TYPE Review PUBLISHED 10 February 2023 DOI 10.3389/fmed.2023.1118585 Molecular autopsy: Twenty years of OPEN ACCESS post-mortem diagnosis in sudden EDITED BY Muneeb A. Faiq, Langone Medical Center, New York University, cardiac death United States REVIEWED BY Estefanía Martínez-Barrios1,2,3 , Simone Grassi4 , María Brión5,6,7,8 , Laura Arbour, The University of British Columbia, Canada Rocío Toro9 , Sergi Cesar1,2,3 , José Cruzalegui1,2,3 , Mònica Coll10,11 , Hiroki Takanari, Tokushima University Hospital, Japan Mireia Alcalde10,11 , Ramon Brugada8,10,11,12 , Andrea Greco3,13 , *CORRESPONDENCE María Luisa Ortega-Sánchez14,15 , Eneko Barberia14,16 , Georgia Sarquella-Brugada georgia@brugada.org Antonio Oliva17 , Georgia Sarquella-Brugada1,2,3,10*† and Oscar Campuzano Oscar Campuzano8,10,11*† oscar@brugada.org 1 † These Pediatric Arrhythmias, Inherited Cardiac Diseases and Sudden Death Unit, Cardiology Department, Sant authors share senior authorship Joan de Déu Hospital de Barcelona, Barcelona, Spain, 2 European Reference Network for Rare, Low SPECIALTY SECTION Prevalence and Complex Diseases of the Heart, Amsterdam, Netherlands, 3 Arrítmies Pediàtriques, This article was submitted to Cardiologia Genètica i Mort Sobtada, Malalties Cardiovasculars en el Desenvolupament, Institut de Recerca Translational Medicine, Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain, 4 Forensic Medical Sciences, Department of a section of the journal Health Science, University of Florence, Florence, Italy, 5 Family Heart Disease Unit, Cardiology Service, Frontiers in Medicine Santiago de Compostela University Hospital, Santiago de Compostela, Spain, 6 Cardiovascular Genetics, Santiago de Compostela Health Research Institute, Santiago de Compostela, Spain, 7 Genomic Medicine RECEIVED 07 December 2022 Group, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 8 Centro de Investigación ACCEPTED 30 January 2023 Biomédica en Red en Enfermedades Cardiovasculares, Madrid, Spain, 9 Medicine Department, School PUBLISHED 10 February 2023 of Medicine, University of Cádiz, Cádiz, Spain, 10 Medical Science Department, School of Medicine, University CITATION of Girona, Girona, Spain, 11 Cardiovascular Genetics Center, Institut d’Investigacions Biomèdiques de Girona Martínez-Barrios E, Grassi S, Brión M, Toro R, (IDIBGI), University of Girona, Girona, Spain, 12 Cardiology Department, Hospital Josep Trueta, Girona, Spain, 13 Cesar S, Cruzalegui J, Coll M, Alcalde M, Department of Medical and Surgical Sciences of the Mother, Children and Adults, University of Modena Brugada R, Greco A, Ortega-Sánchez ML, and Reggio Emilia, Modena, Italy, 14 Forensic Pathology Department, Institut de Medicina Legal i Ciències Barberia E, Oliva A, Sarquella-Brugada G and Forenses de Catalunya (IMLCFC), Barcelona, Spain, 15 School of Medicine, Universitat Autònoma Campuzano O (2023) Molecular autopsy: de Barcelona, Cerdanyola del Vallés, Spain, 16 School of Medicine and Health Sciences, Universitat Rovira i Twenty years of post-mortem diagnosis Virgili, Reus, Spain, 17 Section of Legal Medicine, Department of Health Surveillance and Bioethics, in sudden cardiac death. Fondazione Policlinico A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy Front. Med. 10:1118585. doi: 10.3389/fmed.2023.1118585 COPYRIGHT In the forensic medicine field, molecular autopsy is the post-mortem genetic © 2023 Martínez-Barrios, Grassi, Brión, Toro, Cesar, Cruzalegui, Coll, Alcalde, Brugada, analysis performed to attempt to unravel the cause of decease in cases remaining Greco, Ortega-Sánchez, Barberia, Oliva, unexplained after a comprehensive forensic autopsy. This negative autopsy, classified Sarquella-Brugada and Campuzano. This is an open-access article distributed under the terms as negative or non-conclusive, usually occurs in young population. In these cases, in of the Creative Commons Attribution License which the cause of death is unascertained after a thorough autopsy, an underlying (CC BY). The use, distribution or reproduction in inherited arrhythmogenic syndrome is the main suspected cause of death. Next- other forums is permitted, provided the original author(s) and the copyright owner(s) are generation sequencing allows a rapid and cost-effectives genetic analysis, identifying credited and that the original publication in this a rare variant classified as potentially pathogenic in up to 25% of sudden death journal is cited, in accordance with accepted academic practice. No use, distribution or cases in young population. The first symptom of an inherited arrhythmogenic reproduction is permitted which does not disease may be a malignant arrhythmia, and even sudden death. Early identification comply with these terms. of a pathogenic genetic alteration associated with an inherited arrhythmogenic syndrome may help to adopt preventive personalized measures to reduce risk of malignant arrhythmias and sudden death in the victim’s relatives, at risk despite being asymptomatic. The current main challenge is a proper genetic interpretation of variants identified and useful clinical translation. The implications of this personalized translational medicine are multifaceted, requiring the dedication of a specialized team, including forensic scientists, pathologists, cardiologists, pediatric cardiologists, and geneticists. KEYWORDS molecular autopsy, genetics, sudden cardiac death, inherited arrhythmogenic syndromes, forensic Frontiers in Medicine 01 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 1. Introduction ventricular tachycardia (CPVT) (19, 20). Both groups predispose to disruption of electrical activity, leading to ventricular fibrillation, Twenty years ago, molecular autopsy (post-mortem genetic syncope, and SCD. analysis or post-mortem molecular analysis) was proposed by the first Incomplete penetrance, variable expressivity, and genetic overlap time (1). It refers to a method used in forensic medicine, focused on between diseases are hallmarks of IAS. These facts hinder genetic the application of genetic diagnostic in post-mortem samples without diagnosis in a family as well as risk stratification, especially in a conclusive diagnosis, therefore classified as sudden unexplained