Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Bagnall RD, Weintraub RG, Ingles J, et al. A prospective study of sudden cardiac death among children and young adults. N Engl J Med 2016;374: DOI: /NEJMoa

2 A Prospective, Population-Based Study of Sudden Cardiac Death in the Young SUPPLEMENTARY APPENDIX Richard D. Bagnall, Ph.D., Robert G. Weintraub, MBBS, Jodie Ingles, Grad.Dip.Gen.Couns., Ph.D., M.P.H., Johan Duflou, MBChB., M.Med., Laura Yeates, B.Sc., Grad.Dip.Gen.Couns., Lien Lam, Ph.D., Andrew Davis, MBBS, M.D., Tina Thompson, B.Nurs, Vanessa Connell, Dip. App. Sci., Jenny Wallace, R.N., Charles Naylor, M.A., MBBChir., Jackie Crawford, RN, Donald R. Love, Ph.D., Lavinia Hallam, B.Sc., MB BCh., Jodi White, MBBS, Christopher Lawrence, MBBS, Matthew Lynch, L.L.B., MBBS, Natalie Morgan, RN, Grad.Dip.Genetic.Couns., Paul James, Ph.D., M.D., Desirée du Sart, Ph.D., Rajesh Puranik M.B. Ph.D., Neil Langlois MBBChir M.A., M.D., Jitu Vohra, M.D., Ingrid Winship, MBChB., M.D., John Atherton, MBBS, Ph.D, Julie McGaughran, MBChB, M.D., Jonathan R. Skinner, MBChB., M.D., and Christopher Semsarian, MBBS, Ph.D., M.P.H. 1

3 TABLE OF CONTENTS Supplementary Methods Pages Postmortem criteria for diagnosis 3 Genetic analysis 6 Classification of variants 6 CNV and MLPA analysis 7 Statistical analysis 7 Supplementary Tables Supplementary Appendix Table S1 9 Supplementary Appendix Table S2 14 Supplementary Appendix Table S3 15 Supplementary Appendix Table S4 16 Supplementary Appendix Table S5 17 Supplementary Appendix Table S6 18 Supplementary Appendix Table S7 19 Supplementary Appendix Table S8 20 Supplementary Appendix Table S9 21 Supplementary Appendix Table S10 22 Supplementary Appendix Table S11 23 Supplementary Figures Figure Legends 24 Supplementary Appendix Figure S1 25 Supplementary Appendix Figure S2 26 Supplementary Appendix Figure S3 27 References 28 2

4 SUPPLEMENTARY METHODS 1. POSTMORTEM CRITERIA FOR DIAGNOSIS The following criteria were used for postmortem diagnosis of cardiovascular disease and further discussed by the adjudication committee before reaching a final cause of death. Specifically: Coronary artery disease/atherosclerotic cardiovascular disease/ischaemic heart disease: Autopsy diagnosis was based on the presence of typical atherosclerotic lesions involving epicardial coronary arteries, with stenosis of a major epicardial vessel in excess of 70% cross sectional area, and/or the presence of plaque haemorrhage or rupture in an atherosclerotic vessel, and/or the presence of occlusive or partially occlusive thrombus in the lumen of an atherosclerotic vessel. Areas of replacement fibrosis or acute myocardial infarction may also be present. Acute myocardial infarction: Autopsy diagnosis was based on the presence of localised geographic areas of any or all of the following in ventricular myocardium: coagulation necrosis, myocytolysis, contraction band necrosis, an acute inflammatory cell infiltrate consisting predominantly of neutrophils. Myocarditis: Autopsy diagnosis was based on the identification of myocarditis with/without fibrosis as defined in the Dallas Classification System for myocarditis. Hypertrophic cardiomyopathy: Autopsy diagnosis was based on the presence of left ventricular and/or interventricular septal hypertrophy, with or without the presence of outflow obstruction and mitral valve changes, and associated characteristic ventricular 3

5 myocardial histologic changes of myocyte hypertrophy with bizarre nuclei, myocyte disarray and/or myofibre disarray involving at least 5% of the histologically examined myocardium, often with associated abnormalities of the intima and media of small intramural arteries. Arrhythmogenic right ventricular cardiomyopathy: Autopsy diagnosis was based on the revised ARVC/D International task Force criteria, including pronounced generalised or segmental dilatation of the right ventricle, and histologically identified fibrofatty replacement of myocytes in the right ventricular free wall with or without associated chronic inflammation (Marcus FI, et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: Proposed modification of the Task Force criteria. Circulation 2010;121: ). Dilated cardiomyopathy: Autopsy diagnosis was based on ventricular dilatation, with or without atrial dilatation in the absence of significant valvular, atherosclerotic, hypertensive and congenital heart disease. Histologic appearances are generally non-specific, but can include limited myocardial interstitial fibrosis and variation in myocyte morphology. Myocardial inflammation, if present must be limited in amount, and not sufficient for a diagnosis of myocarditis. Idiopathic cardiac hypertrophy: Macroscopically, a heart weight in excess of normal for age and height or weight, as detailed in commonly used heart weight tables (References: Scholz DG, et al. Age-related changes in normal human hearts during the first 10 decades of life. Part I (Growth): A quantitative anatomic study of 200 specimens from subjects from birth to 19 years old. Mayo Clinic Proceedings 1988;63(2): ); Kitzman DW, et al. Age-related changes in normal human hearts during the first 10 decades of life. Part II 4

