Sudden death is defined as death occurring within 1 hour of the onset of symptoms or within 24 hours of the victim being seen alive.1 Sudden death due to an underlying heart disease is known as sudden cardiac death (SCD). The worldwide annual incidence of SCD is about 4 to 5 million cases per year.2 A tragic and devastating complication of a number of heart diseases, SCD is most often unexpected and has major implications for the surviving family and the community. The majority of SCD cases in middle-aged and older individuals are caused by coronary artery disease; however, SCD is rare in young people and the causes are more diversified.3 4 Autopsy studies have shown no structural heart disease was found in up to 30% of young SCD victims.5 6 7 8

Molecular autopsy was first described by Ackerman et al9 in 1999, to determine the cause of death in uncertain cases after conventional autopsy by genetic analysis. Post-mortem genetic studies have shown that SCD in these victims can be caused by fatal arrhythmias secondary to a group of inheritable cardiac electrical disorders collectively known as sudden arrhythmia death syndrome (SADS).10 11 12 13 14 These include Brugada syndrome (BrS), long QT syndrome (LQTS), short QT syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), hypertrophic cardiomyopathy (HCM), and other cardiomyopathies.

Because SADS-related conditions are genetic diseases, there are two different approaches to identify SADS among young sudden unexplained death (SUD) victims. The first approach involves detailed clinical and targeted genetic examination of the surviving relatives of SCD victims. Studies using this approach suggest that SADS may account for approximately 40% of autopsy-negative sudden death in young people.10 15 However, this approach may not be able to identify subjects with concealed form of SADS due to incomplete penetrance and variable expressivity of the pathological mutations. The second approach is to perform molecular autopsy on SCD victims, which involves post-mortem genetic testing for SADS. A landmark study on molecular autopsy by the Mayo Clinic showed over one third of SCD cases hosted a presumably pathogenic mutations of cardiac ion channel diseases.8 16 Thus a combined approach using both cardiac evaluation of surviving relatives and molecular autopsy on SUD victims should give a higher yield on elucidating the underlying causes of SUD.

In Hong Kong, there are scarce data on the prevalence and types of SADS underlying SCD or SUD in young people. The present study aimed to investigate the prevalence and types of SADS in an East Asian population, and to perform a pilot study on incorporating cardiac evaluation of surviving relatives and molecular autopsy by next-generation sequencing (NGS) into conventional forensic investigations.

In the present study, first, we carried out a 5-year retrospective review of the records of all autopsies for young sudden death victims performed in public mortuaries in Hong Kong. Second, we conducted a 2-year study to determine the prevalence and types of SADS as the underlying causes of SCD among local young victims through conventional and molecular autopsy, and evaluated their first-degree relatives.

The Forensic Pathology Service, Department of Health, provides all public autopsy services for over 7 million people in Hong Kong. We performed this retrospective review of the records of all autopsies in public mortuaries for young sudden death victims (aged 5-40 years) between 1 January 2008 and 31 December 2012. Sudden death was defined as death occurring within 1 hour of the onset of symptoms or within 24 hours of the victim being seen alive.1 Sudden death victims were recruited into the study for a detailed review of their autopsy records. Data including age, height, weight, sex, circumstances of death, clinical history of cardiac disease, and pathologic findings at autopsy were collected and analysed. Sudden death victims whose deaths were caused by trauma, accidents, drowning, and drug toxicity, and those whose autopsy records were either incomplete or not accessible for retrospective review were excluded.

