Confirmation of Cause and Manner of Death Via a Comprehensive Cardiac Autopsy Including Whole Exome Next-Generation Sequencing

* Annually, the sudden death of thousands of young people remains inadequately explained despite medicolegal inves- tigation. Postmortem genetic testing for channelopathies/ cardiomyopathies may illuminate a potential cardiac mechanism and establish a more accurate cause and manner of death and provide an actionable genetic marker to test surviving family members who may be at risk for a fatal arrhythmia. Whole exome sequencing allows for simultaneous genetic interrogation of an individual's entire estimated library of approximately 30 000 genes. Follow- ing an inconclusive autopsy, whole exome sequencing and gene-specific surveillance of all known major cardiac channelopathy/cardiomyopathy genes (90 total) were performed on autopsy blood-derived genomic DNA from a previously healthy 16-year-old adolescent female found deceased in her bedroom. Whole exome sequencing analysis revealed a R249Q-MYH7 mutation associated previously with familial hypertrophic cardiomyopathy, sudden death, and impaired b-myosin heavy chain (MHC-b) actin-translocating and actin-activated ATPase (adenosine triphosphatase) activity. Whole exome se- quencing may be an efficient and cost-effective approach to incorporate molecular studies into the conventional postmortem examination.

(Arch Pathol Lab Med. 2014;138:1083-1089; doi: 10.5858/arpa.2013-0479-SA)

In the United States, an estimated 300 000 to 400 000 individuals die suddenly each year, mostly involving the elderly and cardiac abnormalities identifiable on autopsy.1 With an incidence of between 1.3 and 8.5 per 100 000 patient years, sudden death in infants, children, adolescents, and young adults is relatively uncommon.2 Yet, annually, an estimated 1000 to 5000 young people between 1 and 35 years of age die suddenly.

While the cause and manner of many sudden deaths in the young are explained after a comprehensive medicolegal investigation that includes a conventional autopsy exami- nation, up to 50% of these cases of sudden death in the young remain unexplained, with no definite cardiac etiology identified after gross and microscopic inspection of the heart.3 Such deaths are often termed autopsy-negative sudden unexplained death (SUD).4

Potentially lethal and heritable cardiac channelopathies, such as long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and Brugada syndrome (BrS), are typically associated with grossly and histologically normal hearts. This often leaves medical examiners and coroners in a position to postulate only that a fatal cardiac arrhythmia was responsible for the sudden death in an otherwise healthy young individual, leaving the family with little or no insight into the ramifications for family members.3,5 In the case of sudden death-associated cardiomyopathies, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmo- genic cardiomyopathy (ACM), the lack of uniform diagnos- tic criteria has led to uneasiness in reporting with confidence when these conditions are present, given the serious implications for the decedent's family members. This is made even more complicated by the fact that in their earlier forms, these cardiomyopathies may manifest only subtle features.3

Postmortem genetic testing for channelopathies/cardio- myopathies may illuminate a potential cardiac mechanism and thereby establish a more accurate cause and manner of death and provide an actionable genetic marker by which to test surviving family members who may be potentially at risk for their own fatal arrhythmia. In fact, recently proposed guidelines for autopsy investigations of SUD in the young have suggested that postmortem genetic testing, for both structural and nonstructural genetically determined heart disease, should become the new standard of care in the evaluation of young SUD cases.6-8

While molecular autopsies involving the 4 major cardiac channelopathy genes (KCNQ1 [LQT1], KCNH2 [LQT2], SCN5A [LQT3, BrS1], and RYR2 [CPVT1]) have implicated LQTS, CPVT, and BrS as the underlying pathogenetic basis for an estimated 25% to 30% of SUD cases,9-11 to date there are at least 28 channelopathy-susceptibility genes. In addition, at least 64 genes have been associated with the major cardiomyopathies, that is, HCM, DCM, and ACM. Given the current financial landscape of the typical medical examiner's/coroner's office and the current lack of partici- pation among major insurance companies and third-party payers for postmortem genetic testing, the daunting task of a comprehensive cardiac channelopathy/cardiomyopathy workup appears to be out of reach.

However, next-generation whole exome sequencing (WES), which allows for simultaneous genetic interrogation of an individual's entire estimated library of 30 000 genes, using a small amount of DNA, can be completed at a research cost of $1000 to $2000 in a matter of weeks to months. Whole exome sequencing may represent an efficient and cost-effective approach for postmortem genetic testing, compared to the estimated 10-fold higher cost of performing standard ''1 gene, 1 exon'' at-a-time approach of Sanger sequencing. Presently, there are more than 90 known cardiac channelopathy- and cardiomyopathy-sus- ceptibility genes that could be included in a ''comprehen- sive'' sudden death gene panel. Moreover, WES can generate data that may be interrogated in the future as research uncovers new genetic mutations responsible for sudden death. Herein, we illustrate how WES established the definitive pathogenic cause and likely manner of death for a young victim of sudden death.

