The Secrets of Molecular Autopsy Reshape Sudden Unexpected Death Investigations in the Genomic Era

Abstract

Sudden unexpected death is a tragic event that strikes without warning, leaving families and communities in shock. Uncovering the underlying causes of such deaths has long been challenging for forensic medical professionals. However, with the advancements in genomics and molecular biology, the molecular autopsy can unravel the hidden triggers behind these perplexing cases of sudden unexpected death. The molecular autopsy uses genetic analysis to inquire about sudden death. It is valuable, especially in a negative autopsy or inconclusive autopsy findings, i.e., a known hereditary illness feature frequently brings on sudden unexplained deaths (SUDs). In SUD situations, post-mortem molecular testing assists with forensic investigations and provides specific genetic assessment for the sufferer's nearest biological relatives.[1] Every individual's genomic blueprint is the foundation for developing, functioning and regulating various biological processes. Slight variations or mutations within these genes can lead to significant alterations in the body's function, potentially culminating in fatal consequences. A molecular autopsy involves a comprehensive analysis of an individual's deoxyribonucleic acid (DNA) to identify potential genetic anomalies that might have played a role in their sudden unexpected death. This method relies on advanced genomic sequencing technologies that allow scientists to scrutinise the entire genome or specific gene regions for irregularities. Distinguishing between inherited mutations and sporadic mutations is crucial during a molecular autopsy. Inherited mutations are passed down vertically from parents and could suggest a genetic predisposition toward sudden death. Conversely, sporadic mutations arise spontaneously and may result from diverse environmental factors. SAMPLE COLLECTION In obtaining DNA extraction for genetic analysis, fresh-frozen tissues and blood are the preferred resources [Figure 1]. The Heart Rhythm Society (HRS)/European Heart Rhythm Association consensus document regarding genetic screening for cardiomyopathies and channelopathies vastly suggests the assortment of ‘DNA-compatible samples (5–10 mL whole blood kept in ethylenediaminetetraacetic acid tube, a blood spot card or a frozen sample from the heart, liver or spleen) to be used for future genetic testing purposes’.[2] Such specimens must be frozen or stored in cold storage for about 4 weeks at temperatures ranging from −20 C to −80 C to ensure the preservation of DNA quality.[3] The recently released consensus paper by the HRS and the Asia-Pacific HRS concerning the examination of deceased individuals with SUD and individuals experiencing unexpected stoppage of heart-beating contains similar recommended guidelines.[4] While storing blood samples for potential future reanalysis has become a standard procedure in evaluating sudden cardiac death (SCD), the absence of this option in past SCD cases poses constraints in their retrospective cases for re-evaluation. Contrarily, formalin-fixed and paraffin-embedded tissue specimens, usually employed for histological investigation, remain easily accessible even in earlier SCD cases. These samples could potentially serve as an appropriate replacement. However, formalin fixation alters DNA by causing crosslinking and fragment disintegration.[5]Figure 1: Schematic diagram showing the molecular autopsy process. This figure has been drawn with the premium version of BioRender (https://biorender.com/Accessed on 2023 Aug 13) with the license number FG25Q5YHX7. Image credit: Susmita SinhaCARDIOVASCULAR DISEASES An SCD of an individual resulting from an unrevealed heart condition occurs within an hour of symptoms in a seemingly healthy person or, if unwitnessed, in a person known to have been in good health up to 24 h before the incidence.[6] SCD contributes from 15 to 20% of all deaths in the community, with an annual incidence of 40–100 cases/100,000 person-years.[7] One critical aspect of molecular autopsies in SCD revolves around identifying channelopathies – genetic disorders that impact ion channels in cell membranes. Ion channels are essential for maintaining the flow of charged particles, like ions, in and out of cells. Disruptions in these channels can lead to abnormal heart rhythms (arrhythmias), contributing to SCD. Conventional forensic procedures might overlook abnormalities in the conduction system of the heart and microstructural myocardiopathy despite the potential for lethal arrhythmias.