asymptomatic carriers of a genetic alteration (21). Incomplete death (SUD) (2). These post-mortem molecular studies arise as a penetrance concerns carriers of a pathogenic genetic variant who complement to traditional autopsies, with the potential to detect do not express the phenotype. Variable expressivity refers to carriers genetic alterations that may be responsible for to the pathology of the same pathogenic variants expressing the diseases in different that caused the SUD. A complete forensic autopsy of a deceased degrees of severity, as well as the different age of onset, evolution, person is no-conclusive (called negative autopsy) in nearly 5% of all and outcome (22). In IAS, overlap between phenotypes concerning autopsies performed (3). In SUD cases, an inherited arrhythmogenic one single gene may also occur, known as pleiotropy. Therefore, one syndrome (IAS) is highly suspected as the most plausible cause gene is not related to only one IAS (23). In addition, SCD has also of death (4), classified as sudden death of cardiac origin-sudden been associated with oligogenic models of cardiac disease, in which cardiac death (SCD). potentially pathogenic rare variants may contribute synergistically Currently, nearly 30% of SCD cases in young population remains to the risk of sudden death (24). Consistent with this hypothesis, without a conclusive cause of death after a comprehensive autopsy an overrepresentation of rare variants in cardiac genes has been examination (5, 6). Molecular autopsy has become a fundamental identified in cohorts of young decedents compared to control tool in the current forensic area when an IAS is suspected. Since population (25, 26). All these phenotypical phenomena are likely most pathologies are of genetic origin, family members can be carriers due to a combination of genetic, environmental, and lifestyle factors. of pathogenic genetic alterations, so there is a risk that they will Hence, in addition to pathogenic variants, usually rare in IAS, also suffer from the same malignant arrhythmogenic entity. Taking common variants (called second hits), could be modulating factors of all data into account, unraveling the genetic alteration is crucial for the causative rare variant, leading to a more or less severe phenotype. diagnosis, helping to clarify the most plausible cause of unexpected Variants in non-coding regions such as intronic regions, UTR or in decease but also for the prevention of arrhythmogenic episodes in regions that code for microRNAs can modulate or play a key role in victims’ relatives. The first manifestation of any of IAS may be the IAS (27). Concerning non-genetic factors, we find the patient’s age, sudden death, thus early identification of genetic carriers at risk sex, ethnicity, the presence of other comorbidities and exogenous allows the adoption of preventive personalized therapies (7). Next factors such as fever or taking certain drugs (28, 29). generation sequencing (NGS) technologies allow a cost- and time- effective approach in genetic analysis. Molecular autopsy using NGS reveals a definite pathogenic genetic alteration, which explains the 3. Channelopathies SCD in near 20% of cases, especially in young population (6, 8–14). However, most SCD cases remain with a negative or inconclusive The identification of pure arrhythmogenic disease (or cardiac genetic diagnosis, mainly due to new genes not currently known to be channelopathies) as the cause of death during a comprehensive the cause of IAS or rare variants identified in known genes associated forensic autopsy is difficult due to the fact that they are with IAS remain classified with an ambiguous role or also named characterized by non-structural heart alterations (30). Despite a of unknown significance (VUS). Despite this fact, current clinical detailed macroscopic and microscopic study, in around 5–10% of guidelines recommend molecular autopsy in SUD cases with a highly cases no cause of death is found, defining this event as negative suspected cause of death due to IAS (7, 15, 16). autopsy with IAS as the main suspected cause of death (3, 4, 31). At present, main entities concerning cardiac channelopathies are LQTS, SQTS, BrS, and CPVT (Figure 1). 2. Inherited arrhythmogenic syndromes 3.1. Long QT syndrome Sudden cardiac death accounts for 15–20% of all deaths worldwide, with an overall incidence of 40–100 per 100,000 person- Long QT syndrome is a congenital rare entity (1:2500) years (17). In the young population less than 35 years of age, the mainly characterized by prolongation of the QT interval on the main cause of SCD is IAS (18). These malignant arrhythmogenic electrocardiogram (ECG) (QTc > 460 ms in women and >450 ms entities can be divided in two main groups: first, cardiomyopathies in men) and usually also associated with T-wave abnormalities (7). It which are characterized by structural heart impairment and caused may lead to ventricular tachyarrhythmias-torsade de pointes (TdP)-, by genetic alterations in genes encoding structural proteins, such syncope and even SCD in the context of a structural normal heart. as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy However, a group of patients may remain asymptomatic, with SCD (DCM), and arrhythmogenic cardiomyopathy (ACM). Second, a being the first phenotypic manifestation of this arrhythmogenic group of cardiac channelopathies or purely electrical disease, disease. In clinical diagnosis, it is important to discard a longer characterized by a structurally normal heart and caused by genetic than normal QT interval induced by a pharmacological treatment alterations in genes encoding ion channels or associated proteins, or electrolyte imbalances. Lethal episodes usually occur in infants such as long QT syndrome (LQTS), short QT syndrome (SQTS), and young population, precipitated by specific triggers, including Brugada syndrome (BrS), and catecholaminergic polymorphic exercise, swimming, emotional stress, or sudden loud noises (32). Frontiers in Medicine 02 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 FIGURE 1 Genes involved in the main inherited channelopathies (IC) and their frequency in each syndrome. LQTS, long QT syndrome; CPVT, catecholaminergic polymorphic ventricular tachycardia; SQTS, short QT syndrome; BrS, Brugada syndrome. Despite this fact, SCD during sleep may also occur, but less frequently. 