Key role of the molecular autopsy in sudden unexpected death

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1 CONTEMPORARY REVIEW Key role of the molecular autopsy in sudden unexpected death Christopher Semsarian, MBBS, PhD,* Robert M. Hamilton, MD, CCDS From *Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Sydney, Australia, Sydney Medical School, University of Sydney, Sydney, Australia, Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia, and Labatt Heart Centre, The Hospital for Sick Children and Research Institute, University of Toronto, Ontario, Canada. Sudden Cardiac Death (SCD) is a major and tragic complication of a number of cardiovascular diseases. While in the older populations, SCD is most frequently caused by underlying coronary artery disease and heart failure, in those aged under 40 years, the causes of SCD commonly include genetic disorders, such as inherited cardiomyopathies and primary arrhythmogenic diseases. As part of the evaluation of families in which SCD has occurred, the role of genetic testing has evolved as an important feature in both establishing an underlying diagnosis and in screening at-risk family relatives. Specifically, in cases where no definitive cause is identified at postmortem, i.e. Sudden Unexpected Death (SUD), the molecular autopsy has emerged as a key process in the investigation of the cause of death. The combination of clinical and genetic evaluation of families in which SUD has occurred provides a platform for early initiation of therapeutic and prevention strategies, with the ultimate goal to reduce sudden death among the young in our communities. KEYWORDS Genetics; Ion channels; Molecular autopsy; Multidisciplinary care; Sudden cardiac death ABBREVIATIONS BrS Brugada syndrome; CPVT catecholaminergic polymorphic ventricular tachycardia; LQTS long QT syndrome; SCD sudden cardiac death; SUD sudden unexpected death (Heart Rhythm 2012;9: ) 2012 Heart Rhythm Society. All rights reserved. Introduction Sudden cardiac death (SCD) is a tragic and devastating complication of a number of cardiovascular diseases. The death is most often unexpected and has major implications for the surviving family and the community. SCD is defined as a death occurring usually within an hour of the onset of symptoms, due to an underlying cardiac disease. SCD can occur at all ages. The prevalence of SCD is significant, with at least 3 million people worldwide dying suddenly each year. 1,2 In the United States, SCD occurs in up to 450,000 people each year, translating to over 1000 deaths per day or 1 death every 1.5 minutes. 3,4 Whereas SCD in the older populations is most frequently due to underlying coronary artery disease and heart failure, in individuals aged 40 years, the causes of SCD span a number of diverse diseases often with an underlying genetic cause, such as the inherited cardiomyopathies and primary arrhythmogenic diseases. 5 7 Identification of the cause of SCD in the young is a critical step in determining whether there is an underlying genetic etiology, and, if so, how this impacts on the clinical and genetic evaluation of surviving Dr. Semsarian is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship. Address reprint requests and correspondence: Dr. Chris Semsarian, Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown NSW 2042 Australia. address: c.semsarian@centenary.org.au. family members who are at risk for the same disease. This is particularly important in the evaluation of SCD cases where no cause of death is identified at postmortem, socalled sudden unexpected death (SUD), which occurs in up to 30% of young SCD populations. 5 This review focuses specifically on the role of postmortem evaluation and of genetic testing in SUD cases (ie, the key role of the molecular autopsy ) in identifying the genetic causes of death as well as the utility of the genetic result in the evaluation of surviving relatives. The review highlights the critical role of a multidisciplinary approach in the care of SCD families, which often encompasses the cardiologist, the coroner, the forensic pathologist, genetic counselors, genetic testing centers, and patient support groups. Causes of SCD in the young SCD in the young, defined as those aged 40 years, is caused by a variety of disorders that can broadly be categorized into structural and arrhythmogenic causes. Structural causes of SCD include the genetic cardiomyopathies, such as hypertrophic cardiomyopathy, dilated and restrictive cardiomyopathies, arrhythmogenic right ventricular cardiomyopathy, and left ventricular noncompaction. Other structural causes of SCD in the young include myocarditis, congenital heart diseases, and coronary artery disease. Hypertrophic cardiomyopathy remains the most common structural cause of SCD in the young, including competitive /$ -see front matter 2012 Heart Rhythm Society. All rights reserved. doi: /j.hrthm