In this prospective study, young SCD victims (aged 5-40 years) were identified and recruited into the study by forensic pathologists after a finding of either an inheritable arrhythmogenic cardiomyopathy or no anatomical cause of death (including other structural heart disease) on autopsy, and a negative toxicology screening. Clinical history, including personal history of arrhythmic events and history surrounding the sudden death event of the SCD victim was collected during identification interviews with next-of-kin as far as possible by forensic pathologists. DNA-friendly blood samples were collected for molecular autopsy. Written informed consent for a molecular autopsy was obtained from the next-of-kin of each victim. The first-degree relatives of the victims were referred by forensic pathologists to the study centre for genetic counselling and recruitment into the study. Clinical history, including personal history of arrhythmic events and history surrounding the sudden death event of the SCD victim was collected from family members. All recruited first-degree relatives underwent clinical evaluation including 12-lead electrocardiogram (ECG), signal-averaged ECG, echocardiogram, 24-hour Holter analysis and treadmill exercise testing. Additional investigations were used only as required, such as flecainide provocation testing (if BrS is suspected in subjects >18 years) and targeted genetic screening (if positive molecular autopsy findings in the index SUD victim). All first-degree relatives provided written informed consent for these procedures and for publication of the results.

A panel of 35 genes implicated in SADS for BrS, LQTS, short QT syndrome, CPVT, ARVC, and HCM was tested using NGS (Table 1). The NGS was performed using targeted gene capture technique on a MiSeq Sequencing System (Illumina, Inc, San Diego [CA], United States). Target regions of interest were restricted to the coding regions and the 10-bp flanking regions. Target rates indicated percentage of bases with a minimum depth of coverage of 20×. Alignments to the February 2009 (GRCh37/hg19) human genome assembly and variant calls were generated using dual pipelines, SoftGenetics NextGENe (v2.4.1) and an in-house one where sequencing reads were aligned by Burrows-Wheeler Aligner (v0.7.5a-r405) to hg19 genome and processed with picard-tools (v1.114). Local realignment for indels and base quality recalibration were performed with Genome Analysis Toolkit (GATK, v3.2). Variant calls were made with UnifiedGenotyper (GATK v3.2). Only those genes included in the requested gene panel were processed for variant calling. Variants identified were annotated and analysed with VariantStudio (v2.2.1; Illumina, Inc); in general, variants with an allele frequency of <0.1% for dominant disorders or <1.0% for recessive disorders were reported. Pathogenic and likely pathogenic variants were confirmed by Sanger sequencing; benign and likely benign variants were not reported.

The genetic variant pathogenicity was established according to the Practice Guidelines for the Interpretation and Reporting of Unclassified Variants in Clinical Molecular Genetics by the Clinical Molecular Genetics Society (http:// www.acgs.uk.com/media/774853/evaluation_and_reporting_of_sequence_variants_bpgs_june_2013_-_finalpdf.pdf). Major criteria include degree of conservation, population allele frequencies, co-segregation pattern, literature data, functional studies and in silico prediction. The pathogenicity of novel missense variants was analysed by PolyPhen-2, SIFT, MutationTaster and Assessing Pathogenicity Probability in Arrhythmia by Integrating Statistical Evidence (https://cardiodb.org/APPRAISE/) and that of novel splicing variants was by Splice Site Finder-like, MaxEntScan, GeneSplicer and Human Splicing Finder, wherever appropriate. Splicing variants were considered to be damaging if the score was more than 10% lower than the wild-type prediction. Allele frequencies among populations were as reported in the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/).

A diagnosis of SADS was established when molecular autopsy in SCD victims identified a positive genetic variant implicated in SADS or generally accepted clinical criteria for a particular disease were fulfilled in the SCD victims or their first-degree relatives. The descriptive statistics were analysed using Excel 2016 (Microsoft Corp, Redmond [WA], United States).

There were 17 187 autopsies performed during the study period and 2748 (16%) deaths were aged 5 to 40 years. There were 420 sudden death victims. Among them, 289 (69%) were SCD (male:female ratio=9:2). The median age was 32 years (range, 7-40 years). Coronary artery diseases accounted for 35% of the causes of death; other causes of death were aortic dissection (6%), myocarditis (6%), left ventricular hypertrophy (4%), dilated cardiomyopathy (9%), other structural heart diseases (9%), HCM (4%), ARVC (2%), and unexplained (25%).