3.2. Brugada syndrome The current main treatment is the administration of beta-blockers (β- blockers) although do not provide full protection (16). In patients Brugada syndrome is an inherited rare entity (1:2500) with recurrent syncope while on β-blocker therapy or survivors of characterized by ST-segment elevation in the right precordial an aborted SCD, the prescription of an implantable cardioverter leads (often referred to as type-1 Brugada pattern) on the ECG defibrillator (ICD) is mandatory (33). In high-risk patients with (16). Currently, BrS is diagnosed in patients with ST-segment symptomatic LQTS, left cardiac sympathetic denervation (LCSD) elevation with type I morphology > 2 mm in > 1 lead among should be considered; it includes patients in whom β-blockers are the right precordial leads V1, V2, positioned in the 4th intercostal ineffective or not tolerated, and in patients with recurrent appropriate space (standard ECG) or the 2nd and 3rd intercostal spaces (high ICD shocks despite β-blockers administration (34). parasternal leads) (36). It is considered a disorder involving mainly In recent years, several genes have been potentially associated young male adults (about 40 years of age), and SCD typically occurs with LQTS, usually following an autosomal dominant pattern of during sleep or at rest (16). The diagnostic ECG pattern can be inheritance. A comprehensive genetic analysis including all genes baseline or intermittent, and it can be unmasked during a drug test related to LQTS, may unravel the genetic cause of the disease in using class I sodium channel-blockers or modulating factors such up to 90% of cases (35). Current guidelines recommend KCNQ1, as fever, exercise or drugs. In diagnosis, it is important to discard KCNH2, and SCN5A as the main genes responsible for 45–50, 30– “BrS phenocopies” induced by myocardial ischemia, electrolyte 35, and 5–10% of all cases, respectively; other secondary genes are disturbances, and drug intoxications (37). Patients usually remain also included with a strong association to LQTS but with a frequency asymptomatic and triggers may unmask the diagnostic ECG, which of less than 1% (7). In SUD cases of young population who die may lead to malignant polymorphic ventricular tachyarrhythmias, while exercising, swimming, and even sleeping, LQTS should be sometimes associated with conduction disease and atrial arrhythmias. considering. Molecular autopsy in combination with circumstances It is considered a disorder involving mainly young male adults (about of death may help to clarify the unexpected lethal episode. 40 years of age), and SCD typically occurs during sleep or at rest. Frontiers in Medicine 03 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 The only treatment having any proven effect on the prevention of tachycardias (typically bidirectional but also polymorphic) in young SCD is the implantation of an ICD, recommended if syncope is population with structurally normal hearts (45). CPVT is responsible present (38). However, the recommendation of ICD implantation for SUD in young population, and not rarely the first symptom in asymptomatic patients is not free from controversy, especially in of the disease, especially in infants (46). Individuals may remain children (39). asymptomatic at rest and adrenergic situations (exercise, stress, or Recent guidelines highlight only one definitive gene associated acute emotion) are the trigger for malignant arrhythmias leading with BrS: SCN5A (7). It is accountable for 20–25% of all cases. to syncope and SCD (16). The first therapeutic approach is the Other minor genes have been suggested as cause of BrS, but a administration of beta-blockers (mainly nadolol) but left cardiac comprehensive analysis of all potential genes unravel the cause of sympathetic denervation (LCSD), and/or implantation of an ICD is disease in no more than 30% of cases (40). The low genetic yield indicated if aborted SCD occurs or arrhythmias are not adequately after a comprehensive analysis suggest that other genes remain to controlled by drug therapy (47). CPVT is responsible for SUD in be discovered. However, alterations in regulatory zones or different young population, and not rarely SCD is the first symptom of the patterns of inheritance may play a role in BrS (24). A SUD case in the disease, especially in infants. young and adult-young population, especially if male, may be due to Concerning genetics, pathogenic variants mainly follow an BrS if it occurs during sleep or rest. autosomal dominant pattern of inheritance. In 60–70% of cases, these rare pathogenic alterations are situated in the RYR2 gene. Other minor genes have been related to CPVT, but in less than 5% all 3.3. Short QT syndrome together, showing most of them an autosomal recessive pattern of inheritance (7). Hence, current guidelines recommend performing Short QT syndrome is an extremely rare entity with no more a genetic analysis of only RYR2 in CPVT cases. In consequence, a than 250 cases reported since its first description in 2000 (41). negative autopsy in a young patient who died suddenly during an It is characterized by a shortened QT interval (QTc < 340 ms), adrenergic situation could be due to CPVT, despite it being less and T waves abnormalities in a structural normal heart (16). This common than other conditions causing SCD. highly lethal IAS can be also diagnosed with a QTc interval between 340 and 360 ms and the presence of a rare definite pathogenic variant, family history of SQTS, family history of SCD at <40 years old or survival after ventricular tachycardia/ventricular fibrillation 4. Cardiomyopathies (VT/VF) episode in structural normal heart. SQTS usually appears nearly 30 years of age but it may also occur in infant and young The main group of IAS responsible for SUD in young population. Actually, SQTS is being considered a main cause of population are cardiomyopathies. They are characterized by death in the first year of life (Sudden Infant Death Syndrome, SIDS) structural