Journal Club

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

　　牧野佑子　　髙嶋正利　　　　　 Makino, Yuko Takashima, Masatoshi

2013 年 6 月 6 日　 8:30-8:55８階　医局

Risk and Prevention Study Collaborative Group, Roncaglioni MC, Tombesi M, Avanzini F, Barlera S, Caimi V, Longoni P, Marzona I, Milani V, Silletta MG, Tognoni G, Marchioli R.n-3 fatty acids in patients with multiple cardiovascular risk factors.N Engl J Med. 2013 May 9;368(19):1800-8. doi: 10.1056/NEJMoa1205409.

Margolin DH, Kousi M, Chan YM, Lim ET, Schmahmann JD, Hadjivassiliou M, Hall JE, Adam I, Dwyer A, Plummer L, Aldrin SV, O'Rourke J, Kirby A, Lage K, Milunsky A, Milunsky JM, Chan J, Hedley-Whyte ET, Daly MJ, Katsanis N, Seminara SB.Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination.N Engl J Med. 2013 May 23;368(21):1992-2003. doi: 10.1056/NEJMoa1215993. Epub 2013 May 8.

平成 22 年水産白書

魚介類供給量と平均寿命の関係

0 10 20 30 40 50 60 70 80 90

84

82

80

78

76

74

72

70

ポルトガル韓国

ノルウェースペイン

フランス

フィンランド

ポーランド

トルコ

スロバキア

メキシコ

チェコ米国

英国

オランダギリシア

デンマーク

アイルランド

カナダ

スイス

ドイツ

オーストリア

スウェーデンニュージーランド

豪州

イタリア日本

アイスランド

（歳）

（ kg/ 人 / 年）

平均寿命

FAO 「 Food balance sheets 」（日本以外の国）、農林水産省「食料需給表」、 WHO 「 Statistical Information System（ WHOSIS ）」に基づき水産庁で作成。

http://www.google.co.jp/url?sa=i&rct=j&q=%E3%82%A8%E3%82%B9%E3%82%AD%E3%83%A2%E3%83%BC%E3%80%80%E3%82%AA%E3%83%A1%E3%82%AC3&source=images&cd=&cad=rja&docid=BXLfTVDiai7h8M&tbnid=Zz332-j4lufv-M:&ved=0CAUQjRw&url=http://www.chiropratica.jp/?p=2495&ei=xb9UUY25M4nfkAXI1YGICg&psig=AFQjCNFDVXQPyhIaoL7iV2BsYecM0orZ4A&ust=1364594997827413

Adler AI, Boyko EJ, Schraer CD, Murphy NJ.: Lower prevalence of impaired glucose tolerance and diabetes associated with daily seal oil or salmon consumption among Alaska Natives. Diabetes Care. 1994 Dec;17(12):1498-501.

In our study, the average daily consumption of ω-3 PUFAs(polyunsaturated fatty acid) from seal oil was ~8 g. We based this estimate on a 30% ω-3 PUFA content of seal oil.

Compared with less-than-daily consumption, both daily seal oil (odds ratio [OR] 0.2, 95% confidence interval [CI] 0.1-0.8) and daily salmon consumption (OR 0.5, CI 0.2-1.1) were associated with a lower prevalence of glucose intolerance, controlling for age, ethnicity, body mass index, and sex. The effects were similar when limited to newly discovered cases: OR 0.3, CI 0.1-1.3 for seal oil and OR 0.4, CI 0.1-1.3 for salmon. Consumption of seal oil at least five times per week was required to reduce risk.

