letters
John C Chambers1,2,11, Jing Zhao3,11, Cesare M N Terracciano4,11, Connie R Bezzina5,6,11, Weihua Zhang1,2, Riyaz Kaba4, Manoraj Navaratnarajah4, Amol Lotlikar3, Joban S Sehmi2,4, Manraj K Kooner2,4, Guohong Deng2,7, Urszula Siedlecka4, Saurabh Parasramka2, Ismail El-Hamamsy4, Mark N Wass8, Lukas R C Dekker6, Jonas S S G de Jong6, Michael J E Sternberg8, William McKenna9, Nicholas J Severs4, Ranil de Silva2,4, Arthur A M Wilde5,6, Praveen Anand10, Magdi Yacoub4,11, James Scott4,11, Paul Elliott1,11, John N Wood3,11 & Jaspal S Kooner2,4,11
Coronary heart disease is the leading cause of global mortality, accounting for almost 7 million deaths per year world wide; over 80% of these deaths occur in low- and middle-income countries1,2. More than half of coronary deaths are sudden, with most resulting from ventricular arrhythmia3,4. Risk of arrhythmia is strongly influenced by genetic factors; mutations in the genes encoding cardiac ion channels and sarcomeric and cytoskeletal proteins underlie multiple arrhythmia syndromes, including sudden death3–5. Electrocardiographic time intervals (that is, the PR, QRS and corrected QT (QTc) intervals) predict cardiac arrhythmia4,6–9. We carried out a genome-wide association study of the PR, QRS and QTc intervals to identify new genetic mechanisms influencing cardiac conduction and arrhythmic risk. Genome-wide association was done in 6,543 Indian Asian men and women from the UK participating in the London Life Sciences
Population (LOLIPOP) study (Supplementary Table 1 and Online Methods); this comprises an ethnic group with high cardiovascular disease mortality rates10. Participants classified as having Indian Asian ancestry reported having all four grandparents born PR interval
15
–log10(P)
SCN10A
10
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0 QRS interval
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To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 × 10–15) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10–5 to 10–20). SCN10A encodes NaV1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a–/– mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.
5
0 QT interval
10 –log10(P)
© 2010 Nature America, Inc. All rights reserved.
Genetic variation in SCN10A influences cardiac conduction
NOS1AP C6orf204
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Figure 1 Manhattan plot showing the association of SNPs with PR interval, QRS interval and QTc interval in the genome-wide association study.
1Department
of Epidemiology and Public Health, Imperial College London, London, UK. 2Ealing Hospital National Health Service (NHS) Trust, Middlesex, UK. Institute for Biomedical Research, University College London, London, UK. 4National Heart and Lung Institute, Imperial College London, London, UK. 5Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands. 6Department of Cardiology, Academic Medical Center, Amsterdam, The Netherlands. 7Department of Gastroenterology and Hepatology, Imperial College London, London, UK. 8Structural Bioinformatics Group, Imperial College London, London, UK. 9Institute of Cardiovascular Science, University College London, London, UK. 10Department of Clinical Neuroscience, Imperial College London, London, UK. 11These authors contributed equally to this work. Correspondence should be addressed to J.C.C. (
[email protected]) or J.S.K. (
[email protected]). 3Wolfson
Received 30 September 2009; accepted 20 November 2009; published online 10 January 2010; doi:10.1038/ng.516
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association of the SCN10A locus with PR interval. In a regression analysis conditioned on rs6795970, there was no evidence for an independent relationship of rs6599257 or rs6800541 with PR interval (both P > 0.05). Replication in an independent sample of 6,243 Indian Asians from the LOLIPOP study (Table 1, Supplementary Table 1 and Online Methods) confirmed association of rs6795970 with P-wave duration (P = 4.0 × 10–8) and QRS interval (P = 1.2 × 10–7) but did not find association with QTc interval or heart rate. The association of rs6795970 with PR interval, QRS interval and P-wave duration remained significant after removing individuals receiving treatment with beta-blockers or other drugs that influence cardiac conduction; and was also not accounted for by population stratification (Supplementary Table 3). To assess whether the relationship of SCN10A with cardiac conduction was specific to Indian Asians, we tested for replication of this association in 978 Europeans from the LOLIPOP study. Participants of European ancestry were self-reported as white and born in Europe. Genotyping was performed using the Illumina 610 BeadChip. The LD structure of the locus was similar in Europeans and Indian Asians (Fig. 2). Among Europeans, we found that two of the 145 SNPs located 100 kb on either side of SCN10A were associated with PR interval at P < 10–4 and that the false discovery rate at this P value is 1.45%. rs6795970 was the most closely associated SNP to cardiac conduction (P = 3.7 × 10–5; Fig. 2). Single-SNP genotyping in an additional independent sample of 5,370 Europeans (Supplementary Table 1) replicated the association of rs6795970 with PR interval (P = 1.4 × 10–20), as well as with P-wave duration (P = 1.2 × 10–5) and QRS interval (P = 4.8 × 10–6), (Table 1
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© 2010 Nature America, Inc. All rights reserved.