MATERIALS AND METHODS

Medical Examiner's Case of Sudden Death in an Adolescent Female

An autopsy was performed on a previously healthy 16-year-old white young woman (weight, 78.3 kg; height, 165.0 cm; body mass index, 28.2 kg/m2) who was found deceased in her bedroom. Significant findings from her autopsy included a moderately enlarged heart (352 g; expected range, 119-290 g) but with normal ventricular septum to left ventricular free wall ratio (1.0; normal, ,1.3). Histologically, the myocardium exhibited marked myocyte hypertrophy, myocyte disarray, and mild interstitial fibrosis, most notably in the anterolateral left ventricle. There was no identifiable mitral valve contact lesion in the left ventricular outflow tract. Additional findings included a patent foramen ovale (6 mm, potential diameter), mild cerebral edema, and fatty streaks present in the thoracic aorta. Of particular note, the postmortem toxicologic screen results were all negative.

Because of the absence of overt septal hypertrophy, the medical examiner deemed the autopsy inconclusive for a specific cardiomy- opathy and specifically did not render a necropsy diagnosis of HCM. Instead, the autopsy pathologist and local medical examiner submitted a specimen to Mayo Clinic'sWindland Smith Rice Sudden Death Genomics Laboratory in Rochester, Minnesota, for postmortem genetic mutational analysis in hopes of finding a cardiomyopathy mutation that might establish probable/definitive pathogenic cause.

Whole Exome Next-Generation DNA Sequencing

Three micrograms of genomic DNA isolated from 10 mL of blood, by using the Gentra Puregene Blood Kit (Qiagen, Germantown, Maryland) and following the manufacturer's protocol, was submitted to Mayo Clinic's Medical Genome Facility (Rochester, Minnesota), supported by the Mayo Center for Individualized Medicine for WES. Following exome capture with the SureSelect XT Human All Exon V4 plus UTR Target Enrichment System (Agilent, Santa Clara, California), 71-Mb paired-end sequencing at 96% coverage with a read depth of 353 was carried out on the Illumina HiSeq 2000 platform (San Diego, California) using V3 reagents. Variant alignment to the latest available human genome (hg19), Mapping and Assembly with Quality (Maq) single-nucleotide variant (SNV) detection,12 Burrows-Wheeler alignment insertion/deletion (INDEL) detec- tion,13 Maq and Genome Analysis Toolkit-based SNV/INDEL calling, SeattleSeq/Sorting Intolerant From Tolerant (SIFT) annotation, and allele frequencies for variants in the Single Nucleotide Polymorphism Database (dbSNP) and 1000 genomes were carried out by using the automated Targeted RE- sequencing Annotation Tool (TREAT) analytic pipeline devel- oped at Mayo Clinic (Rochester, Minnesota).14

An annotated list of all SNVs/INDELs that met quality control standards was provided in an Excel (Microsoft, Redmond, Washington) spreadsheet with links for variant visualization, tissue expression, and biologic pathway/process provided. Following WES and variant annotation, variant filtration involving the exclusion of all noncoding regions and synonymous variants (ie, a DNA nucleotide alteration that does not alter the amino acid sequence of the protein) and gene-specific surveillance of all known LQTS, CPVT, BrS, HCM, DCM, and ACM genes (90 total; Table 1 and Figure 1) were performed to identify possible pathogenic mutation(s).

To be considered a possible pathogenic mutation that may have contributed to the sudden death, any variant, discovered within the cardiomyopathy/channelopathy-associated gene subset, must be absent in a large panel of ethnically matched controls (595 white, 319 African American, 134 Asian, and 118 Hispanic persons) and absent in 3 publicly available exome databases including the 1000 Genomes Project (n ¼ 1094 subjects; 381 white, 246 African American, 286 Asian, and 181 Hispanic subjects),15 the National Heart, Lung, and Blood Institute (NHLBI) Grand Opportunity Exome Sequencing Project (n ¼ 5379 subjects; 3510 white and 1869 African American subjects),16 and the Exome Chip Design ( n ¼ 12 000 subjects).17

Possible pathogenic mutations were confirmed in the SUD case's genomic DNA by using standard polymerase chain reaction and Sanger DNA sequencing methods. Polymerase chain reaction primers, conditions, and sequencing methods are available upon request.