abnormalities leading to malignant arrhythmias. These (42). Clinical manifestations may range from asymptomatic to both heart alterations are progressive and may be identified during a atrial and ventricular arrhythmias, leading to palpitations, syncope comprehensive forensic autopsy, at macroscopic or microscopic and even SCD, sometimes the first symptom of the disease. Life- level. Structural impairment occurs first at the microscopic level threatening arrhythmias may appear at rest or during exercise with no and progress to gross heart alterations. Thus, it is not rare that apparent initiating cause. Implantation of an ICD is recommended a comprehensive autopsy may identify alterations at microscopic in high-risk patients and, sometime, the ICD may be combined level but not in anatomic examination. It is widely-accepted in with pharmacological treatment. In patients with relatively benign recent years that the malignant electrical disturbance may occur phenotype, only pharmacological measures may be used, mainly prior to evident structural defects in heart (14). In addition, several Quinidine or Hydroxyquinidine (43). In asymptomatic patients and studies have identified clinically relevant genetic mutations in genes relatives carrying a pathogenic variant, pharmacological treatment is associated with structural heart disease in cases of SUD with a effective in prolonging QT intervals, though its efficacy in preventing negative comprehensive autopsy (6). Therefore, molecular autopsy life-threatening arrhythmias remains to be proven (44). in SUD cases should include genes associated with cardiomyopathies Due to limited families worldwide, few comprehensives’ studies (Figure 2). The identification of a rare pathogenic variant in any have been published to date. Genetic advances have identified two of these genes should be interpreted with caution and as potential main genes following an autosomal dominant pattern of inheritance, cause of death in a case of cardiomyopathy in early stages of the KCNH2 and KCNQ1. Both genes are responsible for 10 and 5% of disease, without an evident structural heart defect, especially in cases, respectively. Three other minor genes have been related to young population (48). Thus, negative autopsy of infant and young SQTS despite being responsible for <1% of cases (7). Molecular population carrying alterations in genes encoding cardiomyopathies autopsy of an infant who died suddenly could be caused by this should not be ruled out without a comprehensive analysis of the index malignant entity despite being very rare. If rare variant identified, case and surviving relatives (49). genotype-phenotype segregation in relatives should be performed to identify potential genetic carriers at risk. 4.1. Hypertrophic cardiomyopathy 3.4. Catecholaminergic polymorphic Hypertrophic cardiomyopathy is the most common IAS and ventricular tachycardia the only one that is not rare (1:500). It is characterized by asymmetric hypertrophy of the left ventricle (LV) wall, confined Catecholaminergic polymorphic ventricular tachycardia is a rare to the intraventricular septum and not explained by other cardiac (1:10.000) lethal entity, mainly characterized by fast ventricular conditions. In an adult, HCM is defined by an end-diastolic wall Frontiers in Medicine 04 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 FIGURE 2 Genes involved in the main cardiomyopathies (CM) and their frequency in each syndrome. HCM, hypertrophic cardiomyopathy; ACM, arrhythmogenic cardiomyopathy; DCM, dilated cardiomyopathy. thickness of ≥15 mm in one or more LV myocardial segments, in (14, 53–55). More recently, molecular autopsy in SUD cases should the absence of any other cause of hypertrophy—as measured by any include genes associated with HCM despite no structural alteration imaging technique (50). HCM includes diastolic dysfunction, heart identified after comprehensive autopsy (56). However, the role of failure, atrial arrhythmias and malignant ventricular arrhythmias rare variants in HCM-related genes still remains controversial and to leading to syncope and SCD. It is the most frequent cause of SCD avoid diagnostic miscues in cases without overt phenotypes, as well in young people, with exercise being the main inductor of malignant as ambiguity in victims’ relatives, a careful interpretation is crucial arrhythmias (51). before clinical translation, especially if the variants remain classified Hypertrophic cardiomyopathy is also known as sarcomere as VUS (57). disease because hundreds of pathogenic variants have been documented in the genes encoding sarcomere proteins, all following an autosomal dominant inheritance (52). These alterations are 4.2. Dilated cardiomyopathy located mainly in two genes (MYBPC3 and MYH7), which code for the cardiac myosin-binding protein C and cardiac beta-myosin heavy Dilated cardiomyopathy is characterized by the presence of LV chain, respectively. Both are responsible for 60% of cases. However, or biventricular dilatation, leading to contractile dysfunction in the more than 20 genes have been associated with HCM, despite being absence of abnormal heart conditions or coronary artery disease responsible for <15% of cases all together (7). Currently, the role sufficient to cause global systolic impairment (16). The clinical course of the genetic test result in the determination of risk in SCD is variable; a large part of patients remains asymptomatic for a long remains uncertain and is, therefore, not clinically useful. An autopsy time. The worst prognosis involves very low ejection fractions or usually identifies gross cardiac alterations related to HCM or at severe diastolic dysfunction, which may lead to terminal heart failure least typical disarray in microscopic analysis. However, molecular with subsequent need for a left ventricular assist device implantation autopsy may unravel a pathogenic variant in a gene associated or even heart transplantation (58). It encompasses a broad range of with HCM but in a SUD case. It is important to remark that in genetic or acquired disorders. A careful diagnostic work-up should young population, “concealed cardiomyopathy” may occur whereby be performed to