【主要評価項目】• 全死亡• 非致死性心筋梗塞、非致死性脳卒中• 心血管死、非致死性心筋梗塞、非致死性脳卒中

【副次評価項目】• 主要評価項目の各因子

GISSI-Prevenzione Ｉ nvestigators., Lancet 1999;354:447 より作図

GISSI-Prevenzione の試験概要

観察期間： 3.5 年間

プラセボ群（ n=2,828 ）

EPA ・ DHA 製剤投与群（ n=2,836 ）

心筋梗塞急性期＊患者（ n=11,324 ）

＊心筋梗塞発現後 3 ヵ月以内

EPA ・ DHA 製剤＋ビタミン E 投与群（ n=2,830 ）

ビタミン E 投与群（ n=2,830 ）

Marchioli R et al., Circulation 2002; 105, 1897 より作図

心筋梗塞発現後 3 ヵ月以内の 11,323 例を対象に、EPA ・ DHA 製剤 1g 群、ビタミン E300mg 群、両薬剤併用群、コントロール群（両薬剤非投与群）の 4 群に無作為に分け、 3.5 年間追跡した GISSI-Prevenzione から、心血管系イベントへの影響について検討した。

オメガ 3 系脂肪酸の心血管系イベントへの影響

突然死

0 3 6 9 12 （月）

5.0

4.0

3.0

2.0

1.0

0

5.0

4.0

3.0

2.0

1.0

00 3 6 9 12 （月）

（ % ）（ % ）

発現率発現率

0 3 6 9 12 （月）

5.0

4.0

3.0

2.0

1.0

0

発現率

EPA ・ DHA 製剤投与群（ n-5,665 ）EPA ・ DHA 製剤非投与群（ n=5,658 ）

* ： p ＜ 0.05 　 vs. EPA ・ DHA 製剤非投与群、 log-rank 検定

-57%*

-47%*

-28%*

-34%*

総死亡

心血管死

-34%

-30%*

（ % ）

GISSI-Prevenzione

Marchioli R et al., Circulation 2002;105:1897 より改変

TG

0 6 12 18 30 42（月）

170

160

150

140

0

（ mg/dL ）

EPA ・ DHA 製剤投与群（ n=5,665 ）EPA ・ DHA 製剤非投与群（ n=5,658 ）

心筋梗塞発現後 3 ヵ月以内の 11,323 例を対象に、 EPA ・ DHA 製剤 1g 群、ビタミン E300mg 群、両薬剤併用群、コントロール群（両薬剤非投与群）の 4 群に無作為に分け、 3.5 年間追跡した GISSI-Prevenzione から、心血管系イベントへの影響について検討した。