6 (Maturity): A quantitative anatomic study of 765 specimens from subjects 20 to 99 years old. Mayo Clinic Proceedings 1988;63(2):137-46). Microscopically, normal ventricular myocardium or myocyte hypertrophy without myocyte disarray, inflammation or significant fibrosis. Anomalous coronary artery: Includes coronary arteries originating from the pulmonary trunk and those originating normally with a course passing between the aorta and pulmonary trunk, but does not include conditions such as tunnelling, high take-off of a coronary artery or other minor morphologic abnormalities. Thoracic aortic dissection: A tear in the thoracic aorta with or without aneurysm formation with resultant hemothorax or hemopericardium and/or ischemic lesions. Predisposing causes include natural inherited and acquired disease such as hypertension, Marfan syndrome, bicuspid aortic valve, familial thoracic aneurysm dissection syndrome and various arteritides, but excludes non-natural etiologies such as trauma and temporally associated cocaine use. Morphologically normal heart: A heart which on macroscopic and microscopic examination at autopsy does not appear significantly diseased. Specifically, there is no significant hypertensive, atherosclerotic or valvular disease, no structural congenital heart disease, no inflammatory, fibrotic or other infiltrate and no evidence of conditions such as hypertrophic or dilated cardiomyopathy. 5

7 2. GENETIC ANALYSIS Raw sequence reads were aligned to the human genome reference sequence (GRCh37/hg19/b37), using BWA-mem v0.7.8 with default parameters. 1 Sequence alignment files were compressed and sorted using SamTools v and duplicate reads were removed using Picard Tools v The Genome Analysis Tool Kit v was used for read realignment around short indels, base quality score recalibration, and genotyping of single nucleotide variants (SNVs) and indels, using HaplotypeCaller in gvcf mode. SNVs were filtered for QD <2.0, ReadPosRankSum <-8.5, Mapping Quality <35.0 and MQRankSum < Indels were filtered for FisherStrand >200.0, QD <1.8, ReadPosRankSum <-20.0 and InbreedingCoefficient <-0.8. Variants were annotated using SeattleSeq Annotation v8.07 tool ( and compared against the September 2014 release of the 1000 Genomes Project, phase 3, data ( and the Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP) ( [Nov, 2013]. 3. CLASSIFICATION OF VARIANTS Pathogenic variants were variants previously reported as disease causing in a patient with a relevant clinical diagnosis and demonstrated to co-segregate with disease, or with functional evidence of pathogenicity. Likely pathogenic variants were variants at canonical splice dinucleotides, nonsense variants and insertion and deletions; and missense variants at conserved nucleotides (GERP score 2) that are predicted to be damaging by at least 2 out of 3 in silico pathogenicity prediction tools (SIFT, PolyPhen2 and Mutation Taster). Missense variants at conserved nucleotides and predicted to be damaging by fewer than 2 in silico tools were considered variants of uncertain significance (VUS). Missense variants 6

8 at non-conserved nucleotides were considered benign. All relevant variants with a read depth <15 were validated using standard Sanger DNA sequencing. 4. CNV ANALYSIS AND MULTIPLE LIGATION-DEPENDED PROBE AMPLIFICATION (MLPA) ANALYSIS Exome depth of coverage was calculated using GATK with the arguments -- minmeantargetrd 10, --maxmeantargetrd 500, --minmeansamplerd 25, and -- maxmeansamplerd 100. Exome targets with length <10 bp or >10 kb, or GC content >90% or <10%, or with >10% of bases masked by RepeatMasker were excluded. CNV calling was performed using XHMM with default parameters, and filtered with PLINK software to remove common CNVs and CNVs overlapping in more than 10% of all samples. CNVs with a quality score 60, and spanning 3 exome targets, with length 1 kb, and spanning or disrupting 125 autosomal cardiac disease genes were retained. MLPA was performed on 71 SUD cases as previously described using the SALSA MLPA LQTS P114 probe kit, which screens for large insertions and deletions in five LQTS genes (SCN5A, KCNH2, KCNQ1, KCNE1 and KCNE2). MLPA products were resolved on the ABI3730xl sequencer (Macrogen), sized using Peak Scanner TM v1.0 software (Applied Biosystems, CA, USA), and analysed using Coffalyser v2 software (MRC-Holland). Peak height ratios of <0.65 and >1.35 indicated a deletion and duplication, respectively. 5. STATISTICAL ANALYSIS The first step was to identify incidence rate of all SCD in the population then describe the cases where a known cause of death was ascertained. Characteristics of the disease groups were compared using Chi-square analysis and unpaired t-tests. SUD cases were 7