identify the underlying cause and then consider an arrhythmic phase of disease occurs prior to structural alteration an etiology-oriented approach to therapy. DCM often results from Frontiers in Medicine 05 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 myocarditis, exposure to alcohol, drugs or other toxins and metabolic death and family history should be carefully analyzed to conclude if or endocrine disturbances. In nearly 40% of cases, DCM can be arrhythmia occurred before structural alteration. classified as familial, therefore, of genetic origin. Familial DCM is due to pathogenic mutations in genes encoding cytoskeletal and sarcomeric proteins of the myocyte (7). Almost 100 genes have been reported as a potential cause of 5. Genetic analysis/Interpretation DCM but, to date, main genes are TTN (20%), LMNA (10%), The first studies focused on post-mortem genetic analysis and MYH7 (5%), following an autosomal dominant pattern of included limited genes (9–11, 31, 65), following recommendations inheritance. All other genes are responsible for no more than 10% available then (15). Up to now, despite main genes associated to all together. These genes are most often related to sarcomeric IAS remaining similar (7), NGS technology allows a broad genetic genes, z-disk/cytoskeleton, and intercalated disk, indicating partial analysis in a rapid and cost-effective way in comparison to traditional overlap with other cardiomyopathies (59). Hence, a detailed Sanger technology; however, Sanger sequencing still remains as gold- diagnostic is necessary to identify the precise underlying origin standard due to high fidelity, and is used mainly for validating and exclude other conditions with overlap phenotypes. A complex uncertain NGS findings (mainly insertion/deletion sequences), polygenic architecture with a combination of rare and common cascade segregation of variants in relatives and amplification of variants, in addition to epigenetic factors, has been proposed as regions not covered by NGS technology (mainly regions of the origin of DCM cases, although further studies are needed to genome rich in cytosine and guanine nucleotides). explain the pathophysiological mechanism involved (60, 61). As More recently, molecular autopsy should use NGS technology abovementioned in HCM cases, molecular autopsy should include due to high genetic yield at low-effective cost, limited amount of genes associated with DCM, despite no structural alteration identified DNA used and reduced time of sequencing (66, 67). Current NGS after comprehensive autopsy (7, 56). Variants in LMNA, RBM20, and approaches may include personalized panels, including main and FLNC have been reported as highly arrhythmogenic phenotypes with minor genes associated with IAS, whole exome sequencing (WES), minimal structural defects (62). In addition, if SUD occurs in a young and even whole genome sequencing (WGS). In addition, all these individual, further investigation should be performed to conclude if NGS approaches have a similar economic price, not superior to arrhythmia occurred before structural alteration. 500 euros/dollars, thus, which one should be chosen? For genetic diagnosis, only panels have been used regularly to date. In the last few years, hospitals and centers of genetic diagnosis use WES approach, 4.3. Arrhythmogenic cardiomyopathy but the final report is only performed focused on a list of concrete genes associated with the suspected or diagnosed IAS; the main Arrhythmogenic cardiomyopathy is a rare entity (1:5000) reason is the similar economic cost and potential use of genetic characterized by the progressive replacement of the myocardium by data in diagnosis, as well as for research purposes (68–77). However, fibrous or fibrofatty tissue. It mainly affects the right ventricle, but it ethical issues should be considered and patient consent should has been seen that it can also affect the LV, giving rise to forms called include all data concerning genetic analysis that will be performed, as biventricular, up to 50% of cases. On more sporadic occasions, cases well as potential clinical implications. Recently, the American College of only left involvement have been seen. Hence, three recognized of Medical Genetics (ACMG) published a statement focused on a list phenotypic variants have been reported: the dominant-right (“the of genes that they recommend reporting after a WES to facilitate classic arrhythmogenic right ventricular cardiomyopathy”—ARVC-) the identification and/or management of risks for selected genetic variant, the biventricular variant, and the dominant-left variant disorders through established interventions aimed at preventing or (also known as “arrhythmogenic left ventricular cardiomyopathy”— significantly reducing morbidity and mortality (78). Finally, WGS ALVC-). The diagnosis of ACM is made using a combination of non- approach is used mainly for research proposes, as well as other recent invasive and invasive tests to evaluate cardiac structure and rhythm, analysis of RNA sequencing (RNA-seq) or integration of multi-omics initially proposed by an international task force (Task Force Criteria, data focused on transcriptome and proteome modifications but not TFC) (63), and revised in 2010 (64). This myocardial substitution currently translated yet to clinical practice. causes progressive ventricular dysfunction, and a high burden Focused on genes associated with IAS (no more than 20 major of ventricular arrhythmias, syncope, and SCD (16). Management genes -ACTC1, DSC2, DSG2, DSP, HCN4, FLNC, KCNH2, KCNQ1, is individualized and focused on prevention through the use of LMNA, MYBPC3, MYH7, PKP2, PLN, RyR2, SCN5A, TNNI3, and antiarrhythmic medication and an ICD in most severe cases, with TTN-, and no more than 100, including minor ones) (7), the technical documented sustained VT/VF, syncope, and aborted SCD (16). approach to screen a large number of genes is not the limitation to A comprehensive genetic analysis of all genes associated with date. It is widely accepted that increasing the number of genes imply ACM identifies the cause of disease in up to 65% of cases, mainly a greater number of rare variants, remaining the most part