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Figure 2 Structure of the SCN10A locus on chromosome 3 among Indian Asians in the genome-wide association study and the 978 Europeans in the replication sample, genotyped with the Illumina 610 array. Pairwise LD between SNPs is shown using Haploview’s standard color scheme. Association with PR interval is given as −log10(P). The nonsynonymous SNP rs6795970 carried forward for replication and further testing is highlighted pink.
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on the Indian subcontinent. ECGs were recorded using Cardiosoft software (GE Healthcare), and time intervals were measured electronically using the Marquette 12SL algorithm (see Online Methods). Genotyping was carried out using Illumina Hap300 and Hap610 BeadChips. We found one locus associated with PR interval (SCN10A on chromosome 3), one locus associated with QTc (NOS1AP on chromosome 1) and no loci associated with QRS at genome-wide significance (P < 5 × 10–8; Fig. 1 and Supplementary Table 2). There were three SNPs in SCN10A associated with PR interval at P < 5 × 10–8 Table 1 Association of rs6795970 (G>A) in SCN10A with ECG parameters in the (rs6795970, rs6599257 and rs6800541; replication sample Supplementary Table 2), and all were in Indian Asians Europeans high pairwise linkage disequilibrium (LD) Effect s.d. P Effect s.d. P Phetero (r2 > 0.5; Fig. 2). The SNP rs6795970 (assoHeart rate (bpm) –0.25 0.21 0.23 –0.05 0.23 0.85 0.51 ciated at P = 2.8 × 10–15) is nonsynonyPR interval (ms) 3.0 0.40 2.1 × 10–13 4.74 0.51 4.3 × 10–20 0.01 mous and the other two associated SNPs are P wave duration (ms) 1.47 0.27 4.0 × 10–8 1.43 0.33 1.2 × 10–5 0.93 intronic. There is no other known coding –7 –6 QRS duration (ms) 1.04 0.20 1.2 × 10 1.16 0.25 4.8 × 10 0.70 variant in LD with rs6795970 at r2 > 0.2 in QTc interval (ms) –0.66 0.39 0.09 –0.75 0.44 0.09 0.88 the HapMap CEU, CHB/JPT or YRI populaThe replication sample comprised 6,243 Indian Asians and 5,370 Europeans. Effect sizes are unit change per copy tions. The nonsynonymous SNP rs6795970 of A allele under additive genetic model, adjusted for age and sex. Phetero is for the comparison of effect sizes between is therefore a candidate for the observed Indian Asians and Europeans. s.d., standard deviation.