EPA ･ DHA 製剤投与後の TG の推移GISSI-Prevenzione

42 ヵ月後のリスク低下率

0

-5

-10

-15

-20

-25

-30

-35

-40

-45

-50

-15*

-20**-21**

-30***

-35***

-32**

-45***

死亡非致死

心筋梗塞・脳卒中

心血管死非致死


総死亡心血管死心臓死冠動脈死突然死


* ： p ＜ 0.05 、 ** ： p ＜ 0.01 、 *** ： p ＜ 0.001

Marchioli R et al., Circulation 2002;105:1897 より作図

GISSI-Prevenzione

リスク変化率

（ % ）

EPA ･ DHA 製剤によるイベント低下は、投与早期から 42 ヵ月後まで認められた

0

-10

-20

-30

-40

-50

-60

-70

†

§

3 ヵ月後 6 ヵ月後

9 ヵ月後

12 ヵ月後

42 ヵ月後

Marchioli R et al., Circulation 2002;105:1897 より作図

* †

†

†† ‡

†

†

†

‡

‡

‡

†

§†

§

†

‡

‡

†

† §

‡

* ： p ＜ 0.1 、†： p ＜ 0.05 、 ‡： p ＜ 0.01 、 § ：p ＜ 0.001

死亡非致死


心血管死非致死


総死亡心血管死心臓死冠動脈死突然死

GISSI-Prevenzione

リスク変化率

*

*

*

（ % ）


• 心血管死• 心血管死および入院• 心臓突然死• 入院• 心血管による入院• 心不全、心筋梗塞、脳卒中による入

院

副次評価項目• 全死亡• 全死亡および心血管系イベントに

よる入院

GISSI-HF Investigators., Lancet 2008;372:1223 より作図

主要評価項目

観察期間： 3.9 年間（中央値）

プラセボ群

EPA ・ DHA 製剤（ 1g/ 日）投与群慢性心不全患者（ n=7,046 ）

• 18 歳以上• NYHA 分類Ⅱ～Ⅳ度• 1 年以内に慢性心不全により入院（ LVEF≧40% の場合）

（ n=3,529 ）

（ n=3,517 ）

GISSI-HF の試験概要

EPA ・ DHA 製剤による慢性心不全患者への影響

GISSI-HF investigators et al., Lancet 2008; 372: 1223.

NYHAⅡ ～Ⅳ度の慢性心不全患者を対象に、 EPA ・ DHA 製剤 1g 投与群またはプラセボ群に無作為に分け、心血管系イベントに及ぼす影響について検討した（平均観察期間： 3.9 年）。※ 補正因子：心不全治療歴、ペースメーカーの使用、大動脈弁狭窄症

総死亡総死亡＋心血管系イベントによる入院

0 6 12 18 24 30 36 42 48 54

70

60

50

40

30

20

10

0p=0.124 （ log-rank 検定）

プラセボEPA ・ DHA 製剤

Adjusted HR* 0.91 （ 95.5% CI 0.833-0.998 ） ; p=0.041Unadjusted HR* 0.93 （ 95.5% CI 0.852-1.021 ） ; p=0.124

0 6 12 18 24 30 36 42 48 54

p=0.059 （ log-rank 検定）

プラセボEPA ・ DHA 製剤

Adjusted HR* 0.92 （ 99% CI 0.849-0.999 ） ; p=0.009Unadjusted HR 0.94 （ 99% CI 0.869-1.022 ） ; p=0.059

GISSI-HF

総死亡

総死亡

+

心血管系イベントによる入院

70

60

50

40

30

20

10

0

（ % ）（ % ）

（月）（月）

オメガ 3 系脂肪酸による心機能への影響

プラセボ 1g/ 日 4g/ 日

30

29

28

27

25

24

22

21

20

26

23

投与前3 ヵ月後


6

5

4

3

1

0

2

1.0±3.8

2.5±3.7

5.5±5.9

ANOVA p=0.03

（％）

LVE

F変

化率

非虚血性心不全患者 43 例を対象に、 EPA ・ DHA 製剤 1g/ 日投与群、 EPA ・ DHA 製剤 4g/ 日投与群、または、プラセボ群に無作為化し、 3 ヵ月間観察した。

GISSI-HFLV

EF

LVEF

Moertl D et al., Am Heart J 2011;161:915.e1 より作図

30

20

10

6

4

2

0

8


4.5

4.0

3.5

3.0

2.5

1.5

1.0

0.5

0

2.0


（ pg/mL ）

p=0.64

プラセボ

0 ヵ月 3 ヵ月3.39 -＞ 3.67

（ 0.22, 3.85 ）（ 0.53, 3.83 ）

0 ヵ月 3 ヵ月

p=0.12

EPA ・ DHA 製剤 1g/ 日

30

20

10

6

4

2

0

8

4.09 -＞ 1.64（ 0.44, 5.87 ）（ 0.02, 3.97 ）

0 ヵ月 3 ヵ月2.98 -＞ 0.68

（ 0.49, 5.26 ）（ 0.02, 1.23 ）

p=0.03

EPA ・ DHA 製剤 4g/ 日


慢性心不全患者におけるオメガ 3 系脂肪酸の炎症（ IL-6 ）への影響


GISSI-HF

IL-6

IL-6

IL-6（ pg/mL ）（ pg/mL ）

30

20

10

6

4

2

0

8

（ pg/mL ）

左室駆出率（ EF ） FMD IL-6

GISSI-HF/3 ヵ月後の変化

30

29

28

27

26

25

24

23

22

21

201g/ 日 4g/ 日プラセボ 1g/ 日 4g/ 日

12

11.5

11

10.5

10

9.5

9

8.5

8プラセボ 1g/ 日 4g/ 日

5

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0



GISSI-HF

プラセボ


GISSI-HF 試験に登録された慢性心不全患者のうち、心エコーにより心機能を評価した 608 例を対象に、EPA ・ DHA 製剤による心機能への影響について検討した。