without following an autosomal pattern of inheritance. Main genes encode for a conclusive role in IAS; however, a recent study has suggested desmosomal proteins, such as PKP2 (35%), DSP (10%), DSG2 (10%), that combined cardiomyopathy and arrhythmia genetic testing is DSC2 (5%), and other minor genes (7). However, recessive conditions able to identify a 10.9% gain in genetic diagnoses that would have such as Naxos disease (JUP) and Carvajal syndrome (DSP), have been been missed if testing had been limited to genes associated with also reported. As occurs in HCM and DCM cases, molecular autopsy a single cardiomyopathy or arrhythmia panel of genes (79, 80). If in SUD cases should include genes associated with ACM despite no clinical evaluation is included in genetic diagnosis, the diagnostic structural alteration identified after comprehensive autopsy (7, 56). yield of molecular autopsy increases to nearly 35% (81). The current In cases in which a pathogenic variant in genes associated with ACM challenge is a proper genetic interpretation of data obtained after is identified, further investigations including autopsy, situation of molecular autopsy and, most importantly, useful clinical translation. Frontiers in Medicine 06 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 A study published in 2013 showed that nearly 30% of all disease- specific protocols, so before carrying out an NGS study, the samples causing genetic variants in the literature may have been reported must follow the concrete specifications of each system. Despite incorrectly (82). In order to standardize the items for classification following guidelines, a percentage of sample cannot be analyzed of rare variants, the ACMG recommendations were published in correctly due to existence of several variables such as collection, 2015 (83). storage, and time post-mortem among others. Focused on this point, our group has suggested to reclassifying To retain at least 5–10 ml of blood and stored in Ethylene urgently all rare variants analyzed in IAS not following ACMG Diamine Tetra Acetic acid (EDTA) tubes is recommended. Collection recommendations (84). In our post-mortem cohort with suspected of blood less than 48 h post-mortem is the optimal time to avoid a IAS, more than 90% of the rare variants that were not classified progressive DNA degradation, then impeding a proper NGS analysis. according to the ACMG recommendations, modified their If no cold temperature is available for storage, tubes can be retained previous classification when applying the aforementioned ACMG at room temperature and analyzed during first 48 h after collection. recommendations (84). In addition, a time-period of 5 years has More than 2 days for DNA extraction, it is recommended store tubes been recently proposed by our group as maximum time to perform at 4◦ C (maximum 2–4 weeks) (103). Freezing at −20◦ C is an option a re-analysis of variants in IAS cohorts (85, 86). We recommend if DNA extraction will be performed after more than 2–4 weeks, in this time-period of reclassification also in variants identified after order to preserve DNA integrity (92). However, it is important to note performing molecular autopsy. Therefore, items included in the that freezing the EDTA tube should be avoided as much as possible, ACMG recommendations imply more accuracy in the classification as the freezing and thawing process damages the DNA structure. of variants, but also greater stringency. In addition, the lack of Concerning fresh/frozen tissue, it is equally useful to take 5 g of available data and controversial published results in the IAS leads heart, liver, muscle or spleen tissue, which should be quickly analyzed to attribute the role of VUS to a large part of these rare variants in order to avoid DNA degradation. If not analyzed immediately (83, 87, 88). At the same time, the constant increase and updating after extraction, it should be frozen by immersion in liquid nitrogen of data concerning population frequency of variants1 , new evidence for 1 min, and to keep this material at a freezing temperature (−20 available from case reports in the literature, segregation, and to −80◦ C) until DNA extraction (92). In this situation, defrosting functional studies, as well as the implementation of the new in silico should be also done progressively before DNA extraction to avoid prediction technologies (89), could help to discern the role of variants broken DNA sequence. previously classified as having uncertain significance in SUD cases. In routine protocols performing autopsy, FFPE tissue samples are Before clinical translation, a group of experts in each area should stored. However, this system is not routinely recommended because discuss the role of variants included in the final report due to it may DNA extractions in FFPE tissue are usually highly variable, and on alter the personalized therapeutic measures adopted in each victim’s many occasions neither the minimum quantity nor the minimum relatives, especially if a variant remains as VUS (90). HRS/EHRA quality is obtained to carry out an adequate NGS study (104). guidelines recommend clinical screening of family members in The DNA is damaged during the paraffin embedding process with cases when a VUS is identified and phenotypic features of family chemical crosslinking that ensues from formalin fixation, which can members may lead to reclassification of rare variants (7). Currently, lead to errors in the sequencing output. Despite this, interesting only pathogenic and likely pathogenic variants are usually used for studies have been carried out obtaining proper DNA from FFPE cascade testing of surviving relatives of a SUD episode (91–93). The tissue (105–107), ensuring that the “primers” used for generating reports of screening of relatives of SUD report varying yields and libraries produce amplicons that are short and, therefore, well-suited have ranged from 18 to 31% (94–100). In recent recommendations, to the fragmented DNA extracted from FFPE and thus allowing a Society of Cardiovascular Pathology state that the autopsy report is molecular autopsy in IAS with confidence (108). the appropriate record to include all findings. Results of