9 compared to those with a known cause of death to identify factors associated with this outcome, using univariate and multivariate logistic regression models. A final analysis to determine whether factors are associated with identification of a genetic diagnosis amongst SUD cases was also performed, however was based on small sub-groups of cases. Activity at time of death was converted in to a number of binary variables, including exercise (yes/no), which included death during or immediately post exercise. Sleep (yes/no) included any cases where they were presumed to be sleeping at time of death. Time of death was grouped in to a day/night binary variable, with night deaths being death between the hours 6pm and 6am. Due to the non-linear nature of age at death, 5-year age group categories were created for regression analysis, with year age group as the reference category. For regression analysis where gene result was the dependent variable, the outcome was defined as a pathogenic or likely pathogenic variant identified in cases where genetic testing was performed. Further analysis was performed where the outcome was defined as a pathogenic or likely pathogenic variant identified in arrhythmia genes, however due to the very small sample only minimal data is shown from this analysis. 8

10 SUPPLEMENTARY TABLES SUPPLEMENTARY APPENDIX TABLE S1. Target Gene List. Gene Symbol Gene Category OMIM Entry KCNH2 Molecular autopsy Long QT syndrome 2, (3); Short QT syndrome 1, (3) KCNQ1 Molecular autopsy Long QT syndrome 1, (3) RYR2 Molecular autopsy Ventricular tachycardia, catecholaminergic polymorphic, 1, (3) SCN5A Molecular autopsy Long QT syndrome-3, (3); Brugada syndrome 1, (3) AKAP9 Cardiac arrhythmia Long QT syndrome-11, (3) ANK2 Cardiac arrhythmia Long QT syndrome 4, (3); Cardiac arrhythmia, ankyrin-b-related, (3) CACNA1C Cardiac arrhythmia Brugada syndrome 3, (3); Timothy syndrome, (3) CACNB2 Cardiac arrhythmia Brugada syndrome 4, (3) CASQ2 Cardiac arrhythmia Ventricular tachycardia, catecholaminergic polymorphic, 2, (3) CAV3 Cardiac arrhythmia Long QT syndrome 9, (3) GPD1L Cardiac arrhythmia Brugada syndrome 2, (3) HCN4 Cardiac arrhythmia Brugada syndrome 8, (3); Sick sinus syndrome 2, (3) KCNE1 Cardiac arrhythmia Long QT syndrome 5, (3); Jervell and Lange-Nielsen syndrome 2, (3) KCNE2 Cardiac arrhythmia Long QT syndrome 6, (3); Atrial fibrillation, familial, 4, (3) KCNE3 Cardiac arrhythmia Brugada syndrome 6, (3) KCNJ2 Cardiac arrhythmia Andersen syndrome, (3); Atrial fibrillation, familial, 9, (3); Short QT syndrome 3, SCN1B Cardiac arrhythmia (3) Brugada syndrome 5, (3), Atrial fibrillation, familial, 13, (3); Cardiac conduction defect, nonspecific, (3) SCN3B Cardiac arrhythmia Brugada syndrome 7, (3); Atrial fibrillation, familial, 16, (3) SCN4B Cardiac arrhythmia Long QT syndrome-10, (3); Atrial fibrillation, familial, 17, (3) SNTA1 Cardiac arrhythmia Long QT syndrome 12, (3) DSC2 Major cardiomyopathy Arrhythmogenic right ventricular dysplasia 11, (3) DSG2 Major cardiomyopathy Arrhythmogenic right ventricular dysplasia 10, (3) DSP Major cardiomyopathy Arrhythmogenic right ventricular dysplasia 8, (3); Dilated cardiomyopathy with woolly hair and keratoderma, (3) 9

11 LDB3 Major cardiomyopathy Cardiomyopathy, dilated 1C, (3) LMNA Major cardiomyopathy Cardiomyopathy, dilated, 1A, (3) MYBPC3 Major cardiomyopathy Cardiomyopathy, familial hypertrophic, 4, (3) MYH7 Major cardiomyopathy Cardiomyopathy, familial hypertrophic, 1, (3) PKP2 Major cardiomyopathy Arrhythmogenic right ventricular dysplasia 9, (3) TNNI3 Major cardiomyopathy Cardiomyopathy, familial hypertrophic, 7, (3); Cardiomyopathy, dilated, 2A, (3) TNNT2 Major cardiomyopathy Cardiomyopathy, familial hypertrophic, 2, (3); Cardiomyopathy, dilated, 1D, (3) ACTC1 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 11, (3); Cardiomyopathy, dilated, 1R, (3); Left ventricular noncompaction 4, (3) MYH6 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 14, (3); Cardiomyopathy, dilated, 1EE, (3) MYL2 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 10, (3) MYL3 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 8, (3) TNNC1 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 13, (3); Cardiomyopathy, dilated, 1Z, (3) TPM1 Minor cardiomyopathy Cardiomyopathy, familial hypertrophic, 3, (3); Cardiomyopathy, dilated, 1Y, (3) ABCC9 Rare cardiomyopathy Cardiomyopathy, dilated, 1O, (3) ACTN2 Rare cardiomyopathy Cardiomyopathy, dilated, 1AA, (3) CALR3 Rare cardiomyopathy Cardiomyopathy, familial hypertrophic, 19, (3) CSRP3 Rare cardiomyopathy Cardiomyopathy, familial hypertrophic, 12, (3); Cardiomyopathy, dilated, 1M, (3) DES Rare cardiomyopathy Cardiomyopathy, dilated, 1I, (3) FKTN Rare cardiomyopathy Cardiomyopathy, dilated, 1X, (3) GLA Rare cardiomyopathy Fabry disease, cardiac variant, (3) JPH2 Rare cardiomyopathy Cardiomyopathy, familial hypertrophic 17, (3) JUP Rare cardiomyopathy Arrhythmogenic right ventricular dysplasia 12, (3) LAMP2 Rare cardiomyopathy Danon disease, (3) MYLK2 Rare cardiomyopathy Cardiomyopathy, hypertrophic, midventricular, digenic, (3) MYOZ2 Rare cardiomyopathy Cardiomyopathy, familial hypertrophic, 16, (3) NEXN Rare cardiomyopathy Cardiomyopathy, dilated, 1CC, (3); Cardiomyopathy, familial hypertrophic, 20, (3) PLN Rare cardiomyopathy Cardiomyopathy, dilated, 1P, (3); Cardiomyopathy, familial hypertrophic, 18, (3) PRKAG2 Rare cardiomyopathy Cardiomyopathy, familial hypertrophic 6, (3); Wolff-Parkinson-White syndrome, (3) RBM20 Rare cardiomyopathy Cardiomyopathy, dilated, 1DD, (3) SGCD Rare cardiomyopathy Cardiomyopathy, dilated, 1L, (3) TAZ Rare cardiomyopathy Barth syndrome, (3) 10