28.5

29.5

30.5

31.5

32.5

33.5

34.5

（%）

ベースライン 1 2 3

p = 0.005

平均値、 95%CI

EPA ･ DHA 製剤投与群（ n=312 ）

プラセボ投与群（ n=296 ）

（年）

左室駆出率

Ghio S et al., Eur J Heart Fail 2010;12:1345.

EPA ･ DHA 製剤による左室駆出率への影響GISSI-HF

0

eicosapentaenoic acid (EPA). 1800mg/day

Figure 2. Kaplan–Meier Curves for Primary and Secondary End Points. Kaplan–Meier curves are shown for the cumulative incidence of major cardiovascular events (the primary end point) and fatal coronary heart disease (a secondary end point) among 4837 patients who had had a myocardial infarction and were assigned to receive a study margarine containing supplemental eicosapentaenoic acid (EPA) combined with docosahexaenoic acid (DHA), a margarine containing alpha-linolenic acid (ALA), a margarine containing both EPA–DHA and ALA, or a placebo margarine.

Kromhout D, Giltay EJ, Geleijnse JM; Alpha Omega Trial Group.: n-3 fatty acids and cardiovascular events after myocardial infarction. N Engl J Med. 2010 Nov 18;363(21):2015-26.

Low-dose supplementation with EPA–DHA or ALA did not significantly reduce the rate of major cardiovascular events

ORIGIN Trial Investigators, Bosch J, Gerstein HC, Dagenais GR, Díaz R, Dyal L, Jung H, Maggiono AP, Probstfield J, Ramachandran A, Riddle MC, Rydén LE, Yusuf S.: n-3 fatty acids and cardiovascular outcomes in patients with dysglycemia. N Engl J Med. 2012 Jul 26;367(4):309-18.

The members of the writing group are as follows: Maria Carla Roncaglioni, M.Sc., Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)–Istituto di Ricerche Farmacologiche Mario Negri, Milan; Massimo Tombesi, M.D., Centro Studi e Ricerche in Medicina Generale, Monza; Fausto Avanzini, M.D., IRCCS–Istituto di Ricerche Farmacologiche Mario Negri, Milan; Simona Barlera, M.Sc., IRCCS– Istituto di Ricerche Farmacologiche Mario Negri, Milan; Vittorio Caimi, M.D., Centro Studi e Ricerche in Medicina Generale, Monza; Paolo Longoni, M.D., Centro Studi e Ricerche in Medicina Generale, Monza; Irene Marzona, M.Sc., IRCCS–Istituto di Ricerche Farmacologiche Mario Negri, Milan; Valentina Milani, M.Sc., IRCCS–Istituto di Ricerche Farmacologiche Mario Negri, Milan; Maria Giuseppina Silletta, M.Sc., Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti; Gianni Tognoni, M.D., Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti; and Roberto Marchioli, M.D., Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti — all in Italy.

N Engl J Med 2013;368:1800-8.

BACKGROUND Trials have shown a beneficial effect of n−3 polyunsaturated fatty acids in patients with a previous myocardial infarction or heart failure. We evaluated the potential benefit of such therapy in patients with multiple cardiovascular risk factors or atherosclerotic vascular disease who had not had a myocardial infarction.