molecular autopsy should be included on the death certificate, including cases where the pathologist suspects that a VUS may actually play a 7. Recommendations/Guidelines pathogenic role (101). In August 2008, the members of Trans-Tasman Response AGAinst sudden Death in the Young (TRAGADY), also endorsed by 6. Samples the Royal College of Pathologists of Australia and officially endorsed by the National Heart Foundation of New Zealand, proposed a A crucial point in order to perform a genetic analysis is obtaining guide to standardize the practice of autopsies in SUD in young sample in proper conditions. Currently, up to 40% of samples are people and attention to family members2 . One year after, in 2009, not collected adequately for post-mortem genetic study (102). In Michaud et al. published a multidisciplinary Swiss collaboration, large part of cases, it impedes a proper diagnosis and results may with the goal of properly informing families of these pathologies not be conclusive due to technical impediments and any postmortem and their implications for surviving family members, recommending changes to the quality of the sample collected during autopsy could molecular autopsy in SUD cases (30). have an impact on results (102). Currently, blood is the optimum The first recommendations concerning the use of genetic specimen for molecular genetic studies despite fresh/frozen tissues diagnosis for the study of channelopathies and cardiomyopathies in and formalin-fixed and paraffin-embedded (FFPE) tissue samples are clinical practice were proposed in 2011, including SUD cases (15). In also sources for DNA extraction (101). At this time, there are various 2015, Wilhelm et al. published the Swiss Society of Forensic Medicine technical platforms for genetic analysis. Each genetic platform has 2 https://www.rcpa.edu.au/getattachment/b72c2a4c-0e36-4656-8704- 1 https://gnomad.broadinstitute.org/ f2e8a7b7bc20/Guidelines-on-Autopsy-Practice.aspx Frontiers in Medicine 07 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 recommendations for SUD cases (109). The main considerations despite controversy in clinical translation of genetic results due to related to molecular autopsy were (1) all cases of SCD in those lack of data allowing a proper interpretation of variants identified under 40 years of age, (2) the collection and adequate storage of (93). In recent years, a consensus statement focused on SUD cases samples for the study, (3) communication with the family, and (4) and victims’ relatives has been published, and a molecular autopsy is a multidisciplinary approach that includes genetic counseling. Also, highly recommended in SUD cases. The evolution of the guidelines in 2015, international guidelines of the European Heart Rhythm on genetic testing and the standardization of molecular autopsy for Association, Heart Rhythm Society, Latin American Heart Rhythm the study of patients with SUD and their relatives is summarized in Society, American College of Cardiology (ACC), and American Heart Figure 3. Association (AHA), were published, recommending the performance A comprehensive forensic autopsy should include a protocol for of a comprehensive genetic study when IAS was suspected (16). One the collection and storage of tissue suitable for molecular autopsy. year after that, the Centers for Disease Control and Prevention, in Both implications of the significance of genetic testing and counseling collaboration with the US National Institutes of Health, launched should be previously discussed with families. In this process, a a massive effort to address the devastating impact of SUD on detailed family history is essential to unravel the cause of SUD. young people, including comprehensive registration of cases and If a genetic analysis is not feasible in the deceased, the clinical the introduction of a standardized autopsy protocol, as well as the assessment of first-degree relatives is recommended with subsequent collection of biological samples for DNA analysis (110). In 2018, focused genetic analysis for family members, including asymptomatic the Swiss Working Group on SCD underlined the need to update ones, in order for early identification of an IAS. Clinical follow-up and improve the general knowledge concerning the genetic risk of is recommended directed by initial findings (92). This last year, a cardiovascular pathologies, the importance of performing an autopsy current expert consensus statement on the state of genetic testing and post-mortem genetic testing in SCD victims, in addition to for IAS was published. It is recommended to retain samples of SUD developing standardized post-mortem disclosure policy at national cases in proper conditions for molecular autopsy. In SUD cases, and international levels for SCD cases and relatives (111). In 2019, clinical assessment in relatives should be performed to discard any genetic post-mortem analysis in cases of SUD were included in the IAS and, in parallel, if a potentially pathogenic genetic alteration is European recommendations of legal medicine and the forensic area, identified in a victim, a cascade of genetic analysis in relatives should FIGURE 3 Timeline of guidelines and recommendations. Evolution of guidelines and recommendations on genetic testing and the standardization of molecular autopsy for the study of patients affected with SUD and their relatives. FIGURE 4 Workflow diagram for the multidisciplinary team and the implementation of molecular autopsies in SUD cases. Once sudden death has been reported, a complete forensic autopsy is performed. In cases in which the result is negative or non-conclusive, it is recommended that a tissue sample be obtained and preserved for molecular study (with prior authorization, based on the legislation in force in each country). If the genetic study is positive or variants of uncertain significance are identified (due to lack of available information), segregation of the victim’s relatives should be carried out to clarify the role of VUS and to identify at-risk family members. Genetic counseling and appropriate clinical management should be provided to the victim’s relatives. Frontiers in Medicine 08 