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letters P < 0.05; Table 2). In contrast, rs6795970[A] was protective against risk of ventricular Scn10a Wild type fibrillation in the setting of acute myocardial Wild type 700 infarction among the 976 participants in the arrhythmia genetics in The Netherlands study 35 600 (AGNES20, Table 2). P = 0.03 The mechanisms linking genetic variants 500 in SCN10A with cardiac conduction remain to be determined. rs6795970 (G>A) results 30 400 in the amino acid change A1073V within 0 6 12 18 24 0 6 12 18 24 h. h. the IDII/III intracellular loop of Na V1.8 Figure 3 PR interval and heart rate in conscious Scn10a–/– (n = 6) and wild-type (n = 4) mice (Supplementary Fig. 2). The cytoplasmic recorded by telemetry. Results are provided as means. Error bars, s.e.m. domains of sodium channels mediate inter action with accessory proteins such as PDZD2 and Supplementary Table 3). We found no significant association and also modulate channel function through cAMP-dependent phosbetween rs6795970 and QTc interval or heart rate in Europeans. phorylation21–24. Single amino acid changes within the IDII/III loop To investigate whether SCN10A is involved in cardiac conduc- of other sodium channels are associated with altered electrophysio tion, we studied Scn10a–/– mice. Changes in cardiac conduction logical properties and abnormalities of cardiac conduction23. or cardiac arrhythmias have not previously been reported in these rs6795970, located in SCN10A, is 75.5 kb from SCN5A, a gene mice. ECG time intervals were recorded by telemetry for up to 24 h. known to be involved in cardiac conduction. Rare mutations and We found that the PR interval was shorter in Scn10a–/– mice than common variants in SCN5A are associated with prolongation of QTc in wild-type littermates (Fig. 3). There were no differences in other interval and cardiac arrhythmias4,5,25,26. In contrast, in the present ECG parameters or echocardiographic cardiac dimensions and func- study, rs6795970 in SCN10A was not associated with QTc interval tion between Scn10a–/– and wild-type mice (Supplementary Table 4). (Table 1). Direct genotyping confirmed that rs6795970 in SCN10A These observations suggest that SCN10A in humans acts to lengthen is not in LD with rs7638909 or rs12053903, common SCN5A cardiac conduction, and that rs6795970 in SCN10A is a gain-of- variants that are associated with variation in cardiac conduction function variant. (rs6795970 r 2 0.01 in the CEU, CHB/JPT or YRI populations. These observaknown to have a central role in the propagation of action poten- tions support the view that the relationship of rs6795970 with PR and tials in nociceptive nerve fibers13,15,16. The NaV1.8 ion channel is QRS intervals is not mediated through SCN5A. characterized by a long-duration action potential and preservaIn addition to our findings for SCN10A, we also replicate the association of excitability during rapid and sustained stimulation 17,18. tion of common variants in the NOS1AP, ATP1B1, SCN5A, C6orf204, NaV1.8 has not previously been reported to be involved in cardiac KCNH2, LITAF and KCNJ2 with QTc interval among Indian Asians; these conduction. We found expression of SCN10A in the atria and ven- are loci previously reported to be associated with QT interval in individ tricles of mouse and human heart tissue (Supplementary Fig. 1). uals of European ancestry (all P < 0.05, Supplementary Table 5)27,29. We SCN10A expression was separately confirmed in isolated atrial and did not identify any associations with QRS interval. ventricular myocytes from wild-type mice (Supplementary Fig. 1). Our findings that common genetic variants in SCN10A influence the Our results extend previous observations of SCN10A expression in the PR, P-wave and QRS intervals provide new insight into the mechanisms left ventricle of individuals with dilated cardiomyopathy19. underlying cardiac conduction. We further find that common variWe then tested the relationship of rs6795970 with clinical conduc- ants at this locus are susceptibility factors for heart block and serious tion abnormalities. Among Indian Asians and Europeans genotyped ventricular arrhythmia. The role of common and rare variants of in the replication sample, only allele A of rs6795970 was associated SCN10A in other arrhythmia syndromes merits further evaluation. with increased risk of first-degree atrioventricular block, bundlebranch block and bifascicular heart block (odds ratios 1.25–2.30, all Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/. Heart rate
PR interval
Scn10a–/–
© 2010 Nature America, Inc. All rights reserved.