12 TCAP Rare cardiomyopathy Cardiomyopathy, dilated, 1N, (3) TGFB3 Rare cardiomyopathy Arrhythmogenic right ventricular dysplasia 1, (3) TMEM43 Rare cardiomyopathy Arrhythmogenic right ventricular dysplasia 5, (3) TMPO Rare cardiomyopathy Cardiomyopathy, dilated, 1T, (3) VCL Rare cardiomyopathy Cardiomyopathy, familial hypertrophic, 15, (3) ALDH7A1 Epilepsy Epilepsy, pyridoxine-dependent, (3) ARHGEF9 Epilepsy Epileptic encephalopathy, early infantile, 8, (3) ARX Epilepsy Epileptic encephalopathy, early infantile, 1, (3) ASAH1 Epilepsy Spinal muscular atrophy with progressive myoclonic epilepsy, (3) CACNA1H Epilepsy {Epilepsy, childhood absence, susceptibility to, 6}, (3); {Epilepsy, idiopathic generalized, CACNB4 Epilepsy susceptibility to, 6}, (3) {Epilepsy, idiopathic generalized, susceptibility to, 9}, (3); {Epilepsy, juvenile myoclonic, susceptibility to, 6}, (3) CASR Epilepsy {Epilepsy idiopathic generalized, susceptibility to, 8}, (3) CARS2 Epilepsy Neurology Oct 31; PMID: CDKL5 Epilepsy Epileptic encephalopathy, early infantile, 2, (3) CHD2 Epilepsy Epileptic encephalopathy, childhood-onset, (3) CHRNA2 Epilepsy Epilepsy, nocturnal frontal lobe, type 4, (3) CHRNA4 Epilepsy Epilepsy, nocturnal frontal lobe, 1, (3) CHRNB2 Epilepsy Epilepsy, nocturnal frontal lobe, 3, (3) CLCN2 Epilepsy {Epilepsy, idiopathic generalized, susceptibility to, 11}, (3); {Epilepsy, juvenile absence, susceptibility to, 2}, (3);{Epilepsy, juvenile myoclonic, susceptibility to, 8}, (3) CLN8 Epilepsy Ceroid lipofuscinosis, neuronal, 8, Northern epilepsy variant, (3) CNTN2 Epilepsy?Epilepsy, familial adult myoclonic, 5, (3) CNTNAP2 Epilepsy Cortical dysplasia-focal epilepsy syndrome, (3) CPA6 Epilepsy Epilepsy, familial temporal lobe, 5, (3) CSTB Epilepsy Epilepsy, progressive myoclonic 1A (Unverricht and Lundborg), (3) DEPDC5 Epilepsy Epilepsy, familial focal, with variable foci, (3) DOCK7 Epilepsy Epileptic encephalopathy, early infantile, 23, (3) DNM1 Epilepsy Am J Hum Genet Oct 2;95(4):360-70; PMID: EFHC1 Epilepsy {Epilepsy, juvenile absence, susceptibility to, 1}, (3); {Myoclonic epilepsy, juvenile, susceptibility to, 1}, (3) EPM2A Epilepsy Epilepsy, progressive myoclonic 2A (Lafora), (3) 11