METHODS In this double-blind, placebo-controlled clinical trial, we enrolled a cohort of patients who were followed by a network of 860 general practitioners in Italy. Eligible patients were men and women with multiple cardiovascular risk factors or atherosclerotic vascular disease but not myocardial infarction. Patients were randomly assigned to n−3 fatty acids (1 g daily) or placebo (olive oil). The initially specified primary end point was the cumulative rate of death, nonfatal myocardial infarction, and nonfatal stroke. At 1 year, after the event rate was found to be lower than anticipated, the primary end point was revised as time to death from cardiovascular causes or admission to the hospital for cardiovascular causes.

ADVERSE EVENTS Gastrointestinal side effects (abdominal pain, nausea, diarrhea, and other symptoms) were the most frequently reported adverse drug reactions, but the incidence did not differ significantly between the two groups (Table 3).

The investigators attributed two cases of severe epistaxis, both in patients who were also receiving anticoagulant or antiplatelet therapy, to the experimental treatment. Among the serious adverse events, there were 490 diagnoses of cancer among patients who received n−3 fatty acids (7.9% of patients) and 453 among those who received placebo (7.2%, P = 0.19); bleeding occurred in 16 patients who received n−3 fatty acids (0.3%) and in 12 who received placebo (0.2%, P = 0.44).

The beneficial effect of n−3 fatty acids in those two trials was due to a reduction in sudden deaths from cardiac causes. It is conceivable that the effects of n−3 fatty acids become manifest primarily in patients who are particularly prone to ventricular arrhythmic events (e.g., those with a myocardial scar or left ventricular dysfunction). Our trial had extremely limited power to detect a reduction in sudden deaths from cardiac causes or arrhythmic events. The safety profile of n−3 fatty acids in this population of older persons who are already receiving many treatments for chronic disease could be of interest for their use in patient populations that are more prone to fatal and nonfatal arrhythmic events.

RESULTS Of the 12,513 patients enrolled, 6244 were randomly assigned to n−3 fatty acids and 6269 to placebo. With a median of 5 years of follow-up, the primary end point occurred in 1478 of 12,505 patients included in the analysis (11.8%), of whom 733 of 6239 (11.7%) had received n−3 fatty acids and 745 of 6266 (11.9%) had received placebo (adjusted hazard ratio with n−3 fatty acids, 0.97; 95% confidence interval, 0.88 to 1.08; P=0.58). The same null results were observed for all the secondary end points.

CONCLUSIONSIn a large general-practice cohort of patients with multiple cardiovascular risk factors, daily treatment with n−3 fatty acids did not reduce cardiovascular mortality and morbidity.

(Funded by Società Prodotti Antibiotici and others; ClinicalTrials.gov number, NCT00317707.)

Message心血管リスクまたは動脈硬化を有するが心筋梗塞の既往はない患者コホート（約 1万人）を対象に、 n－ 3 脂肪酸の効果を無作為化プラセボ対照試験で検討。追跡期間中央値 5 年で、心血管疾患による死亡または入院の発生率は n－ 3脂肪酸群 11.7％、プラセボ（オリーブオイル）群 11.9％だった（調整後ハザード比0.97 、 P＝ 0.58 ）。

オリーブオイルが対象となってフィシュオイルと比較している。同じくらい有効ということであろう。

Ubiquitin is a small regulatory protein that has been found in almost all tissues (ubiquitously) of eukaryotic organisms. It directs proteins to compartments in the cell, including the proteasome which destroys and recycles proteins. Ubiquitin can be attached to proteins and label them for destruction. This discovery won the Nobel Prize for chemistry in 2004. Ubiquitin tags can also direct proteins to other locations in the cell, where they control other protein and cell mechanisms.

https://en.wikipedia.org/wiki/Ubiquitin

In the ubiquitination cascade, E1 can bind with dozens of E2s, which can bind with hundreds of E3s in a hierarchical way. Other ubiquitin-like proteins (ULPs) are also modified via the E1–E2–E3 cascade.E3 enzymes possess one of two domains:• The HECT (Homologous to the E6-AP