40
–/–
Ms
Bpm
800
Table 2 Association of rs6795970 (G>A) in SCN10A with ECG conduction block and ventricular fibrillation LOLIPOP replication sample
OR
95% CI
P
First-degree heart block
1.25
1.03, 1.52
0.02
Bundle-branch block
1.37
1.11, 1.70
0.003
Bifascicular heart block
2.30
1.18, 4.47
0.01
0.79
0.66, 0.95
0.01
AGNES study Ventricular fibrillation
Shown are the association data for ECG conduction block among the 6,243 Indian Asians and 5,370 Europeans in the LOLIPOP replication sample and the association with ventricular fibrillation among the 976 participants of the AGNES study. Results are expressed as odds ratio (OR) per copy of A allele under an additive genetic model, with adjustment for age, sex and race in the replication sample and for age and sex in the AGNES study. CI, confidence interval.
Nature Genetics volume 42 | number 2 | february 2010
Note: Supplementary information is available on the Nature Genetics website. Acknowledgments We thank the participants involved in the research. The LOLIPOP study was supported by the British Heart Foundation (SP/04/002) and by the Wellcome Trust (084723/Z/08/Z). Studies in the AGNES population were supported by the Netherlands Heart Foundation (Grant 2007B202) and the Leducq Foundation (Grant 05-CVD). J.Z. was supported by a BBSRC LOLA award (BB/F000227/1). R.K. was supported by the British Heart Foundation (Grant F/99089). M.N.W. is supported by the Biotechnology and Biological Sciences Research Council grant BB/F020481/1. W.M. is funded by the National Institute for Health Research Biomedical Research Centres scheme. J.N.W. is a member of the Wellcome Trust-funded London Pain Consortium. We thank G. Turner and M. Minett for maintaining the Scn10a-null mutant mouse colony, M.W. Tanck and R. Pazoki for help in statistical analyses and S. Rothery for technical assistance.
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letters AUTHOR CONTRIBUTIONS Design and scientific rationale: J.C.C., J.Z., C.M.N.T., C.R.B., R.K., W.M., N.J.S., R.de.S., A.A.M.W., P.A., M.Y., J.S., P.E., J.N.W. and J.S.K. Obtained funding: J.C.C., C.M.N.T., C.R.B., R.K., N.J.S., A.A.M.W., P.A., M.Y., P.E., J.N.W. and J.S.K. Recruitment and characterization of participants: J.C.C., C.R.B., R.K., J.S.S., L.R.C.D., J.S.S.G.de.J., N.J.S., A.A.M.W., P.E. and J.S.K. Analyzed ECG readings: J.C.C., C.M.N.T., C.R.B., M.N., A.L., J.S.S., S.P., M.K.K., L.R.C.D., J.S.S.G.de.J. and A.A.M.W. Performed genotyping: J.C.C., C.R.B., W.Z., J.S.S., L.R.C.D., J.S.S.G.de.J., R.de.S., A.A.M.W., P.E. and J.S.K. PCR experiments: J.Z., C.M.N.T., R.K., U.S., I.E-H., N.J.S., J.C.C., J.N.W. and J.S.K. Mouse ECG studies: C.M.N.T., R.K., M.N., U.S., R.de.S., P.E., J.N.W., J.C.C. and J.S.K. Performed statistical analysis: J.C.C., C.M.N.T., C.R.B., W.Z., M.N., J.S.S., G.D., M.N.W., J.S.S.G.de.J., M.J.E.S., R.D.S., A.A.M.W., P.E. and J.S.K. Drafted manuscript: J.C.C., J.Z., C.M.N.T., J.S.S., R.de.S., J.S., P.E., J.N.W. and J.S.K. Critically revised manuscript: J.C.C., J.Z., C.M.N.T., C.R.B., W.Z., R.K., M.N., A.L., J.S.S., M.K.K., G.D., U.S., I.E-H., M.N.W., L.R.C.D., J.S.S.G.de.J., M.J.E.S., W.M., N.J.S., R.de.S., A.A.M.W., P.A., J.S., M.Y., P.E., J.N.W. and J.S.K.
