Presentation on theme: "Journal Club 2013年6月6日 8:30-8:55 ８階 医局 埼玉医科大学 総合医療センター 内分泌・糖尿病内科"— Presentation transcript:
1 Journal Club 2013年6月6日 8:30-8:55 ８階 医局 埼玉医科大学 総合医療センター 内分泌・糖尿病内科 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 May 9;368(19): doi: /NEJMoaMargolin 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 May 23;368(21): doi: /NEJMoa Epub 2013 May 8.2013年6月6日 8:30-8:55８階 医局埼玉医科大学 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
3 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] ) and daily salmon consumption (OR 0.5, CI ) 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 for seal oil and OR 0.4, CI 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 Dec;17(12):
5 オメガ3系脂肪酸の心血管系イベントへの影響 GISSI-Prevenzioneオメガ3系脂肪酸の心血管系イベントへの影響（%）（%）5.04.03.02.01.0総死亡5.04.03.02.01.0突然死-28%*発現率発現率-34%*-47%*-57%*EPA・DHA製剤による心血管系イベントへの影響について検討した成績です。EPA・DHA製剤投与により、投与6ヵ月後、1年後と早期から有意な心血管系イベント抑制が認められました。【試験方法】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。36912 （月）36912 （月）（%）心血管死5.04.03.02.01.0EPA・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-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%36912 （月）Marchioli R et al., Circulation 2002; 105, 1897より作図
6 EPA･DHA製剤投与後のTGの推移 GISSI-Prevenzione TG 170 160 150 140 （mg/dL）TG170160150140EPA・DHA製剤による心血管系イベントへの影響について検討した試験において、脂質代謝について解析した結果です。EPA・DHA製剤を投与した群、非投与群の2群に分けて検討したところ、EPA・DHA製剤投与群のTG推移は、試験期間を通じて有意に低下したことが示されました。【試験方法】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製剤投与群（n=5,665）EPA・DHA製剤非投与群（n=5,658）612183042（月）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 42ヵ月後のリスク低下率 GISSI-Prevenzione リスク変化率 死亡 非致死 心筋梗塞・ 脳卒中 心血管死 非致死 心筋梗塞・ 総死亡心血管死心臓死冠動脈死突然死（%）-5-10-15-20-25-30-35-40-45-50-15*リスク変化率EPA・DHA製剤による心血管系イベントへの影響について検討した成績です。EPA・DHA製剤投与により、投与早期から有意な心血管系イベント抑制が認められましたが、その効果は42ヵ月後も持続していることが示されました。【試験方法】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。-20**-21**-30***-32**-35****：p＜0.05、 **：p＜0.01、 ***：p＜0.001-45***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 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 GISSI-PrevenzioneEPA･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（%）-10-20-30-40-50-60-70†*EPA・DHA製剤による心血管系イベントへの影響について検討した成績です。EPA・DHA製剤投与により、投与早期から有意な心血管系イベント抑制が認められましたが、その効果は42ヵ月後も持続していることが示されました。【試験方法】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。†‡†‡リスク変化率†††*†‡†*†‡††‡‡†*：p＜0.1、†：p＜0.05、 ‡：p＜0.01、 §：p＜0.001*†3ヵ月後6ヵ月後9ヵ月後12ヵ月後42ヵ月後‡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
16 Kromhout D, Giltay EJ, Geleijnse JM; Alpha Omega Trial Group Kromhout D, Giltay EJ, Geleijnse JM; Alpha Omega Trial Group.: n-3 fatty acids and cardiovascular events after myocardial infarction. N Engl J Med Nov 18;363(21):Low-dose supplementation with EPA–DHA or ALA did not significantly reduce the rate of major cardiovascular eventsFigure 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 Jul 26;367(4):
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:
19 BACKGROUNDTrials 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 METHODSIn 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.
31 ADVERSE EVENTSGastrointestinal 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).
32 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.
33 RESULTSOf 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.
34 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, NCT )
37 https://en.wikipedia.org/wiki/Ubiquitin 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 Ubiquitin tags can also direct proteins to other locations in the cell, where they control other protein and cell mechanisms.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.2009E3 enzymes possess one of two domains:The HECT (Homologous to the E6-AP Carboxyl Terminus) domainThe RING (Really Interesting New Gene) domain (or the closely related U-box domain)https://en.wikipedia.org/wiki/Ubiquitin
38 From the Department of Neurology (D. H. M. , J. D. S 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 mgh.harvard.edu; or to Dr. Katsanis at the Center for Human Disease Modeling, Duke University, Durham NC 27710, or atN Engl J Med 2013;368:
39 BackgroundThe combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive.
40 MethodsWe 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.
42 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.
48 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.
49 Figure 3. Epistatic Effects of the OTUD4 p. G333V Allele 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.
50 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).
55 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.
56 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.
57 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.
58 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.)