There were 32 SCD victims aged 5 to 40 years between 1 July 2014 and 30 June 2016 with a finding of either an inheritable arrhythmogenic cardiomyopathy or no anatomical cause of death (including other structural heart disease) on autopsy and negative toxicology results. Eleven individuals were excluded: two who had no Hong Kong identity card and nine whose families refused consent. Finally, 21 SCD victims (18 male, 3 female) were recruited into the study. Table 2 shows the clinical and forensic data of the 21 SCD victims. The median age was 31 years (range, 14-39 years). From the conventional autopsy, 18 (86%) were unrevealing; two (10%) SCD victims had ARVC and one (5%) had HCM. The majority died during resting (48%) or sleeping (24%). Only three SCD victims (14%) died during exercise. Three (14%) SCD victims had family history of SCD and seven (33%) had a history of syncope. Many (71%) of the SCD victims had unremarkable past health. Genetic analysis showed 29% with positive heterozygous genetic variants (Table 3). Seven variants were identified in seven genes, six variants of which were novel. The genetic background was heterogeneous without any common mutations found. Combining conventional and molecular autopsy findings, the cause of death in 12 (57%) still remains unknown; cause of death was confirmed in four (19%) as ARVC, in two (10%) as BrS, and one (5%) each in HCM, LQTS, and CPVT.

Overall, more than half of first-degree relatives (6 of 11 individuals) who underwent genetic testing carried the positive genetic variants. Among them, SADS-related clinical features were detected in three first-degree relatives from two families (cases 14 and 16). The true clinical phenotype can sometimes be detected in surviving relatives upon appropriate clinical evaluation, which in turn significantly aids the interpretation of genetic findings. Cases 14 and 16 illustrate the importance of this (Fig 1). The first-degree relatives of these two cases showed SADS-related clinical features after cardiologist’s assessment.

Case 14 was a 17-year-old male victim who died of sudden nocturnal death. He had an episode of syncope few months prior to his death but he did not seek medical attention. Family screening by clinical evaluation found no structural heart disease but ECG revealed prolonged QTc interval in his asymptomatic father and sister (530 ms and 480 ms, respectively) suggesting a diagnosis of LQTS (Fig 1). Molecular autopsy found heterozygous NM_199460.2(CACNA1C):c.2276C>G NP_955630.2:p.(Ala759Gly). This novel variant is predicted to be damaging and absent in the gnomAD. CACNA1C is the gene encoding for the alpha-1c subunit of the type 1 voltage-dependent calcium channel involved in the cardiac myocyte membrane polarisation. Its mutations have been reported in BrS and LQTS.

Case 16 was a 19-year-old male victim who also died of sudden nocturnal death. There was no known syncope or preceding symptoms ahead of the collapse. He had history of abnormal heart beat, although ECG was not done. His family history was unremarkable. Autopsy revealed pulmonary hypertension. Molecular autopsy detected heterozygous NM_198056.2(SCN5A):c.2893C>T (p.Arg965Cys) which has been reported as a disease causing mutation of BrS.17 18 19 The same variant has also been reported in patients with LQTS (with digenic mutation in KCNH2 in one case).20 21 The allele frequency of this variant (rs199473180) is reported to be 0.058% in East Asians (gnomAD). In silico analyses by SIFT, MutationTaster, and PolyPhen-2 have revealed this variant to be damaging, disease causing, and probably damaging, respectively. Functional study showed that the p.(Arg965Cys) mutant led to slower recovery from inactivation as a result of channels with a more negative potential in steady state inactivation.18 Family screening found his father carrying the same SCN5A mutation and a type 2 Brugada ECG pattern (Fig 1). However, no type 1 Brugada ECG feature was revealed by a flecainide provocation test. The clinical phenotype in this case supports the pathogenicity of this novel genetic variant.