© 2010 Nature America, Inc. All rights reserved.
COMPETING INTERESTS STATEMENT The authors declare no competing financial interests. Published online at http://www.nature.com/naturegenetics/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.
1. Murray, C.J. & Lopez, A.D. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 349, 1269–1276 (1997). 2. Lopez, A.D., Mathers, C.D., Ezzati, M., Jamison, D.T. & Murray, C.J. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet 367, 1747–1757 (2006). 3. Huikuri, H.V., Castellanos, A. & Myerburg, R.J. Sudden death due to cardiac arrhythmias. N. Engl. J. Med. 345, 1473–1482 (2001). 4. Noseworthy, P.A. & Newton-Cheh, C. Genetic determinants of sudden cardiac death. Circulation 118, 1854–1863 (2008). 5. Knollmann, B.C. & Roden, D.M. A genetic framework for improving arrhythmia therapy. Nature 451, 929–936 (2008). 6. Schnabel, R.B. et al. Development of a risk score for atrial fibrillation (Framingham Heart Study): a community-based cohort study. Lancet 373, 739–745 (2009). 7. Zimetbaum, P.J. et al. Electrocardiographic predictors of arrhythmic death and total mortality in the multicenter unsustained tachycardia trial. Circulation 110, 766–769 (2004). 8. Vrtovec, B. et al. Prolonged QTc interval and high B-type natriuretic peptide levels together predict mortality in patients with advanced heart failure. Circulation 107, 1764–1769 (2003). 9. Schouten, E.G. et al. QT interval prolongation predicts cardiovascular mortality in an apparently healthy population. Circulation 84, 1516–1523 (1991).
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ONLINE METHODS Genome-wide association and replication study. Participants. Genome-wide association was carried out in 6,543 Indian Asians participating in the London Life Sciences Population study (LOLIPOP), a population based cohort of Indian Asian and European self-reported white men and women aged 35–75 years living in West London, UK30. Replication testing was performed in a further 6,243 Indian Asian and 5,370 European self-reported white LOLIPOP participants not included in the genome-wide study. The characteristics of participants are summarized in Supplementary Table 1. An interviewer-administered questionnaire was used to collect data on ethnicity, medical history, family history, current prescribed medication and cardiovascular risk factors. Indian Asians were identified as having all four grandparents born on the Indian subcontinent. Europeans were self-reported white and born in Europe. Physical assessment included anthropometric measurements (height, weight, waist and hip) and blood pressure. Twelve lead ECGs were recorded using Cardiosoft software (GE Healthcare) and ECG intervals were measured electronically using the Marquette 12SL algorithm; this algorithm incorporates adjustment of QT interval for heart rate by Bazett’s formula [QTc = measured QT duration/√(RR interval)], where the RR interval is the time interval between R waves. Blood was collected for biochemical analysis after an 8-h fast; the analysis included glucose, total and HDL cholesterol and triglycerides. Whole blood was also taken for extraction of DNA. All participants gave written consent including for genetic studies. The study is approved by the Ealing Hospital and St. Mary’s Hospital research ethics committees. Genotyping. For the genome-wide association stage, genotyping was carried out using the Illumina Hap300 (n = 1,868) and Hap610 (n = 4,675) BeadChips, according to standard methodology. In brief, each sample was whole-genome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared HumanHap610 BeadChips for a minimum of 16 h at 48 °C. After hybridization, the BeadChips were processed for the single-base extension reaction and staining and imaging on an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina Beadstudio 2.0 software, which converted fluorescence intensities into SNP genotypes. Samples with call rates 90% of cases developed ventricular fibrillation within 2 h after onset of complaints. Signed informed consent was obtained for all patients. Exclusion criteria for both cases and controls included non-STEMI, prior myocardial