13 GABRA1 Epilepsy Epileptic encephalopathy, early infantile, 19, (3); {Epilepsy, childhood absence, susceptibility to, 4}, (3); {Epilepsy, juvenile myoclonic, susceptibility to, 5}, (3) GABRB3 Epilepsy {Epilepsy, childhood absence, susceptibility to, 5}, (3) GABRD Epilepsy {Epilepsy, generalized, with febrile seizures plus, type 5, susceptibility to}, (3); {Epilepsy, GABRG2 Epilepsy idiopathic generalized, 10}, (3); {Epilepsy, juvenile myoclonic, susceptibility to}, (3) Epilepsy, generalized, with febrile seizures plus, type 3, (3); {Epilepsy, childhood absence, susceptibility to, 2}, (3) GNAO1 Epilepsy Epileptic encephalopathy, early infantile, 17, (3) GOSR2 Epilepsy Epilepsy, progressive myoclonic 6, (3) GRIN2A Epilepsy Epilepsy, focal, with speech disorder and with or without mental retardation, (3) HCN1 Epilepsy Epileptic encephalopathy, early infantile, 24, (3) IER3IP1 Epilepsy Microcephaly, epilepsy, and diabetes syndrome, (3) KCNB1 Epilepsy Epileptic encephalopathy, early infantile, 26, (3) KCNA1 Epilepsy Epilepsia Nov;55(11): (PMID: ) KCNMA1 Epilepsy Generalized epilepsy and paroxysmal dyskinesia, (3) KCNQ2 Epilepsy Epileptic encephalopathy, early infantile, 7, (3) KCNT1 Epilepsy Epilepsy, nocturnal frontal lobe, 5, (3); Epileptic encephalopathy, early infantile, 14, (3) KCTD7 Epilepsy Epilepsy, progressive myoclonic 3, with or without intracellular inclusions, (3) LGI1 Epilepsy Epilepsy, familial temporal lobe, 1, (3) ME2 Epilepsy {Epilepsy, idiopathic generalized, susceptibility to}, (3) MEF2C Epilepsy Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations, (3) NECAP1 Epilepsy?Epileptic encephalopathy, early infantile, 21, (3) NHLRC1 Epilepsy Epilepsy, progressive myoclonic 2B (Lafora), (3) PAFAH1B1 Epilepsy Lissencephaly 1, (3) PCDH19 Epilepsy Epileptic encephalopathy, early infantile, 9, (3) PLCB1 Epilepsy Epileptic encephalopathy, early infantile, 12, (3) PNKP Epilepsy Nat Genet Mar;42(3):245-9 (PMID: ) PRICKLE1 Epilepsy Epilepsy, progressive myoclonic 1B, (3) PRICKLE2 Epilepsy Epilepsy, progressive myoclonic 5, (3) PRRT2 Epilepsy Neurology Nov 20;79(21): (PMID: ) SCARB2 Epilepsy Epilepsy, progressive myoclonic 4, with or without renal failure, (3) SCN1A Epilepsy Epilepsy, generalized, with febrile seizures plus, type 2, (3) 12

14 SCN1B Epilepsy/cardiac arrhythmia Epilepsy, generalized, with febrile seizures plus, type 1, (3) SCN2A Epilepsy Epileptic encephalopathy, early infantile, 11, (3) SCN8A Epilepsy Epileptic encephalopathy, early infantile, 13, (3) SCN9A Epilepsy Epilepsy, generalized, with febrile seizures plus, type 7, (3) SIAT9 Epilepsy Amish infantile epilepsy syndrome, (3) SLC13A5 Epilepsy Epileptic encephalopathy, early infantile, 25, (3) SLC25A22 Epilepsy Epileptic encephalopathy, early infantile, 3, (3) SLC2A1 Epilepsy {Epilepsy, idiopathic generalized, suscpetibility to, 12}, (3) SNIP1 Epilepsy Psychomotor retardation, epilepsy, and craniofacial dysmorphism, (3) SPTAN1 Epilepsy Epileptic encephalopathy, early infantile, 5, (3) SRPX2 Epilepsy?Rolandic epilepsy, mental retardation, and speech dyspraxia, (3) ST3GAL3 Epilepsy Epileptic encephalopathy, early infantile, 15, (3) STRADA Epilepsy Polyhydramnios, megalencephaly, and symptomatic epilepsy, (3) STXBP1 Epilepsy Epileptic encephalopathy, early infantile, 4, (3) SYN1 Epilepsy Epilepsy, X-linked, with variable learning disabilities and behavior disorders, (3) SYNGAP1 Epilepsy Nat Genet Jul;45(7):825-30; PMID: SZT2 Epilepsy Epileptic encephalopathy, early infantile, 18, (3) TBC1D24 Epilepsy Epileptic encephalopathy, early infantile, 16, (3); Myoclonic epilepsy, infantile, familial, (3) TNK2 Epilepsy Ann Neurol Sep;74(3): (PMID: ) 59 cardiac disease genes common to all sequencing panels were screened for rare variants in SUD cases. Genes were divided into Molecular Autopsy genes, Cardiac Arrhythmia genes, Major and Minor Cardiomyopathy genes and Rare Cardiomyopathy genes. Frequency of gene mutations in disease populations determined the Major (>5%), Minor (1 to 5%) and Rare (<1%) categories according to Wilde AA, Behr ER. Genetic testing for inherited cardiac disease. Nat Rev Cardiol Oct;10(10): On-line Mendelian Inheritance In Man (OMIM) entries for each gene are shown in right column. Epilepsy genes were defined by a review of recent literature and searching the OMIM database [Nov 01, 2014] for diseases containing the terms epilepsy or epileptic encephalopathy. 13