To elucidate the cause of SCD, a comprehensive post-mortem evaluation is required. Figure 2 shows the combined approach using both cardiac evaluation of surviving relatives and molecular autopsy on SUD victims which would give a higher yield on elucidating the underlying causes of SUD. Any history of syncope, cardiac symptoms especially related to exertion, emotion and stress, previous ECG, circumstances of SCD (activity at the time of death), any family history of cardiac disease, premature or sudden death, near-arrest attack, and epilepsy should be investigated. Patients with SADS can present with or be (incorrectly) labelled as having epilepsy.22 23 24 25 Guidelines on post-mortem examination of SUD in the young have been published by the Royal College of Pathologists of Australasia (https://www.rcpa.edu.au/getattachment/89884c69-f066-411d-a3d1- 39460444db13/Guidelines-on-Autopsy-Practice.aspx). In addition to traditional autopsy, some reports have used whole-body computed tomography and magnetic resonance imaging to identify structural heart abnormalities such as ARVC and HCM.26 27 However, these imaging modalities were not used in the present study.

Technologies applied in molecular autopsy have changed from Sanger sequencing targeting a few major SUD-related genes to NGS targeting an expanded gene panel of up to 200 genes.28 The former approach achieved diagnostic yields of around 15% to 20%.8 16 29 30 31 32 With increasing throughput capacities at more affordable costs, the large gene panel or exome approach by NGS is becoming more appealing in molecular autopsy. A proof-of-principle exome-wide study was carried out among 50 SUD subjects, with likely pathogenic variants identified in 32%.33 The diagnostic yield from various NGS studies, including our own, is up to 35%, despite the different lengths of gene lists.13 34 35 36 However, the spectrum of diseases might be different and rarer causes might be identified, because substantial clinical suspicion is typically lacking among young SCD victims with unremarkable premorbid history. However, a negative genetic result does not necessarily exclude the possibility of a genetic basis for the disease.

The major difficulty of molecular autopsy is establishing the causation between the death and the genetic variants. Applying molecular autopsy to the investigation of death causes involves probabilistic rather than binary “yes or no” answers. Interpretation of variant pathogenicity relies heavily on the characteristics of the genetic variant, allele rarity in population frequencies, in silico predictions, co-segregation patterns among affected family members, previous literature data on reports of similar cases, and available functional data. This approach follows the usual practice by which pathologists deal with genetic reporting.

There are two main advantages of molecular autopsy in SCD. First, molecular autopsy enables a correct diagnosis of SCD to be established, which can bring some level of closure to the family. Findings of a SADS-related condition can differentiate a natural cause from an unnatural cause of death, such as the possibility of drowning precipitated by an inherited arrhythmia during swimming. Second, the ultimate goal of diagnosing SCD is to prevent another tragedy among family members. The majority of SADS-related disorders are autosomal dominant inherited, and family members are at 50% risk of inheriting the mutation, with variable penetrance. Family pedigree for at least three generations should be included in the extended clinical workup, and genetic counselling should be considered for second- or higher-degree relatives wherever appropriate.

There are limitations to our study. First, SCD victims with autopsy conducted in a public hospital were not recruited in this study. Hence, the sample numbers may not be representative for the whole territory. Second, no premorbid ECG findings of the victims were available to correlate with the genetic results. Third, despite multiple efforts, no genetic mutations were found in over two thirds of the SCD victims in our study. Expanding the gene list may be able to increase the yield. The associated phenotypes and pathogenesis of some genes are still yet to be further elucidated.

Sudden arrhythmia death syndrome is a significant cause of SCD in the young. This study is first of its kind in an East Asian population and provides important data on the prevalence and types of SADS among young SCD victims. This is also the first local feasibility study on incorporating cardiac evaluation of surviving relatives and NGS molecular autopsy into the conventional forensic investigations. The interpretation of genetic variants in the context of SCD is complicated and we recommend its analysis and reporting by qualified pathologists. This model may be considered to cover all age-groups of SCD victims, as well as other potential applications such as sudden unexpected death in epilepsy, or sudden infant death syndrome. This local pilot study should be considered an important advance in diagnosing young SCD victims in East Asian populations.

All authors have made substantial contributions to the concept or design of the study, acquisition of data, analysis or interpretation of data, drafting of the manuscript, and critical revision for important intellectual content. All authors had full access to the data, contributed to the study, approved the final version for publication, and take responsibility for its accuracy and integrity.

All authors have disclosed no conflicts of interest.

