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Journal Club

Journal Club. 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.

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Journal Club

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  1. Journal Club 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. MargolinDH, 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. 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8:30-8:55 ８階　医局

  2. 魚介類供給量と平均寿命の関係 （歳） イタリア 日本 ニュージーランド 豪州 スイス スウェーデン 84 フランス ドイツ アイスランド カナダ ノルウェー スペイン アイルランド オーストリア 82 オランダ ギリシア フィンランド ポルトガル 英国 韓国 デンマーク 80 平均寿命 チェコ 米国 メキシコ 78 ポーランド スロバキア 76 トルコ 74 72 （kg/人/年） 70 0 10 20 30 40 50 60 70 80 90 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。 平成22年水産白書

  3. In our study, the average daily consumption of ω-3 PUFAs(polyunsaturated fattyacid) 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. 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.

  4. GISSI-Prevenzioneの試験概要 EPA・DHA製剤投与群（n=2,836） 心筋梗塞急性期＊患者 （n=11,324） EPA・DHA製剤＋ビタミンE投与群（n=2,830） ＊心筋梗塞発現後3ヵ月以内 ビタミンE投与群（n=2,830） プラセボ群（n=2,828） 観察期間：3.5年間 • 【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-PrevenzioneＩnvestigators., Lancet 1999;354:447より作図

  5. GISSI-Prevenzione オメガ3系脂肪酸の心血管系イベントへの影響 （%） （%） 5.0 5.0 5.0 総死亡 突然死 4.0 4.0 4.0 -28%* 3.0 3.0 3.0 発現率 発現率 -34%* 2.0 2.0 2.0 -47%* 1.0 1.0 1.0 -57%* 0 0 0 0 0 0 3 3 3 6 6 6 9 9 9 12 （月） 12 （月） 12 （月） （%） 心血管死 EPA・DHA製剤投与群（n-5,665） EPA・DHA製剤非投与群（n=5,658） *：p＜0.05vs. EPA・DHA製剤非投与群、log-rank検定 -30%* 発現率 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。 -34% Marchioli R et al., Circulation 2002; 105, 1897より作図

  6. GISSI-Prevenzione EPA･DHA製剤投与後のTGの推移 TG （mg/dL） 170 160 150 EPA・DHA製剤投与群（n=5,665） EPA・DHA製剤非投与群（n=5,658） 140 （月） 0 0 6 12 18 30 42 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。 Marchioli R et al., Circulation 2002;105:1897より改変

  7. GISSI-Prevenzione 42ヵ月後のリスク低下率 死亡非致死 心筋梗塞・ 脳卒中 心血管死非致死 心筋梗塞・ 脳卒中 総死亡 心血管死 心臓死 冠動脈死 突然死 （%） 0 -5 -10 -15 -15* リスク変化率 -20 -20** -21** -25 -30 -30*** -32** -35 -35*** -40 -45 *：p＜0.05、**：p＜0.01、***：p＜0.001 -45*** -50 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。 Marchioli R et al., Circulation 2002;105:1897より作図

  8. GISSI-Prevenzione 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 死亡非致死 心筋梗塞・ 脳卒中 心血管死非致死 心筋梗塞・ 脳卒中 総死亡 心血管死 心臓死 冠動脈死 突然死 0 （%） -10 -20 † ‡ † ‡ † * リスク変化率 -30 † † * † † § ‡ -40 * † † § ‡ † † -50 ‡ § ‡ † § -60 * 42ヵ月後 3ヵ月後 6ヵ月後 9ヵ月後 12ヵ月後 *：p＜0.1、†：p＜0.05、 ‡：p＜0.01、 §：p＜0.001 † -70 ‡ 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。 Marchioli R et al., Circulation 2002;105:1897より作図

  9. GISSI-HFの試験概要 慢性心不全患者 （n=7,046） EPA・DHA製剤（1g/日）投与群 （n=3,529） • 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） プラセボ群 （n=3,517） 観察期間：3.9年間（中央値） 主要評価項目 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 • 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 GISSI-HF Investigators., Lancet 2008;372:1223より作図

  10. GISSI-HF EPA・DHA製剤による慢性心不全患者への影響 総死亡 総死亡＋心血管系イベントによる入院 （%） （%） 70 70 プラセボ EPA・DHA製剤 プラセボ EPA・DHA製剤 総死亡+心血管系イベントによる入院 60 60 50 50 40 40 総死亡 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 30 30 20 20 Adjusted HR* 0.92（99% CI 0.849-0.999）; p=0.009Unadjusted HR0.94（99% CI 0.869-1.022）; p=0.059 10 10 p=0.124（log-rank検定） p=0.059（log-rank検定） 0 0 0 0 6 6 12 12 18 18 24 24 30 30 36 36 42 42 48 48 54 54 （月） （月） 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 GISSI-HF investigators et al.,Lancet 2008; 372: 1223.