15 SUPPLEMENTARY APPENDIX TABLE S2. Cause of SCD vs Gender and Age Group Cause of SCD Number of Cases Male Female Age 1-5 Age 6-10 Age Age Age Age Age Average Age SUD (67%) 65 (33%) CAD (85%) 18 (15%) DCM (60%) 14 (40%) Myocarditits (56%) 14 (44%) ARVC (76%) 6 (24%) HCM (85%) 3 (15%) Aortic dissection (72%) 5 (27%) Other (73%) 12 (26%)

16 SUPPLEMENTARY APPENDIX TABLE S3: Activity at time of sudden death based on underlying disease Event Exercise Post exercise Light Activity Rest Sleep NA Total cases Aortic dissection 2 (11%) 0 2 (11%) 4 (22%) 1 (6%) 9 (50%) 18 ARVC 5 (20%) 3 (12%) 6 (24%) 5 (20%) 3 (12%) 3 (12%) 25 CAD 3 (3%) 4 (3%) 18 (15%) 32 (27%) 23 (20%) 38 (32%) 118 CPVT (100%) DCM 2 (6%) 0 4 (12%) 5 (14%) 11 (31%) 13 (37%) 35 HCM 4 (20%) 2 (10%) 3 (15%) 5 (25%) 5 (25%) 1 (5%) 20 LQTS (100%) Myocarditis 3 (10%) 0 1 (3%) 5 (16%) 9 (28%) 14 (43%) 32 Unexplained 18 (9%) 2 (1%) 28 (14%) 32 (17%) 80 (41%) 35 (18%) 195 Other 3 (7%) 3 (7%) 6 (14%) 7 (16%) 7 (16%) 18 (40%) 44 Total cases NA = (information) not available 15

17 SUPPLEMENTARY APPENDIX TABLE S4: Sudden death characteristics by 5-year age brackets 1-5 years 6-10 years years years years years years Exp SUD Total Exp SUD Total Exp SUD Total Exp SUD Total Exp SUD Total Exp SUD Total Exp SUD Total N (%) 12 (24) 37 (76) 49 (10) 2 (50) 2 (50) 4 (1) 13 (65) 7 (35) 20 (4) 26 (37) 45 (63) 71 (14) 38 (60) 25 (40) 63 (13) 70 (64) 40 (36) 110 (22) 131 (76) 42 (24) 173 (35) Male gender 5 (42) 26 (70) 31 (63) 0 (0) 1 (50) 1 (25) 9 (69) 5 (71) 14 (70) 18 (69) 38 (84) 56 (79) 32 (84) 15 (60) 47 (75) 54 (77) 24 (64) 78 (71) 102 (78) 24 (60) 126 (73) Exercise 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 1 (50) 4 (40) 1 (17) 5 (25) 5 (24) 8 (19) 13 (23) 6 (22) 5 (22) 11 (20) 6 (12) 4 (14) 10 (18) 12 (14) 4 (13) 16 (10) Sleep 5 (71) 30 (91) 35 (88) 0 (0) 0 (0) 0 (0) 3 (30) 2 (33) 5 (31) 9 (43) 19 (44) 28 (44) 7 (26) 10 (43) 17 (34) 13 (27) 9 (32) 22 (29) 22 (27) 10 (32) 32 (28) Night 5 (83) 19 (73) 24 (75) 0 (0) 1 (50) 1 (33) 3 (30) 2 (40) 5 (33) 17 (74) 24 (60) 41 (65) 10 (43) 14 (74) 24 (57) 25 (49) 17 (63) 42 (54) 43 (51) 24 (74) 67 (58) SUD = sudden unexplained death; Exp = explained sudden death 16

18 SUPPLEMENTARY APPENDIX TABLE S5: Unadjusted and Adjusted Odds Ratios for Factors Associated with SUD compared to Explained SCD Unadjusted OR (All cases n=490) Adjusted OR (n=349, R 2 =15%) n Unadjusted OR 95% CI p-value Adjusted OR 95% CI p-value Age, years < < ref - ref - Female gender Activity: Exercise Sleep Night death (6pm-6am) NB: Ref - reference age group for comparison, n - number of SCD cases 17

19 SUPPLEMENTARY APPENDIX TABLE S6: Rare Variants in Molecular Autopsy Genes Sample ID Gene cdna variation Amino acid change ESP MAF 1000G MAF ExAC count GERP++ score SIFT Polyphen2 Mutation Taster Pathogenicity Call NSW55A RYR2 NM_ :c.6506A>G Glu2169Gly D,D,D Likely Pathogenic VIC47A RYR2 NM_ :c.9673G>A Gly3225Ser D,P,D Likely Pathogenic VIC29A RYR2 NM_ :c.9680G>C Arg3227Pro D,P,D Likely Pathogenic NZ53A RYR2 NM_ :c.14266A>G Ile4756Val D,D,D Likely Pathogenic VIC56A SCN5A NM_ :c.659C>T Thr220Ile D,D,A Pathogenic NZ36A SCN5A NM_ :c.1637A>G Asp546Gly D,D,D Likely Pathogenic NSW123A SCN5A NM_ :c.1756_1761del Ala586_Leu587del Likely Pathogenic VIC44A SCN5A NM_ :c.5686C>T Arg1896Trp T,D,D Pathogenic NZ50A KCNH2 NM_ :c.784G>A Gly262Ser T,B,N VUS WA12A KCNH2 NM_ :c.889C>T Pro297Ser T,B,N VUS VIC83A KCNH2 NM_ :c.2246del Gly749Ala_fs* Likely Pathogenic WA5A KCNQ1 NM_ :c.1781G>C Arg594Pro D,D,D Pathogenic NB: ESP MAF - Minor allele frequency in ESP data; 1000G MAF - Minor allele frequency in 1000 Genomes Project data; ExAC count - Allele count in Exome Aggregate Consortium data; Rare MAF < 0.1%; T - Tolerated; B - Benign; D - Probably damaging; P - Possibly damaging; N - Neutral; A - Automatic pathogenic; VUS - Variant of uncertain significance. 18