2 146 Heart Rhythm, Vol 9, No 1, January 2012 Figure 1 Causes of sudden cardiac death in the young. Among all sudden cardiac deaths in a young Australian population over a 10-year period, 31% had no abnormalities identified at postmortem (ie, sudden unexpected death [SUD]). HCM hypertrophic cardiomyopathy; LVH left ventricular hypertrophy. (Adapted from Doolan A, Langlois N, Semsarian C. Causes of sudden cardiac death in young Australians. Med J Aust 2004;180: ) athletes. 8 Importantly, in all structural causes of SCD in the young, the postmortem examination has a high probability of identifying the cause of death. In contrast, arrhythmogenic causes of SCD in the young are more difficult to identify as a cause of death at postmortem. These primary arrhythmogenic disorders include familial long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), Brugada syndrome (BrS), and short QT syndrome. 9 Because these disorders rarely cause any structural change to the heart, the postmortem often is negative, that is, no cause of death is identified at postmortem, including normal histopathology and normal toxicology analysis. Such cases in which no abnormalities are found at postmortem have been classified as being of unknown etiology or unascertained, resulting in an underestimation of the incidence of arrhythmogenic causes of SCD. In 2004, a review of SCD cases in an Australian population over a 10-year period identified for the first time that up to 30% of sudden deaths in the young had a normal (negative) postmortem examination, 5 strongly suggesting an underlying arrhythmogenic cause (Figure 1). This subsequently was confirmed in a second independent young Australian population. 10 Compounding this underestimate of these SUD cases, primary arrhythmogenic disorders can predispose to more overt causes of death. For example, young deaths attributed to events such as drowning and motor vehicle accidents may have been directly precipitated by a ventricular arrhythmia, as illustrated by an association between swimming and development of ventricular arrhythmias in patients with familial LQTS. 11,12 In addition to the clinical diagnostic criteria for the primary arrhythmogenic diseases that can lead to SUD, genetic studies over the last 20 years have been crucial in indentifying the specific gene abnormalities that underpin these diseases. Collectively, these primary arrhythmogenic disorders have been defined as ion channelopathies, predominantly involving K, Na, and Ca 2 channels expressed in the heart. 9 The main genes involved in the pathogenesis of familial LQTS1 3, CPVT1, and BrS are listed in Table 1. Approximately 80% of all family members with LQTS studied to date have been found to have mutations in the KCNQ1, HERG, and SCN5A genes, 13,14 whereas approximately 70% of CPVT1 cases have a mutation in the ryanodine receptor gene (RyR2), and approximately 25% of BrS cases have a mutation in the SCN5A gene (Table 1). 9,15,16 Importantly, 95% of mutations in these primary arrhythmogenic diseases are inherited as an autosomal dominant trait, meaning 50% of offspring of an affected individual will also carry the disease-causing gene mutation. This has important implications on the evaluation of families in which a sudden death has occurred. This includes families in which SUD has occurred in a 0- to 1-year-old, that is, sudden infant death syndrome deaths, with gene mutations known to cause familial LQTS identified in up to 10% of cases Role of the postmortem in SUD cases Given the importance of establishing a definitive cause of death in a young SUD case, the postmortem evaluation is a critical first step in all cases. The proper and detailed conduct of the postmortem examination itself, collection of appropriate samples for subsequent analysis (including DNA analysis), careful evaluation of the findings, and a precise and accurate final conclusion all are important aspects of the role of the postmortem. Following a clearly defined process in the postmortem process not only can define the exact cause of death in a young SUD case, but the results also have far-reaching effects in identifying at-risk relatives of the decedent, thereby providing a therapeutic window for disease prevention (Figure 2). The system of postmortem evaluation, including the role of the coroner, varies among countries, and indeed among states within a country, and these potential differences are Table 1 Major genetic causes of SUD in the young Disease Gene Encoded protein Percent of disease Percent of SUD LQTS1 KCNQ1 I Ks potassium channel -subunit LQTS2 HERG I Kr potassium channel -subunit LQTS3 SCN5A I Na sodium channel -subunit 5 10 BrS SCN5A I Na sodium channel -subunit CPVT1 RyR2 Cardiac ryanodine receptor BrS Brugada syndrome; CPVT catecholaminergic polymorphic ventricular tachycardia; LQTS long QT syndrome; SUD sudden unexpected death (age 1 40 y).