  11. GISSI-HF オメガ3系脂肪酸による心機能への影響 ANOVA p=0.03 LVEF （％） 30 6 5.5±5.9 投与前 3ヵ月後 29 5 28 27 4 26 LVEF LVEF変化率 25 3 2.5±3.7 24 2 23 22 1.0±3.8 1 21 20 0 プラセボ 1g/日 4g/日 プラセボ 1g/日 4g/日 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。 Moertl D et al., Am Heart J 2011;161:915.e1より作図

  12. GISSI-HF 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 プラセボ EPA・DHA製剤1g/日 EPA・DHA製剤4g/日 IL-6 30 30 30 4.5 （pg/mL） （pg/mL） （pg/mL） （pg/mL） p=0.64 p=0.12 p=0.03 20 20 20 投与前 3ヵ月後 4.0 10 10 10 3.5 3.0 8 8 8 2.5 6 6 6 2.0 IL-6 IL-6 1.5 4 4 4 1.0 2 2 2 0.5 0 0 0 0 プラセボ 1g/日 4g/日 0ヵ月 3ヵ月 0ヵ月 3ヵ月 0ヵ月 3ヵ月 3.39 -＞ 3.67 4.09 -＞ 1.64 2.98 -＞ 0.68 （0.22, 3.85） （0.53, 3.83） （0.44, 5.87） （0.02, 3.97） （0.49, 5.26） （0.02, 1.23） 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。 Moertl D et al., Am Heart J 2011;161:915.e1より作図

  13. GISSI-HF GISSI-HF/3ヵ月後の変化 左室駆出率（EF） FMD IL-6 30 5 12 投与前 3ヵ月後 4.5 29 11.5 28 4 11 3.5 27 10.5 26 3 2.5 25 10 24 2 9.5 1.5 23 9 22 1 8.5 0.5 21 20 0 8 プラセボ 1g/日 4g/日 プラセボ 1g/日 4g/日 プラセボ 1g/日 4g/日 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。 Moertl D et al., Am Heart J 2011;161:915.e1より作図

  14. GISSI-HF EPA･DHA製剤による左室駆出率への影響 （%） 34.5 EPA･DHA製剤投与群（n=312） 33.5 左室駆出率 32.5 31.5 プラセボ投与群（n=296） 30.5 平均値、95%CI 29.5 p = 0.005 28.5 0 （年） ベースライン 1 2 3 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。 GhioS et al., Eur J Heart Fail 2010;12:1345.

  15. eicosapentaenoic acid (EPA). 1800mg/day

  16. 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 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.

  17. 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.

  18. The members of the writing group are as follows: Maria Carla Roncaglioni, M.Sc., Istituto di Ricovero e Cura a CarattereScientifico (IRCCS)–Istituto di RicercheFarmacologiche Mario Negri, Milan; Massimo Tombesi, M.D., Centro Studi e Ricerche in MedicinaGenerale, Monza; FaustoAvanzini, M.D., IRCCS–Istituto di RicercheFarmacologiche Mario Negri, Milan; SimonaBarlera, M.Sc., IRCCS– Istituto di RicercheFarmacologiche Mario Negri, Milan; Vittorio Caimi, M.D., Centro Studi e Ricerche in MedicinaGenerale, Monza; Paolo Longoni, M.D., Centro Studi e Ricerche in MedicinaGenerale, Monza; Irene Marzona, M.Sc., IRCCS–Istituto di RicercheFarmacologiche Mario Negri, Milan; ValentinaMilani, M.Sc., IRCCS–Istituto di RicercheFarmacologiche Mario Negri, Milan; Maria GiuseppinaSilletta, M.Sc., Consorzio Mario NegriSud, Santa Maria Imbaro, Chieti; Gianni Tognoni, M.D., Consorzio Mario NegriSud, Santa Maria Imbaro, Chieti; and Roberto Marchioli, M.D., Consorzio Mario NegriSud, Santa Maria Imbaro, Chieti — all in Italy. N Engl J Med 2013;368:1800-8.

  19. 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.

  20. 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.

  21. 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).

  22. 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.

  23. 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.

  24. CONCLUSIONS In 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 SocietàProdottiAntibiotici and others; ClinicalTrials.gov number, NCT00317707.)

  25. 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。

  26. 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. 2009 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) https://en.wikipedia.org/wiki/Ubiquitin

  27. 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 katsanis@cellbio.duke.edu. N Engl J Med 2013;368:1992-2003.

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

  29. Methods We performed whole-exome sequencing in a patient with ataxia and hypogonadotropichypogonadism, 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.

  30. 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 hypogonadotropichypogonadism 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 hypogonadotropichypogonadism. 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.

  31. 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.

  32. 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.

  33. 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).

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