Carboxyl Terminus) domain• The RING (Really Interesting New Gene)

domain (or the closely related U-box domain)

2009

From the Department of Neurology (D.H.M., J.D.S.), Harvard Reproductive Sciences Center and Reproductive Endocrine Unit (Y.-M.C., J.E.H., A.D., L.P., S.V.A., J.O., S.B.S.), Analytic and Translational Genetics Unit (E.T.L., A.K., K.L., M.J.D.), Department of Medicine, Pediatric Surgical Research Laboratories (K.L.), and Department of Neuropathology (E.T.H.-W.), Massachusetts General Hospital, Division of Endocrinology, Department of Medicine, Boston Children’s Hospital (Y.-M.C.), and Department of Pathology, Brigham and Women’s Hospital ( J.C.) — all in Boston; Center for Human Genetics, Cambridge, MA (A.M., J.M.M.); Center for Human Disease Modeling, Department of Cell Biology (M.K., N.K.), and Department of Pediatrics (N.K.), Duke University Medical Center, Durham, NC; Department of Neurology, Royal Hallamshire Hospital, Sheffield, United Kingdom (M.H.); Specialty Hospital, Amman, Jordan (I.A.); and Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, and Center for Protein Research, University of Copenhagen, Copenhagen (K.L.). Address reprint requests to Dr. Seminara at the Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, MA 02115, or at seminara.stephanie@ mgh.harvard.edu; or to Dr. Katsanis at the Center for Human Disease Modeling, Duke University, Durham NC 27710, or at [email protected].

N Engl J Med 2013;368:1992-2003.

BackgroundThe combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive.

Methods We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model.

Figure 1. Segregation of RNF216 and OTUD4 Mutations in the Index Pedigree and Identification of Additional RNF216 Mutations in Unrelated Probands. The seven-generation pedigree shown in Panel A includes Patients 1, 2, and 3, all of whom presented with ataxia, dementia, and hypogonadotropic hypogonadism and were homozygous for both RNF216 p.R751C and OTUD4 p.G333V. Double lines indicate consanguineous unions. Genotyped, unaffected family members are shown to be either homozygous for the nonmutated alleles (denoted with a + symbol) or heterozygous for one or both changes. The pedigrees shown in Panel B are for the families of additional RNF216 mutation-positive patients (Patients 4 through 8), all of whom presented with ataxia and hypogonadotropic hypogonadism. Squares denote male family members, circles female family members, solid symbols affected family members, slashes deceased family members, diamonds siblings of either sex, the triangle miscarriages, and Arabic numbers the number of siblings or miscarriages.

Figure 2. Functional Studies of rnf216 in Zebrafish. Panels A through D show dorsal views of control zebrafish embryos (Panel A) and embryos injected with rnf216 morpholino oligonucleotides (MO) (Panel B), rnf216 MO plus nonmutant human RNF216 (Panel C), and rnf216 MO plus mutant human RNF216 (with RNF216 carrying the p.R751C mutation identified in the index pedigree) (Panel D) at 3 days after fertilization (staining with an antibody against α acetylated tubulin). The circles outline the area of the optic tectum, the structure on which all measurements were based. The bar graph in Panel E shows the relative size of the optic tectum in control embryos and the embryos injected with rnf216 MO, rnf216 MO plus nonmutant human RNF216, and rnf216 MO plus mutant human RNF216. P values are based on two-tailed t-tests. I bars indicate standard errors. AU denotes arbitrary units.

Figure 3. Epistatic Effects of the OTUD4 p.G333V Allele. Panels A through F show dorsal views of control zebrafish embryos (Panel A) and embryos injected with rnf216 MO (morpholino oligonucleotides) (Panel B), otud4 MO (Panel C), double MO (DMO, rnf216 MO plus otud4 MO) (Panel D), double MO plus nonmutant human OTUD4 (Panel E), and double (DMO) plus mutant human OTUD4 (OTUD4 carrying the p.G333V mutation identified in the index pedigree) (Panel F) at 3 days after fertilization (anti-α acetylated tubulin stain). The asterisks indicate the optic tecta that were measured to assess the differences between the conditions being evaluated. The bar graph in Panel G shows the mean relative size of the optic tecta in control embryos and the five groups of injected embryos. I bars indicate standard errors. P values are based on two-tailed t-tests.

