGT （ n=71 ）

頸動脈ＩＭＴ

〔 Yamasaki Y et al ： Diabetologia,38,585-591,1995 〕

健常人 55 例、 2 型糖尿病患者 211 例および IGT 71 例を対象に、それぞれの年代別頸動脈 IMT を計測

原始 HDL

HDL3HDL2

カイロミクロンカイロミクロンレムナント VLDL IDL LDL

肝臓HMG-CoA

コレステロール

TG

LPL

＜コレステロール逆転送系＞

LCAT

AⅠ食事

胆汁酸

コレステロール

＜外因性経路＞

LPL

LDL受容体

＜内因性経路＞

小腸

B-48

E

B-100

末梢組織

リポ蛋白の代謝

レムナント受容体

HTGL

LCAT

SR-BⅠ

B-100B-100

E

CⅡ

HTGL

CⅡ

CETP

E

B-48

E

AⅠAⅠ

AⅡ

CⅡ

A pathway analysis using previously published techniques indicated that 34% of efflux to the serum pool was mediated by ABCA1, 20% by SRBI, and 46% by ABCG1, aqueous diffusion, or undiscovered pathways.

20 ％

46 ％

34 ％

capacity of HDL to accept cholesterol from macrophages

cholesterol efflux capacity

assessed by

ILLUMINATE 試験torcetrapib は CETP 阻害剤は LDL-C という悪玉コレステロールを削減するだけでなく、 HDL-C という善玉コレステロールを大幅に増やすことができる。スタチンでは HDL-C は 2 ～ 3 割増やすが 7 割も増やすことができる。が、ハザードレシオが1.25 倍となり、むしろ有害だった（統計的に有意）。死亡リスクも 1.58 倍と有意に高かった。 (N Engl J Med. 2007 Nov 22;357(21):2109-22.)DEFINE 試験Anacetrapib は torcetrapib で見られた血圧上昇や副腎への影響などは認められなかった。強力な脂質改善効果で， 24週後に LDL コレステロール（ LDL-C ）は半減， HDL コレステロール（ HDL-C ）は倍増した。 (New Engl J Med 2010 Nov. on line)

cholesterol ester transport protein

ABC: 　 ATP-binding cassette transporter

ABCA1 was discovered as the mutation causing Tangier's Disease

Background High-density lipoprotein (HDL) may provide cardiovascular protection by promoting reverse cholesterol transport from macrophages. We hypothesized that the capacity of HDL to accept cholesterol from macrophages would serve as a predictor of atherosclerotic burden.

Methods We measured cholesterol efflux capacity in 203 healthy volunteers who underwent assessment of carotid artery intima–media thickness, 442 patients with angiographically confirmed coronary artery disease, and 351 patients without such angiographically confirmed disease. We quantified efflux capacity by using a validated ex vivo system that involved incubation of macrophages with apolipoprotein B–depleted serum from the study participants.

Cholesterol efflux capacity measurementThis assay quantifies total efflux mediated by pathways of known relevance in cholesterol efflux from macrophages (i.e., ATPbinding cassette transporter A1 [ABCA1] and G1 [ABCG1], scavenger receptor B1, and aqueous diffusion). Cholesterol efflux capacity in the coronary disease and pharmacologic-study cohorts was quantified with the use of a slightly modified method designed to increase throughput. J774 cells, derived from a murine macrophage cell line, were plated and radiolabeled with 2 μCi of 3H-cholesterol per milliliter. ABCA1 was up-regulated by means of a 6-hour incubation with 0.3 mM 8-(4-chlorophenylthio)-cyclic AMP. Subsequently, efflux mediums containing 2.8% apolipoprotein B–depleted serum were added for 4 hours. All steps were performed in the presence of the acyl–coenzyme A:cholesterol acyltransferase inhibitor CP113,818 (2 μg per milliliter). Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from the cells. The quantity of radioactive cholesterol incorporated into cellular lipids was calculated by means of isopropanol extraction of control wells not exposed to patient serum. Percent efflux was calculated by the following formula: [(microcuries of 3H-cholesterol in mediums containing 2.8% apolipoprotein B–depleted serum − microcuries of 3H-cholesterol in serum-free mediums) ÷ microcuries of 3H-cholesterol in cells extracted before the efflux step] × 100.

The mean carotid intima–media thickness was 0.66±0.13 mm. Age, systolic blood pressure, and glycated hemoglobin level were each associated with increased carotid intima–media thickness in bivariate analyses (P<0.05 for each comparison). No significant relationship between HDL cholesterol level and carotid intima–media thickness was noted in either unadjusted or adjusted models (P = 0.37 and P = 0.73, respectively).

We noted increased efflux capacity after therapy with pioglitazone, a phenomenon that could be related to enhanced transcription of apolipoprotein A-I.

Results The levels of HDL cholesterol and apolipoprotein A-I were significant determinants of cholesterol efflux capacity but accounted for less than 40% of the observed variation. An inverse relationship was noted between efflux capacity and carotid intima–media thickness both before and after adjustment for the HDL cholesterol level. Furthermore, efflux capacity was a strong inverse predictor of coronary disease status (adjusted odds ratio for coronary disease per 1-SD increase in efflux capacity, 0.70; 95% confidence interval [CI], 0.59 to 0.83; P<0.001). This relationship was attenuated, but remained significant, after additional adjustment for the HDL cholesterol level (odds ratio per 1-SD increase, 0.75; 95% CI, 0.63 to 0.90; P = 0.002) or apolipoprotein A-I level (odds ratio per 1-SD increase, 0.74; 95% CI, 0.61 to 0.89; P = 0.002). Additional studies showed enhanced efflux capacity in patients with the metabolic syndrome and low HDL cholesterol levels who were treated with pioglitazone, but not in patients with hypercholesterolemia who were treated with statins.

Conclusions Cholesterol efflux capacity from macrophages, a metric of HDL function, has a strong inverse association with both carotid intima–media thickness and the likelihood of angiographic coronary artery disease, independently of the HDL cholesterol level.

(Funded by the National Heart, Lung, and Blood Institute and others.)

   

Message/Comments

健常人および冠動脈疾患患者計 996 人を対象に、高比重リポ蛋白（ HDL ）のコレステロール排出能とアテローム性動脈硬化の関連を調査。コレステロール排出能は、 HDL コレステロール値に関係なく、頸動脈内膜中膜厚および動脈硬化リスクと逆相関しており、動脈硬化の予測因子となりうることが示された。

コレステロールの「引き抜き」が大切！