RESULTS

The SUD victim's exome contained 30 008 variants. Of these, 98 genetic variants resided within the 90 channelop- athy/cardiomyopathy-associated genes surveyed (Table 1). Twenty four variants within 15 genes were nonsynonymous variants (ie, a DNA alteration leading to a protein product with an altered amino acid sequence; Table 2). Most of these variants were common polymorphisms with a heterozygote frequency of 1% or greater in the public exome databases. However, 1 missense mutation (R249Q-MYH7; Table 2 and Figure 2) was absent in all publicly available exome databases including the 1000 Genomes Project (n ¼ 1094), the NHLBI Exome Sequencing Project (n ¼ 5379), and the 12 000 Exome Chip Design (n ¼ 12 000).

Figure 1 depicts our WES-based sudden cardiac death stepwise variant filtration process for mutation identifica- tion. The R249Q-MYH7 mutation was confirmed in the decedent's genomic DNA by using standard Sanger DNA sequencing methods. Mutations in the MYH7-encoded b- myosin heavy chain (MHC-b) is one of the most common causes of genetically identifiable HCM and has been reported rarely in DCM also. Specifically, R249Q-MYH7 has been associated previously with familial HCM, sudden death, and impaired MHC-b actin-translocating and actin- activated ATPase (adenosine triphosphatase) activity.18,19

Once the pathogenic mutation was determined, the cause of death was revised to R249Q-MYH7-mediated cardiomy- opathy. The surviving family members were then referred to a board-certified genetic counselor with specific expertise in cardiovascular diseases, for genetic counseling and for the orchestration and commencement of clinical cardiac evalu- ations and family-specific R249Q-MYH7 conformational mutation analysis to identify other family members at risk for sudden cardiac death. Considering the strength of the evidence for its assignment as a definite pathogenic mutation, predictive testing can be offered to the family. Accordingly, a mutation-negative relative with a normal echocardiogram finding can be dismissed from cardiology, whereas yearly cardiac evaluations would be considered for mutation-positive relatives. Further, given the positive family history of premature sudden death, a prophylactic implantable cardioverter defibrillator would be considered for a relative with so-called genotype-positive (ie, mutation positive for R249Q)/phenotype-positive (ie, diagnostic echocardiogram or cardiac magnetic resonance imaging findings) disease.

COMMENT

Potentially lethal cardiac channelopathies (LQTS, CPVT, and BrS) and cardiomyopathies (HCM, DCM, and ACM) are heritable genetic disorders that often exhibit variable expressivity ranging from a lifelong asymptomatic course, to mild cardiac episodes (ie, palpitations or syncope), to sudden cardiac death as the first manifestation, even in a single pedigree. In fact, the postmortem examination may represent the first opportunity to establish the presence and identity of potentially lethal genetic heart disease.20 Tragi- cally, unforeseen sudden deaths especially involving the young, leave a devastating void and exact an overwhelming psychological toll on living family members.

While a conventional autopsy may indicate an underlying pathologic process, a negative or inconclusive autopsy leaves a family without explanation and with anxiety that other family members could see a similar fate. Genetic testing in the postmortem setting can expose the underlying mechanism for the sudden death and identify or confirm the suspicion of an inherited sudden death-predisposing disorder.4,9 Therefore, a more complete cardiac investigation would empower caregivers and families to reduce the risk of additional sudden deaths through preventative measures.6

Recently, the Association for European Cardiovascular Pathology has recommended strongly the use of postmor- tem genetic analysis for both structural and nonstructural genetically determined heart disease as a part of the requirements for an adequate postmortem evaluation of a sudden cardiac death.7 In 2008, guidelines to ensure standardization of autopsy practices for sudden unexpected deaths in the young, and the preservation of appropriate material for all cases of sudden cardiac death to be used for genetic testing, was put forward by members of the Trans- Tasman Response AGAinst Sudden Death in the Young (TRAGADY).6 In 2011, the Heart Rhythm Society and the European Heart Rhythm Association recommended genetic testing in autopsy-negative SUD cases, especially when clinically relevant presentations existed among the decedent or family members.8 However, this level of care has been extremely difficult for medical examiners and coroners to provide owing to the cost and time-consuming nature of current genetic testing efforts.

However, WES is becoming increasingly available at a reasonable price and may be more efficient and cost- effective than previous approaches. In fact, while the cost of performing research-grade WES for this case, using Mayo Clinic's core facility, was only $1528, it would have cost an estimated $16 121 ($9.50/exon) to perform traditional DNA Sanger sequencing for all 1697 coding regions exons of 90 unique genes that have been implicated in sudden death- associated channelopathies/cardiomyopathies.