The 5-year review was presented at the Asia Pacific Heart Rhythm Society Meeting on 3 to 6 October 2013, in Hong Kong. Part of the SADS HK Study results was presented at CardioRhythm on 24 to 26 February 2017, in Hong Kong and was published (J HK Coll Cardiol 2016;24:34).

This work was partly funded by SADS HK Foundation Limited, Hong Kong.

The study was approved by local ethics committees (Ethic Committee of the Department of Health (L/M 601/2013) and Kowloon West Cluster Research Ethics Committee (KWC-REC Reference KW/FR-13-023-67-05)). Consent was obtained from the next-of-kin of the SCD victims and from the first-degree relatives themselves under study.

1. Puranik R, Chow CK, Duflou JA, Kilborn MJ, McGuire MA. Sudden death in the young. Heart Rhythm 2005;2:1277-82. Crossref

2. Chugh SS, Reinier K, Teodorescu C, et al. Epidemiology of sudden cardiac death: clinical and research implications. Prog Cardiovasc Dis 2008;51:213-28. Crossref

3. Basso C, Corrado D, Thiene G. Cardiovascular causes of sudden death in young individuals including athletes. Cardiol Rev 1999;7:127-35. Crossref

4. Link MS. Sudden cardiac death in the young: epidemiology and overview. Congenit Heart Dis 2017;12:597-9. Crossref

5. Chugh SS, Kelly KL, Titus JL. Sudden cardiac death with apparently normal heart. Circulation 2000;102:649-54. Crossref

6. Corrado D, Basso C, Thiene G. Sudden cardiac death in young people with apparently normal heart. Cardiovasc Res 2001;50:399-408. Crossref

7. de Noronha SV, Sharma S, Papadakis M, Desai S, Whyte G, Sheppard MN. Aetiology of sudden cardiac death in athletes in the United Kingdom: a pathological study. Heart 2009;95:1409-14. Crossref

8. Tester DJ, Medeiros-Domingo A, Will ML, Haglund CM, Ackerman MJ. Cardiac channel molecular autopsy: insights from 173 consecutive cases of autopsy-negative sudden unexplained death referred for postmortem genetic testing. Mayo Clin Proc 2012;87:524-39. Crossref

9. Ackerman MJ, Tester DJ, Driscoll DJ. Molecular autopsy of sudden unexplained death in the young. Am J Forensic Med Pathol 2001;22:105-11. Crossref

10. Tester DJ, Ackerman MJ. The role of molecular autopsy in unexplained sudden cardiac death. Curr Opin Cardiol 2006;21:166-72. Crossref

11. Wever-Pinzon OE, Myerson M, Sherrid MV. Sudden cardiac death in young competitive athletes due to genetic cardiac abnormalities. Anadolu Kardiyol Derg 2009;9 Suppl 2:17-23.

12. Hellenthal N, Gaertner-Rommel A, Klauke B, et al. Molecular autopsy of sudden unexplained deaths reveals genetic predispositions for cardiac diseases among young forensic cases. Europace 2017;19:1881-90. Crossref

13. Hertz CL, Christiansen SL, Ferrero-Miliani L, et al. Next-generation sequencing of 100 candidate genes in young victims of suspected sudden cardiac death with structural abnormalities of the heart. Int J Legal Med 2016;130:91-102. Crossref

14. Christiansen SL, Hertz CL, Ferrero-Miliani L, et al. Genetic investigation of 100 heart genes in sudden unexplained death victims in a forensic setting. Eur J Hum Genet 2016;24:1797-802. Crossref

15. Semsarian C, Hamilton RM. Key role of the molecular autopsy in sudden unexpected death. Heart Rhythm 2012;9:145-50. Crossref

16. Tester DJ, Spoon DB, Valdivia HH, Makielski JC, Ackerman MJ. Targeted mutational analysis of the RyR2-encoded cardiac ryanodine receptor in sudden unexplained death: a molecular autopsy of 49 medical examiner/coroner’s cases. Mayo Clin Proc 2004;79:1380-4. Crossref

17. Priori SG, Napolitano C, Gasparini M, et al. Natural history of Brugada syndrome: insights for risk stratification and management. Circulation 2002;105:1342-7. Crossref

18. Hsueh CH, Chen WP, Lin JL, et al. Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations. J Biomed Sci 2009;16:23. Crossref

19. Yu JH, Hu JZ, Zhou H, et al. SCN5A mutation in patients with Brugada electrocardiographic pattern induced by fever [in Chinese]. Zhonghua Xin Xue Guan Bing Za Zhi 2013;41:1010-4.