20 SUPPLEMENTARY APPENDIX TABLE S7. Rare Variants in Cardiac Arrhythmia Genes. Sample ID Gene Symbol cdna variation Amino acid change ESP MAF 1000G MAF ExAC count GERP ++ score SIFT Polyphen2 Mutation Taster Pathogenicity Call VIC83A ANK2 NM_ :c.2474C>G Thr825Arg T,D,D Likely Pathogenic NSW14A ANK2 NM_ :c.8927C>G Ser2976Cys ,D,D Likely Pathogenic NSW12A ANK2 NM_ :c.9657T>G Ser3219Arg T,P,N VUS QLD42A ANK2 NM_ :c.11807A>G Tyr3936Cys D,D,N Likely Pathogenic NSW12A AKAP9 NM_ :c.2972C>T Ser991Leu T,B,N VUS NZ26A AKAP9 NM_ :C.3904del Thr1302Gln_fs* Likely Pathogenic NZ52A AKAP9 NM_ :c.5117A>G Asp1706Gly T,P,N VUS NSW105A AKAP9 NM_ :c.11189A>G Gln3730Arg D,D,D Likely Pathogenic WA7A SCN4B NM_ :c.40T>C Trp14Arg T,D,N VUS WA11A CACNA1C NM_ :c.521C>T Ala174Val D,P,D Likely Pathogenic NSW56A HCN4 NM_ :c.3206G>A Arg1069Gln T,B,N VUS NSW12A SCN1B NM_ :c.50C>G Ala17Gly T,D,N VUS ESP MAF - Minor allele frequency in ESP data; 1000G MAF - Minor allele frequency in 1000 Genomes Project data; ExAC count - Allele count in Exome Aggregate Consortium data; T - Tolerated; B - Benign; D - Probably damaging; P - Possibly damaging; N - Neutral; '.' - No prediction. VUS - Variant of uncertain significance 19

21 SUPPLEMENTARY APPENDIX TABLE S8: Rare Variants in Major and Minor Cardiomyopathy Genes Sample ID Gene cdna variation Amino acid change ESP MAF 1000G MAF ExAC count GERP++ score SIFT Polyphen2 Mutation Taster Pathogenicity Call NSW104A LMNA NM_ :c.1027C>T Arg343Trp D,D,D Likely Pathogenic NSW22A TNNT2 NM_ :c.452G>A Arg151Gln D,D,D Likely Pathogenic VIC72A MYL3 NM_ :c.220G>A Gly74Arg D,B,D Likely Pathogenic VIC11A DSP NM_ :c.2552T>A Leu851Gln T,P,D Likely Pathogenic QLD5A DSP NM_ :c.4352T>C Leu1451Pro D,P,D Likely Pathogenic NSW37A DSP NM_ :c.4565C>T Thr1522Met T,B,N VUS VIC60A DSP NM_ :c.4997G>A Arg1666Gln T,B,D VUS WA16A DSP NM_ :c.6046G>A Gly2016Arg T,D,D Likely Pathogenic VIC47A MYBPC3 NM_ :c.1003C>T Arg335Cys D,D,D Likely Pathogenic WA15A MYBPC3 NM_ :c.2980C>T Leu994Phe D,D,D Likely Pathogenic NSW100A PKP2 NM_ :c.253_256del Glu85Met_fs* Likely Pathogenic NZ14A PKP2 NM_ :c.968_972del Gln323Arg_fs* Likely Pathogenic WA6A MYH6 NM_ :c.440G>A Arg147Lys T,P,D Likely Pathogenic NSW55A MYH7 NM_ :c.1988G>A Arg663His T,P,D Pathogenic QLD50A MYH7 NM_ :c.2427C>A Asp809Glu T,B,D VUS NSW3A MYH7 NM_ :c.4321G>T Ala1441Ser T,B,D VUS VIC3A ACTC1 NM_ :c.961G>A Ala321Thr D,P,D Likely Pathogenic NSW20A TPM1 NM_ :c.161A>T Glu54Val D,D,D Likely Pathogenic QLD42A DSG2 NM_ :c.2563C>A Gln855Lys T,B,N VUS NB: ESP MAF - Minor allele frequency in ESP data; 1000G MAF - Minor allele frequency in 1000 Genomes Project data; ExAC count - Allele count in Exome Aggregate Consortium data; Rare MAF < 0.1%; T - Tolerated; B - Benign; D - Probably damaging; P - Possibly damaging; N Neutral; VUS - Variant of uncertain significance. 20