In this report, we provide proof-of-principle for WES- based postmortem genetic testing for a 16-year-old otherwise healthy young woman who died suddenly. Given the inconclusive gross and histologic findings on autopsy and the lack of a significant family history, a test to help confirm the presence of disease was highly desired. After WES and gene-specific surveillance, we identified an R249Q-MYH7 mutation that has been associated previously with familial HCM and sudden death.18,19

Using WES, we were able to complete our analysis of all 90 genes, verify R249Q-MYH7 in a CLIA (Clinical Labora- tory Improvement Amendments of 1988)-approved labora- tory, commence genetic counseling, cascade genetic testing in surviving family members, and initiate surveillance for those family members at risk for HCM, all within a few months. As a result, 6 additional family members were found to be at risk for HCM and were advised of recommendations for both clinical cardiac and genetic testing. Thus far, 3 family members have been informed that they are negative for the familial R249Q-MYH7 mutation and are therefore not at increased risk for either disease expression or future disease transmission, and have been dismissed from further cardiac follow-up.

To perform WES-based postmortem genetic testing, it is imperative that medical examiners and coroners adhere to current sample collection and retention guidelines. To ensure an adequate amount of DNA is available for WES, it is recommended that at least 5 to 10 mL of blood in EDTA (ie, purple-top tube) or 5 g of fresh tissue from the heart, liver, or spleen be collected at autopsy.5,7 While WES may be performed on DNA isolated from blood spot cards, the amount of DNA isolated from this source is often inadequate for a WES-based strategy. If blood spot cards are the only retainable source of DNA, owing to logistical concerns, then we would recommend collecting at least 4 to 8 ''US quarter''-size spots of blood on a card specifically formulated for DNA extraction.

While the comprehensive nature of WES is beneficial, it also brings the daunting task of sifting through hundreds to thousands of nonsynonymous genetic variants for each individual exome, many of which might be predicted in silico to be deleterious. With WES, there is also great potential for the unintended identification of noncardiac disease-associated genetic variants not responsible for the sudden death, but that may be of substantial concern for surviving family members. Hypothetically, WES analysis of an SUD case could reveal the presence of a cancer- susceptibility BRCA1 or BRCA2 mutation that may be present in surviving family members who are currently unaware of their increased risk for cancer.

However, using bioinformatics and data filtering, one can focus variant analysis on targeted genes and avoid unintend- ed incidental findings while conserving resources only for identifying the cause of sudden cardiac death. This is particularly important in cases of a targeted sudden death WES investigation, as surviving family members would not likely have the opportunity or full ability to make a well- informed decision as to the vast scope of information that may become available through WES (carrier status, risk for late-onset conditions). Focused testing limits the ethical dilemma and responsibility as to what is reported to the family and allows subsequent genetic counseling to concen- trate on the family's understanding and adaptation to the cause of death, and on the implications to other family members, and to support the coping and grieving process.

It is therefore critical that the continued evaluation of a SUD case, using WES-based molecular testing, involve not only the medical examiner-and ideally, when available, also a cardiac pathologist responsible for performing a careful gross and histologic examination and for procurement of appropriate samples for genetic testing-but also a multidis- ciplinary health care team. This team should include a cardiologist with expertise in the clinical management of cardiac channelopathies and cardiomyopathies, who is conversant in the interpretation of cardiac genetic test results as a probabilistic predictor of pathogenicity, and a board- certified genetic counselor specifically trained in cardiovas- cular diseases and in the complex nature of WES.5,21,22

This report details the first utilization of WES to confirm the underlying pathogenic substrate most likely responsible for the sudden unexpected death of an otherwise healthy adolescent. Moreover, it allowed for the rapid identification and triaging of her at-risk living family members and dismissal of her mutation-negative relatives. Whole exome sequencing followed by gene-specific surveillance may be a highly efficient and cost-effective approach for a truly comprehensive cardiovascular autopsy.

References

1. Virmani R, Burke A, Farb A. Sudden cardiac death. Cardiovasc Pathol. 2001;10(6):275-282.

2. Liberthson RR. Sudden death from cardiac causes in children and young adults. N Engl J Med. 1996;334(16):1039-1044.

3. Tester DJ, Ackerman MJ. Cardiomyopathic and channelopathic causes of sudden unexplained death in infants and children. Annu Rev Med. 2009;60(1): 69-84.

4. Tester D, Ackerman M. The molecular autopsy: should their evaluation continue after the funeral? Pediatr Cardiol. 2012;33(3):461-470.