3 Semsarian and Hamilton Molecular Autopsy in Sudden Cardiac Death 147 Figure 2 Flowchart for the evaluation of families in the setting of a sudden cardiac death in the young. Genetic testing is often performed concurrently with clinical evaluation of family members. BrS Brugada syndrome; CPVT catecholaminergic polymorphic ventricular tachycardia; LQTS long QT syndrome; SUD sudden unexpected death. highlighted here. Although coroners were mentioned during the reign of Alfred the Great (AD ), the coroner as known today dates from September 1194 (during the reign of Richard the Lionheart), when the Articles of Eyre proscribed the periodic visitation of the King s Judges to keep the pleas of the Crown (Latin: custos placitorum coronas) from which the word coroner is derived. The first coroners were knights and men of substance. They were elected by a select few voters and were charged with a wide variety of legal responsibilities, collecting significant taxes or fines as part of their duties. The one function that has persisted to date is the investigation of death! Within the United States, a medical examiner system usually either replaces or complements the coroner system; the medical examiner usually is a physician and often a forensic pathologist. Canada also has a mixed system of coroners and medical examiners, depending on the province. Coroners in some jurisdictions such as England and Wales must have a degree in a medical or legal field. In Canada, the United Kingdom, Australia, and other countries, coroners often make recommendations for safety practices, such as advising relatives to undergo clinical screening when there is a suspicion of an inherited heart disease, which may prevent deaths. 21,22 Several jurisdictions now recognize the potential value of investigation when a SCD occurs in young individuals and have developed guidelines for the postmortem examination and management of such cases. These guidelines ensure standardization of autopsy practice, appropriate ancillary testing, and retention of adequate material for genetic testing. These guidelines are initiated for a sudden witnessed collapse in a young person or in other suspicious circumstances (eg, when a drowning occurs in an adequate swimmer without explanation). Key aspects of the postmortem evaluation are summarized in Table 2 and are modified from the Best Practice Guidelines of the Royal Australasian College of Pathologists implemented in The coroner should obtain as much information as possible regarding the decedent, including the following: All available medical records, including cardiac investigations and especially ECGs A detailed family history of any sudden unexplained or cardiac death in first- or second-degree relatives A complete history of the events surrounding the death An assessment of the circumstances and scene A warrant for a forensic autopsy External examination (along with history) should exclude the possibility of commotio cordis. During internal examination, special attention should be paid to the cardiac examination to exclude hemopericardium, pulmonary artery thromboemboli, aortic dissection, valvular thrombi, stenosis or regurgitation, atherosclerotic stenosis, coronary dissection (by sectioning through main coronary arteries), abnormal insertions, takeoff or course of coronary arteries, occlusive atherosclerotic plaque at the level of coronary ostia, and thinning or fat infiltration of the right ventricle (through gross inspection and transillumination). Gross cross-sections through the ventricles should be performed to assess for myocardial infarcts, scars, hypertrophy, and mural thrombi, and the weight of the heart following removal of blood and postmortem clot should be obtained. Samples of major organs and tissues should be collected and retained in formalin, and sections of the heart should include the right ventricle, including the anterior wall and the outflow tract, so that arrhythmogenic right ventricular dyspla- Table 2 Key features of a postmortem evaluation in SCD cases Performed in all cases of sudden unexpected death in the young (0 40 y) Detailed premorbid clinical history and family history obtained Skilled macroscopic and microscopic examination of all organs, particularly of the heart and brain Adequate histologic material for review or referral, if necessary, obtained Collection of 5 10 ml of whole blood and frozen sections of highly cellular tissues (eg, liver or spleen) for future DNA extraction and analysis Liaison with a multidisciplinary specialized cardiac genetics service SCD sudden cardiac death.

4 148 Heart Rhythm, Vol 9, No 1, January 2012 sia can be excluded. The interventricular septum and the anterior and posterior left ventricles should have sections performed to rule out myocarditis, with a minimum of 10 blocks examined histologically. One of the tissue blocks should include the AV node. Consideration should be given to obtaining a cardiac pathology consultation. Nonspecific findings such as lung edema need to be considered in the context of the circumstances of death (eg, apparent drowning in a good swimmer), and caution needs to be taken in the interpretation of findings such as myocarditis, which frequently can be overdiagnosed. In addition to histology, vitreous fluid should be obtained and submitted for biochemical analysis. Comprehensive toxicologic testing should be performed. Sample collections should include blood from the heart and the femoral vessels, stomach contents, urine, and liver. In all SCD cases in the young, a5to10-ml blood sample should be collected for subsequent DNA extraction and analysis. In addition, frozen sections of liver or spleen, which are highly cellular and therefore rich in DNA, should be collected and stored. This can be arranged within the regional coroner s laboratory or through a prior arrangement with a regional clinical genetics laboratory. Paraffinembedded tissues are not suitable for DNA studies because the quality of the DNA is significantly diminished. 