22 SUPPLEMENTARY APPENDIX TABLE S9. Rare Variants in Rare Cardiomyopathy Genes. Sample ID Gene Symbol cdna variation Amino acid change ESP MAF 1000G MAF ExAC count GERP ++ score SIFT Polyphen2 Mutation Taster Pathogenicity Call NSW56A NEXN NM_ :c.1499C>A Ala500Glu T,B,D VUS NZ14A ACTN2 NM_ :c.302A>G Asn101Ser D,D,D Likely pathogenic SA25A ACTN2 NM_ :c.2231C>T Thr744Met T,B,D VUS NZ38A DES NM_ :c.1310del Gly437Val_fs* Likely pathogenic NZ38A TMEM43 NM_ :c.355G>A Val119Met D,D,D Likely pathogenic SA22A PRKAG2 NM_ :c.452C>G Ser151Cys T,D,D Likely pathogenic QLD33A FKTN NM_ :c.743T>C Ile248Thr T,P,D Likely pathogenic WA11A FKTN NM_ :c.869A>T Lys290Ile T,B,N VUS NZ29A RBM20 NM_ :c.98C>A Pro33Gln D,B,N VUS NZ29A RBM20 NM_ :c.103C>G Pro35Ala D,B,N VUS VIC3A RBM20 NM_ :c.2588C>G Pro863Arg D,B,N VUS NZ49A RBM20 NM_ :c.3595G>A Glu1199Lys T,B,D VUS NSW28A CSRP3 NM_ :c.311_312del Thr104Lys_fs* Likely pathogenic VIC24A TCAP NM_ :c.175G>A Val59Met T,B,D VUS NSW22A JPH2 NM_ :c.1033G>C Val345Leu T,B,D VUS ESP MAF - Minor allele frequency in ESP data; 1000G MAF - Minor allele frequency in 1000 Genomes Project data; ExAC count - Allele count in Exome Aggregate Consortium data; T - Tolerated; B - Benign; D - Probably damaging; P - Possibly damaging; N - Neutral. VUS - Variant of uncertain significance 21

23 SUPPLEMENTARY APPENDIX TABLE S10. Likely Pathogenic Variants in Epilepsy Genes Implicated with Dominant Disorders. Sample ID Gene Symbol cdna variation Amino acid change ESP MAF 1000G MAF ExAC count GERP ++ score SIFT Polyphen2 Mutation Taster NZ29A CHD2 NM_ :c.907G>A Gly303Ser T,D,D VIC14A CHRNA4 NM_ :c.296G>A Arg99His D,B,D VIC24A SCN8A NM_ :c.4393C>T Arg1465Cys D,B,D QLD50A SPTAN1 NM_ :c.3604G>A Val1202Met T,P,D Pathogenicity call Likely pathogenic Likely pathogenic Likely pathogenic Likely pathogenic ESP MAF - Minor allele frequency in ESP data; 1000G MAF - Minor allele frequency in 1000 Genomes Project data; ExAC count - Allele count in Exome Aggregate Consortium data; T - Tolerated; B - Benign; D - Probably damaging; P - Possibly damaging; N - Neutral. VUS - Variant of uncertain significance 22

24 SUPPLEMENTARY APPENDIX TABLE S11: Likely pathogenic/pathogenic gene variants associated with SUD characteristics All Genes: Gene - Gene + p-value N (%) 86 (76) 27 (24) - Age, years (10) 6 (22) (1) 0 (0) (2) 1 (4) (29) 5 (19) (15) 3 (11) (23) 4 (15) (19) 8 (30) Male gender 64 (74) 19 (70) Activity Exercise 12 (16) 3 (13) Sleep 41 (53) 11 (48) Night death 52 (69) 18 (72) Arrhythmia Genes Only: Gene - Gene + p-value N (%) 97 (86) 16 (14) - Age, years (11) 4 (25) (1) 0 (0) (3) 0 (0) (30) 1 (6) (14) 2 (13) (22) 3 (19) (19) 6 (38) Male gender 72 (74) 11 (69) Activity Exercise 14 (16) 1 (7) Sleep 45 (52) 7 (50) Night death 60 (70) 10 (71)

25 SUPPLEMENTARY FIGURE LEGENDS SUPPLEMENTARY APPENDIX FIGURE S1: Variant classification scheme. Bioinformatic analysis pipeline used for variant classification. Abbreviations: MAF = minor allele frequency, VUS = variant of uncertain significance. SUPPLEMENTARY APPENDIX FIGURE S2: Clinical and demographic features comparing SUD with explained SCD cases by 5-year age groups. Sudden unexplained death (SUD) cases = grey bars; explained sudden death cases (SCD) = black bars; NC = no cases in the age group. SUPPLEMENTARY APPENDIX FIGURE S3: Clinical follow-up of SUD families. Abbreviations: SCD = sudden cardiac death, SUD = sudden unexplained death. 24

26 SUPPLEMENTARY FIGURES SUPPLEMENTARY APPENDIX FIGURE S1 25

27 SUPPLEMENTARY APPENDIX FIGURE S2 26

28 SUPPLEMENTARY APPENDIX FIGURE S3 27

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