5. Semsarian C, Hamilton R. Key role of the molecular autopsy in sudden unexpected death. Heart Rhythm. 2012;9(1):145-150.

6. Skinner JR, Duflou JA, Semsarian C. Reducing sudden death in young people in Australia and New Zealand: the TRAGADY initiative. Med J Aust . 2008; 189(10):539-540.

7. Basso C, Burke M, Fornes P, et al. Guidelines for autopsy investigation of sudden cardiac death. Virchows Arch. 2008;452(1):11-18.

8. Ackerman M, Priori S, Willems S, et al. HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies: this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). Heart Rhythm. 2011;8(8):1308-1339.

9. Tester D, Medeiros-Domingo A, Will M, Haglund C, Ackerman M. 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(6):524-539.

10. Skinner J, 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(3):412-419.

11. Gladding P, Evans C, Crawford J, et al. Posthumous diagnosis of long QT syndrome from neonatal screening cards. Heart Rhythm. 2010;7(4):481-486.

12. Li H, Ruan J, Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 2008;18(11):1851-1858.

13. Li H, Durbin R. Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics. 2009;25(14):1754-1760.

14. Asmann YW, Middha S, Hossain A, et al. TREAT: a bioinformatics tool for variant annotations and visualizations in targeted and exome sequencing data. Bioinformatics. 2012;28(2):277-278.

15. Clarke L, Zheng-Bradley X, Smith R, et al. The 1000 Genomes Project: data management and community access. Nat Methods. 2012;9(5):459-462.

16. Exome Variant Server NESPE. Seattle, WA.http://evs.gs.washington.edu/ EVS/. Accessed September 18, 2013.

17. Exome Chip Design. http://genome.sph.umich.edu/wiki/Exome_Chip_ Design. Accessed September 18, 2013.

18. Watkins H, Rosenzweig A, Hwang DS, et al. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomy- opathy. N Engl J Med. 1992;326(17):1108-1114.

19. Roopnarine O, Leinwand LA. Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy. Biophys J. 1998;75(6):3023-3030.

20. Crotti L. Genetic predisposition to sudden cardiac death. Curr Opin Cardiol. 2011;26(1):46-50.

21. Hershberger RE, Cowan J, Morales A, Siegfried JD. Progress with genetic cardiomyopathies: screening, counseling, and testing in dilated, hypertrophic, and arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ Heart Fail. 2009;2(3):253-261.

22. Ingles J, Yeates L, Semsarian C. The emerging role of the cardiac genetic counselor. Heart Rhythm. 2011;8(12):1958-1962.

Christina G. Loporcaro, BS; David J. Tester, BS; Joseph J. Maleszewski, MD; Teresa Kruisselbrink, MS; Michael J. Ackerman, MD, PhD

Accepted for publication October 7, 2013.

Published as an Early Online Release December 2, 2013.

From the Mayo Medical School, Rochester, Minnesota (Ms Loporcaro); the Departments of Internal Medicine/Division of Cardiovascular Diseases (Mr Tester and Dr Ackerman), Laboratory Medicine & Pathology, Division of Anatomic Pathology (Dr Maleszewski), Laboratory Medicine & Pathology, Division of Laboratory Genetics (), Molecular Pharmacology & Experimental Therapeutics (Dr Ackerman), and Pediatric and Adolescent Medicine/Division of Pediatric Cardiology (Dr Acker- man), and the Windland Smith Rice Sudden Death Genomics Laboratory (Mr Tester and Dr Ackerman), Mayo Clinic, Rochester, Minnesota.

Dr Ackerman is a consultant for Transgenomic, Inc (Omaha, Nebraska), which provides one of the commercially available clinical genetic tests for long QT syndrome (LQTS) called FAMI- LION-LQTS. In addition, Dr Ackerman (significant) and Mr Tester (modest) receive royalty payments from Mayo Clinic (Rochester, Minnesota) for intellectual property/technology agreements made between Mayo Clinic and Transgenomic, Inc. The other authors have no relevant financial interest in the products or companies described in this article.

This work was supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program, the Sheikh Zayed Saif Mohammed Al Nahyan Fund in Pediatric Cardiology Research, the Dr. Scholl Fund, and the Hannah M. Wernke Memorial Fund.

The abstract was presented in poster format by Mr Tester at the American Society of Human Genetics Annual Meeting; November 8, 2012; San Francisco, California.

Reprints: Michael J. Ackerman, MD, PhD, Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, 501 Guggen- heim,200FirstStSW,Rochester,MN55905(e-mail:ackerman. [email protected]).