infarction and age 80 years, congenital heart defects, known structural heart disease, severe comorbidity, electrolyte disturbances, trauma, surgery, or coronary artery bypass grafts within the previous 4 weeks. Class I and III anti-arrhythmic drug use was also an exclusion criterion. Genotyping was carried out by TaqMan. Characteristics of AGNES participants are summarized in Supplementary Table 6. ECG and echocardiography studies in Scn10a–/– and wild-type mice. The procedure to create Scn10a global-null mutant mice by deletion of exons 4 and 5 and backcrossing onto a C57Bl6 background has been previously described16. We studied seven Scn10a–/– mice and five wild-type littermates, aged 8–34 weeks. ECG recording was performed in conscious mice using a radiotelemetry system (Data Science International) as previously described33. A radiotelemetry transmitter (Data Science International, model EA-F20) for recording electrical activity was implanted in the scruff of the neck under general anesthesia (1.5% isoflurane in 100% oxygen). The electrode wires were tunneled under the skin with the negative lead sutured in the muscle just under the right clavicle and the positive lead placed left of the xyphoid process and below the rib cage. Recording was performed at least 3 d after implantation and continuously for 16–18 h. Up to 3,000 ECG complexes were analyzed during a 5-min period every hour and the values obtained were averaged. Analysis of the recording was performed using ECG-auto software (EMKA technologies). The primary hypothesis was that PR interval would be different between Scn10a–/– animals and their wild-type littermates. Data for heart rate, QRS duration and QTc interval were also available as secondary phenotypes. Echocardiography was carried while the mice were anesthetized and before implantation of the radiotelemetry system. A 15-MHz probe on an Acuson Sequoia 256 system was used to obtain trans-thoracic parasternal short-axis views at the mid-papillary muscle level. M-mode was used for the measurement of left-ventricular systolic and diastolic diameter and calculation of the left ventricular ejection fraction and fractional shortening. All the animal experiments were approved by the UK Home Office under the terms of the Animals (Scientific Procedures) Act 1986. Expression of SCN10A in mouse and human cardiac tissues. Human cardiac tissue was obtained from explanted hearts of transplant recipients. Ethical approval for collection and use of human samples was obtained by the Royal Brompton, Harefield and National Heart and Lung Institute Research Ethics Committee. Mouse tissue was obtained from C57Bl6 animals. Cardiac myocytes were isolated by enzymatic digestion in hyaluronidase- and collagenase-containing solution. Samples were snap-frozen on liquid nitrogen before RNA extraction. RNA was isolated with RNeasy Mini Kit (74104, Qiagen) and was digested with DNase I (79254, Qiagen) the membrane. Complementary DNA was constructed using iScript cDNA Synthesis Kit (170-8891, Bio-Rad) according to the manufacturer’s instructions. PCR amplification was performed with 10–50 ng cDNA, using primer sequences described in Supplementary Table 7. PCR reactions were performed under the following conditions: 94 °C for 30 s, 56 °C for 30 s, 72 °C for 50 s for 35 cycles, and finally 72 °C for 10 min. PCR products were analyzed by DNA electrophoresis in 1.5% agarose gel. URLs. KASPar, http://www.kbioscience.co.uk/. 30. Chambers, J.C. et al. Common genetic variation near MC4R is associated with waist circumference and insulin resistance. Nat. Genet. 40, 716–718 (2008). 31. Price, A.L. et al. Principal components analysis corrects for stratification in genomewide association studies. Nat. Genet. 38, 904–909 (2006). 32. Pe’er, I., Yelensky, R., Altshuler, D. & Daly, M.J. Estimation of the multiple testing burden for genomewide association studies of nearly all common variants. Genet. Epidemiol. 32, 381–385 (2008). 33. Stagg, M.A. et al. Cytoskeletal protein 4.1R affects repolarization and regulates calcium handling in the heart. Circ. Res. 103, 855–863 (2008).
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