23,24 If no cause of death is evident at the time of postmortem examination, consideration should be given, after discussion with next of kin, for further specialized examination of key organs, including the heart and brain. In cases where a genetic cause of SCD is either likely or found at autopsy, the coroner or chief forensic pathologist should recommend clinical follow-up of family relatives (Figure 2). The next of kin should be advised that tissues have been retained and can be made available on authorized request to the appropriate genetic testing facility. Coroners and medical examiners should establish regional referral patterns for genetic assessment, preferably through multidisciplinary clinics experienced in cardiac genetics of highrisk conditions. 23,25 Genetic testing in SUD cases: The molecular autopsy In SUD cases where no cause of death is identified after a complete and thorough postmortem examination, genetic testing of the decedent s blood sample may identify an underlying cause. 26 Figure 3 summarizes the current pickup rate of genetic testing in SUD cases. Specifically, screening of the four genes KCNQ1 (LQTS1), HERG (LQTS2), SCN5A (LQTS3 and BrS), and RyR2 (CPVT1) results in an identification of the cause of SUD in up to 35% of cases. 9,26 The remaining 65% remain unexplained by current genetic testing approaches and suggests that other, as yet unknown genes may be responsible. Recommendations for the role of postmortem genetic testing in SUD cases have been formulated recently by the joint Heart Rhythm Society European Heart Rhythm Association Expert Consensus Statement. 27 A key recommendation is that In the setting of autopsy negative SUDS, comprehensive or targeted (KCNQ1, KCNH2, Figure 3 Current pickup rate of causes of sudden unexpected death (SUD) by genetic testing. Ion channelopathies account for up to 35% of all SUD cases when the 4 main ion channel genes are tested: KCNQ1 (LQTS1), HERG (LQTS2), SCN5A (LQTS3 and BrS), and RyR2 (CPVT1). SCN5A, RYR2) ion channel genetic testing may be considered in an attempt to establish probable cause and manner of death and to facilitate the identification of potentially at-risk relatives and is recommended if circumstantial evidence points towards a clinical diagnosis of LQTS or CPVT specifically (such as emotional stress, acoustic trigger, drowning as the trigger of death). Clearly the selection of genes to be tested may be guided by factors related to the clinical circumstances of the death, the outcomes of family screening, and the costs involved. Identification of an underlying cause of death in a previously defined SUD has at least two major clinical implications. First, the identification of a cause of death has a major influence in families in coming to terms as to why their child or spouse died suddenly. The diagnosis brings some level of closure in this respect. Second, the identification of a genetic cause of SUD provides the family with a diagnostic test for screening other at-risk family members, in conjunction with clinical screening approaches. For these reasons, it is important for the coroner to ensure postmortem blood samples and key heart pathology sections are kept for a minimum period of time. This may range from a minimum of 1 year to 5 years (as is the case in Canada and Australia) and is important because some families may present late after a SCD. Impact of a genetic diagnosis from the molecular autopsy on management of the family The identification of a genetic diagnosis in the decedent has major implications for surviving family relatives. The HRS EHRA Consensus statement recommends the following: Mutation-specific genetic testing is recommended for family members and other appropriate relatives following the identification of a SUDS-causative mutation in the decedent. 27 In all situations where an inherited heart disease is suspected in the setting of a sudden death, appropriate clinical screening is indicated. The cardiac investigation of surviving family members should include a thorough clin-

5 Semsarian and Hamilton Molecular Autopsy in Sudden Cardiac Death 149 Figure 4 Multidisciplinary approach to care of families where a sudden unexpected death (SUD) has occurred in the young. This involves an integrated approach involving key health and allied health professionals, as well as patient support groups. (Adapted from Ingles J, Semsarian C. Sudden cardiac death in the young: a clinical genetic approach. Intern Med J 2007;37:32 37.) ical history, physical examination, 12-lead resting ECG, M-mode and two-dimensional echocardiography, and, in most instances, an exercise ECG stress test. Additional tests may be required for specific cardiac genetic diseases (eg, flecainide challenge in BrS and cardiac magnetic resonance imaging in arrhythmogenic right ventricular cardiomyopathy). This approach to clinical screening alone (without genetic testing) of at-risk relatives may identify the underling cardiac disease in the family in up to 40%. 28,29 Clinical screening usually is undertaken early in families and often is completed prior to genetic studies in the decedent. Establishing a clinical diagnosis in the surviving family is important at many levels, including in assisting the subsequent selection and interpretation of genetic findings in the decedent. The clear goal of both clinical and genetic screening of family members is to identify those who may have the same disease or who may be phenotypically normal but carry the same pathogenic mutation as the decedent. Early identification of these at-risk individuals provides opportunities to initiate therapies aimed at preventing the complications of the particular disease. For example, gene carriers for LQTS may require modification of lifestyle activities, avoidance of QT-prolonging medications, initiation of beta-blocker therapy, and, in some, consideration of implantable cardioverter defibrillator therapy. These strategies all aim to reduce the incidence of sudden death. 