Panels H, I, and J show dorsal views of control embryos (Panel H) and embryos injected with DMO (Panel I) and DMO plus nonmutant human OTUD4 (Panel J) at 3 days after fertilization (anti-α acetylated tubulin stain). The rectangles outline the cerebellar area; maximum disorganization is observed in embryos injected only with DMO (Panel I). The bar graph in Panel K shows the percentage of embryos with cerebellar defects under the conditions being evaluated (as shown in Panels A through F and Panels H, I, and J).

Figure 4. Neuroradiologic and Neuropathological Findings. Panel A shows a sagittal T2-weighted magnetic resonance imaging scan of the brain in Patient 3. Diffuse cerebellar atrophy (arrow) and cortical atrophy can be seen. Panel B shows a transverse image obtained with fluid-attenuated inversion recovery imaging, revealing multiple distinct and confluent foci of hyperintensity in the white matter. In Panel C, immunohistochemical analysis of a hippocampal brain section from Patient 2 shows a neuronal intranuclear inclusion with immunoreactivity (brown) to an antibody against ubiquitin, counterstained with hematoxylin and eosin. An electron micrograph of the hippocampal neurons, in Panel D, also shows an intranuclear inclusion, which consists of aggregates of granular material and fine filaments, 10 to 15 nm in diameter (arrow), that are for the most part randomly oriented. The scale bar corresponds to 1 μm.

RNF216 encodes an E3 ubiquitin ligase that attaches ubiquitin to protein substrates, marking them for proteasome-mediated degradation.

Figure 5. Endocrine Phenotypes. In Panels A through D, the graphs at the left show the endogenous secretion of luteinizing hormone over a period of up to 12 hours. Patient 6 was studied on two occasions, 15 months apart (Panels A and B). Arrowheads indicate pulses of luteinizing hormone secretion, and boxes duration of sleep; the shading indicates the reference range for healthy men and women. Concentrations of estradiol (E2) and testosterone (T), measured from pooled samples obtained during the study, are indicated. In Panels A, B, and D, the graphs at the right show the response to exogenous pulsatile gonadotropin-releasing hormone (GnRH) over the course of up to 7 days. The dose of GnRH was 75 ng per kilogram of body weight, with the exception of the first dose of GnRH on day 1 for Patient 6 (Panel A), which was 165 ng per kilogram. (Note the difference in the y axis scales in Panels A and B.) In Panel C, the graph at the right shows the secretion of luteinizing hormone in response to varying doses of GnRH (black circles and regression line). The data for the patient fall to the right of the 95% confidence interval (dashed red lines) for the mean amplitude of the response to a range of GnRH doses in 6 other men with idiopathic hypogonadotropic hypogonadism (solid red line).

OTUD4 encodes a deubiquitinating enzyme. Deubiquitinases allow target proteins and ubiquitin itself to be recycled and often function in partnership with specific E3 ligases.

ResultsDigenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin- immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis.

ConclusionThe syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease.

(Funded by the National Institutes of Health and others.)

Message低ゴナドトロピン性性腺機能低下を合併した運動失調症患者の遺伝子変異を全エクソン配列解析などで調査。血族同胞 3 人にユビキチン化に関与する遺伝子 RNF216 とOTUD4 のホモ接合変異が、また他の 5 人にRNF216 のヘテロ接合変異が見られた。ゼブラフィッシュにおけるこれらの遺伝子ノックアウトは眼、視蓋と小脳の異常をもたらした。

全エクソン配列解析！をしてコンピュータで異常遺伝子を見つけている！

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