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 be done (7). In our opinion, if lack of available data impedes a definite 9. Conclusion classification of a rare variant and it remains as VUS, segregation should also be done in order to clarify the role, at least in the affected A negative autopsy in cases of sudden death impedes concluding family members (Figure 4). a definite cause of the decease. In these cases, an IAS is highly Finally, despite the fact that molecular autopsy is widely suspected and the molecular autopsy is a critical approach to recommended, it is not included in forensic protocols in most uncover the pathogenic genetic alteration. Unraveling the cause countries. The most recent survey conducted by the European Heart of death allows to give an answer to the family but also help Rhythm Association for the Investigation on Sudden Unexpected clinicians to figure out the early identification of the victim’s relatives Death in the Young (SUDY) in Europe reported that only 37% and the adoption of personalized preventive measures focused of SUD cases with a suspected arrhythmic cause undergo post- on reducing the risk of malignant arrhythmogenic episodes. The mortem genetic testing, revealing a high heterogeneity in adherence molecular autopsy in suspected IAS uses current NGS technologies, to current recommendations for SUDY investigation (92, 112). allowing a rapid and cost-effective analysis of several genes together. There are several reasons to avoid this implementation, such as However, an increased number of genes analyzed does not imply a economic considerations or legal restrictions involved with the higher yield in genetic diagnosis. Currently, the lack of functional sample collection, the storage time and the number of genes and clinical data in IAS impedes a proper genetic interpretation analyzed, as well as the ethical implications of genetic results and a large part of genetic alterations identified remains as obtained after a molecular autopsy (113). In addition, due to VUS after a comprehensive genetic analysis. In these cases, the the progressive number of rare variants which remain with an transition into clinical practice should be done with caution, and ambiguous role after a molecular autopsy, a large part of SUD a close multidisciplinary collaboration including forensic experts, cases remains inconclusive; therefore, it should be useful to develop pathologists, cardiologists, pediatric cardiologists, and specialized recommendations/guidelines focused on variant interpretation in geneticists is crucial. forensic medicine. Recently, Society of Cardiovascular Pathology has published a guide of recommendations to pathologists for a comprehensive and standardized cardiac examination in cases of SCD (101). Author contributions AO, GS-B, OC, EM-B, SG, MB, RT, SC, JC, MC, MA, AG, MO-S, 8. Medico-legal issues and EB: writing. EM-B, SG, MB, RT, SC, JC, MC, MA, AG, MO-S, and EB: data analysis. AO, RB, and OC: supervision. GS-B, RB, and Current guidelines for autopsy investigation of SCD cases OC: funding. All authors contributed to the article and approved the recommends a genetic analysis in post-mortem cases (2), following submitted version. standardized cardiac protocols (101). There are numerous obstacles that impede quality investigation of SCD. Despite recommended in current guidelines (7, 92, 101), performing molecular autopsy usually depends on the authorization received by the competent public Funding authorities (114). If this authorization is granted, acquiring further consent for the analysis is not usually required (110). However, in This work was supported by Obra Social “La Caixa Foundation” certain countries and under specific conditions, relatives can be still (LCF/PR/GN19/50320002) co-funded by Instituto de Salud Carlos asked for authorization to extract any tissue from deceased. As above- III (FIS PI21/00094 and PI19/01283). CIBERCV was an initiative mentioned, when an IAS is suspected, post-mortem genetic testing of the ISCIII, Spanish Ministry of Economy and Competitiveness. should be considered a public health priority, being these disorders Funders had no role in study design, data collection, data analysis, transmitted in a prevailing autosomal dominant fashion (115). First- interpretation, or writing of the report. degree relative, who are directly exposed to this risk, should be carefully counseled, albeit balancing health risks alert and patient rights empowerment should be the main concern of the counselor Conflict of interest (116). In detail, it is advisable to explain to the person who gives their consent the reason or reasons why a genetic study is going to The authors declare that the research was conducted in the be carried out, as well as what is going to be tested and the benefits absence of any commercial or financial relationships that could be for family members, and especially offspring (101, 110). construed as a potential conflict of interest. In our country, creation of multidisciplinary referral units in the post-mortem diagnosis of SCD cases and treatment of relatives with a potential IAS is widely accepted despite not definitively implemented (117). Currently, there is a great legal void in the approach to Publisher’s note prevention of SCD, both at the judicial and healthcare levels due to the variable recognition of acquired skills and to the differences in All claims expressed in this article are solely those of the the forensic organization throughout our country (118). In addition, authors and do not necessarily represent those of their affiliated despite forensic autopsies should be performed according to the organizations, or those of the publisher, the editors and the reviewers. minimum quality standards (2), lack of homogenization among the Any product that may be evaluated in this article, or claim that may different counties exists so far, impeding a proper translation into be made by its manufacturer, is not guaranteed or endorsed by the clinical practice. publisher. Frontiers in Medicine 09 frontiersin.org Martínez-Barrios et al. 10.3389/fmed.2023.1118585 References 1. Ackerman M, Tester D, Driscoll D. 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