20. Jimmy JJ, Chen CY, Yeh HM, et al. Clinical characteristics of patients with congenital long QT syndrome and bigenic mutations. Chin Med J (Engl) 2014;127:1482-6.

21. Lieve KV, Williams L, Daly A, et al. Results of genetic testing in 855 consecutive unrelated patients referred for long QT syndrome in a clinical laboratory. Genet Test Mol Biomarkers 2013;17:553-61. Crossref

22. Medford BA, Bos JM, Ackerman MJ. Epilepsy misdiagnosed as long QT syndrome: it can go both ways. Congenit Heart Dis 2014;9:E135-9. Crossref

23. Partemi S, Cestèle S, Pezzella M, et al. Loss-of-function KCNH2 mutation in a family with long QT syndrome, epilepsy, and sudden death. Epilepsia 2013;54:e112-6. Crossref

24. Goyal JP, Sethi A, Shah VB. Jervell and Lange-Nielson Syndrome masquerading as intractable epilepsy. Ann Indian Acad Neurol 2012;15:145-7. Crossref

25. Tu E, Bagnall RD, Duflou J, Semsarian C. Post-mortem review and genetic analysis of sudden unexpected death in epilepsy (SUDEP) cases. Brain Pathol 2011;21:201-8. Crossref

26. Roberts IS, Benamore RE, Benbow EW, et al. Post-mortem imaging as an alternative to autopsy in the diagnosis of adult deaths: a validation study. Lancet 2012;379:136-42. Crossref

27. Puranik R, Gray B, Lackey H, et al. Comparison of conventional autopsy and magnetic resonance imaging in determining the cause of sudden death in the young. J Cardiovasc Magn Reson 2014;16:44. Crossref

28. Semsarian C, Ingles J. Molecular autopsy in victims of inherited arrhythmias. J Arrhythm 2016;32:359-65. Crossref

29. Liu C, Zhao Q, Su T, et al. Postmortem molecular analysis of KCNQ1, KCNH2, KCNE1 and KCNE2 genes in sudden unexplained nocturnal death syndrome in the Chinese Han population. Forensic Sci Int 2013;231:82-7. Crossref

30. Doolan A, Langlois N, Chiu C, Ingles J, Lind JM, Semsarian C. Postmortem molecular analysis of KCNQ1 and SCN5A genes in sudden unexplained death in young Australians. Int J Cardiol 2008;127:138-41. Crossref

31. Skinner JR, Crawford J, Smith W, et al. Prospective, population-based long QT molecular autopsy study of postmortem negative sudden death in 1 to 40 year olds. Heart Rhythm 2011;8:412-9. Crossref

32. Hofman N, Tan HL, Alders M, et al. Yield of molecular and clinical testing for arrhythmia syndromes: report of 15 years’ experience. Circulation 2013;128:1513-21. Crossref

33. Bagnall RD, Das K J, Duflou J, Semsarian C. Exome analysis-based molecular autopsy in cases of sudden unexplained death in the young. Heart Rhythm 2014;11:655-62. Crossref

34. 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;374:2441-52. Crossref

35. Farrugia A, Keyser C, Hollard C, Raul JS, Muller J, Ludes B. Targeted next generation sequencing application in cardiac channelopathies: analysis of a cohort of autopsy-negative sudden unexplained deaths. Forensic Sci Int 2015;254:5-11. Crossref

36. Tester DJ, Ackerman MJ. Postmortem long QT syndrome genetic testing for sudden unexplained death in the young. J Am Coll Cardiol 2007;49:240-6. Crossref