30 The management of these families with SUD is therefore complex. There are many issues in such families, at different levels. They include direct clinical evaluations and management, coordination of services including forensics and genetic testing, grief and genetic counseling, patient education and support, and awareness of psychological and social issues. The family experiences a range of emotions, from grief following the death of a loved one, to fear that it will happen again in the family, to a feeling of hopelessness not knowing why their loved one died, to anger over why it happened to them. These normal psychological responses need to be managed sensitively and appropriately. Even in families where genetic testing in the decedent does not identify a specific genetic cause, the family s interaction with the cardiologist-led clinic has many benefits in terms of education and counseling, aspects that would not be seen in a purely laboratory-based approach. Therefore, the ideal model that encompasses all these facets of care is the specialized multidisciplinary cardiac genetic clinic, where expertise in all these areas can be provided to families where sudden death in the young has occurred (Figure 4). 25 This type of multidisciplinary model of care has been shown to improve the care of patients with genetic heart diseases because they are generally less worried and have reduced levels of anxiety. 31 This multidisciplinary model approach is particularly important when genetic testing is involved because the testing itself may raise ethical issues. Families with SCD occasionally raise ethical issues common to clinical genetics. The legal next of kin may not be invested in the welfare of all biological family members at risk. Whereas physicians typically are obligated to the privacy and interests of their specific patient and are focused on the risk of surviving family members, the coroner s duty is to the public. Specifically, the coroner has a primary responsibility to ensure that the mode of all deaths is determined to the greatest extent possible, with a secondary purpose of protecting surviving family members as members of the public. Nevertheless, it should be recognized that a familial cause of SCD still is identified in only a minority of cases even when fully evaluated. Thus, given the uncertainty around the investigation of SCD, it is rare that a specific duty to warn can be invoked or justified on an immediate and definite impending risk. To our knowledge, there is no legislation in the Australia, United States, or Europe that allows the treating doctor to directly contact other family members as part of a duty to warn, and this is rarely the case for genetic findings from molecular autopsy. Most next of kin recognize the value of investigation of families when a young SCD has occurred and willingly notify biological relatives, who most often are in-laws and children. However, the willingness of health insurers to pay for genetic testing of decedents varies among jurisdictions. In Ontario, Canada, the common provincial insurance plan currently is willing to fund genetic testing on decedents despite the usual termination of health coverage upon the death of an individual, but it is not obligated by law or regulation. This issue is even more complex in jurisdictions with multiple health payers, such as in the U.S. system. The issue remains as to who pays when it comes to genetic testing in the postmortem setting but hopefully will become more consistent as the benefits and cost-effectiveness of the molecular autopsy approach are realized. Next steps in postmortem genetic testing The current genetic testing strategy in the setting of molecular autopsy is a targeted gene testing approach, which most commonly involves the 4 main genes responsible for LQTS1 3, CPVT1, and BrS (Table 1). Although this has been a productive approach to date, the development of

6 150 Heart Rhythm, Vol 9, No 1, January 2012 next-generation sequencing technologies allowing rapid analysis of large panels of genes in a very short period of time means that within the next 5 years, more expansive genetic testing in the setting of SCD in the young will be feasible. This is likely to range from targeted cardio-gene chips, which may test for genes responsible for cardiac disease and sudden death, to whole genome or exome sequencing approaches, which will provide information on all (approximately) 24,000 human genes. 32,33 Clearly the excitement of these genetic advances is a two-edged sword: higher rates of identification of genetic causes of SCD with benefits to families versus the flood of genetic information, much of which will be uninterpretable or lead to a higher identification of variants of uncertain significance. 34 Because of this, even with genome-wide approaches, clinical and genetic knowledge will be essential in targeting the specific genes to focus on in the setting of a young SCD. Nevertheless, the future is exciting, and any advances that help to identify the causes of SCD, and subsequently translate to early therapy and prevention in at-risk individuals, will be of great benefit in reducing the incidence of SCD in the young. Conclusion SCD is a tragic and devastating complication of a number of cardiovascular diseases. In up to one-third of young sudden deaths, no cause is identified at postmortem. In these SUD cases, the postmortem evaluation, and specifically the collection of appropriate blood and tissues for subsequent DNA analysis, plays a key role in the identification of an underlying genetic cause of death. The identification of the precise gene mutation causing disease leading to sudden death subsequently can be used in screening at-risk family members, with the ultimate aim of diagnosing disease early and therefore providing a platform for the initiation of therapeutic and prevention strategies. 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