[8] In addition, constantly monitoring the histopathology of the conduction system through standard procedures may help identify a variety of inherited as well as acquired abnormalities (such as extra pathways, dispersion, conduction fibre hypoplasia, inflammatory infiltration, degeneration and fibro-fatty substitution) that may be a factor in SUD, especially in infants.[8] Again, molecular autopsies also focus on cardiomyopathies – conditions that affect the heart muscle's structure and function. Genetic mutations affecting the genes accountable for maintaining the integrity of the heart muscle can lead to hypertrophic, dilated or arrhythmogenic cardiomyopathies, all of which can increase the likelihood of sudden death. As a fatal consequence of cardiovascular diseases, including dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (ACM) or channelopathies, SCD frequently develops in young people. In addition, channelopathies include conditions that affect g ion channels, including Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia, short QT syndrome and long QT syndrome. In contrast, among the elderly, the primary contributor to SCD is coronary artery disease which signifies the primary factor behind SCD, trailed by cardiomyopathies, valvular diseases and myocarditis.[9] HCM is identified in the inexplicable thickening of the left ventricle, irregular arrangement of heart muscle cells and the presence of fibrous tissue. HCM is because genetic mutations affect the genes responsible for sarcomere proteins (such as MYH7 and MYBPC3) genetically transmitted through an autosomal dominant inheritance design, showing inconsistent expressivity and partial occurrence.[210] Characteristics commonly seen in DCM encompass enlargement of the left ventricle and its replacement with fibrous tissue, resulting in systolic dysfunction, anomalies in the conduction system and heightened vulnerability to severe irregular heart rhythms that could be fatal. Characteristics commonly found in DCM comprise enlargement of the left ventricle, replacement of normal tissue with fibrotic tissue, resulting in irregularities in the conduction system, and an increased vulnerability to severe and potentially fatal arrhythmias. A genetic substrate can be detected in approximately one-third of instances, primarily involving mutations that impact cytoskeletal proteins. It is important to note that alterations in the genes DES and LMNA are uniquely associated with an arrhythmogenic appearance.[210] The left or right ventricle, or occasionally both, may have fibro-fatty tissues instead of cardiac muscle tissues, which distinguishes ACM. This condition has variable expression and an autosomal dominant inheritance template with inadequate infiltration. Genetic defects typically affect heart desmosomes, with PKP2 and DSP being the most frequently mentioned possibilities.[210] EPILEPSY An abrupt and unforeseen non-drowning and non-traumatic demise characterises Sudden Unexpected Death in Epilepsy (SUDEP). It can happen with or without seizure symptoms in an epileptic person who is otherwise healthy. However, analysis of the post-mortem does not indicate the cause of death.[1112] Individuals diagnosed with epilepsy face an approximately 24-fold more significant likelihood of experiencing SUD in comparison to the overall populace.[13] Furthermore, subjects afflicted with persistent and refractory epilepsy have been demonstrated to be more susceptible to SUDEP.[14] In addition, post-mortem examinations of the molecular autopsy following the death of individuals due to SUDEP revealed variations in genetic coding for subunits of Na+ and K+ ion channels. Furthermore, a potential association with SUDEP has been suggested for 18 genetic factors and four distinct repetitions based on documented results.[15] MULTIDISCIPLINARY COLLABORATION One of the most effective approaches and economically efficient methods in molecular autopsy is next-generation sequencing, specifically whole-exome sequencing (WES). WES can rapidly analyse the sequence of whole human genomic code and find unusual and previously undetected mutations. Specific research involving 25 people who died suddenly and unexpectedly revealed that 18 of these cases harboured mutations that could potentially lead to such unforeseen SUD.[16] Interpreting the findings of a molecular autopsy requires a multidisciplinary approach. Geneticists, forensic pathologists, cardiologists and bioinformaticians collaborate to combine genetic data with clinical information, autopsy findings and family histories. This holistic perspective helps create a comprehensive picture of the factors contributing to sudden unexpected deaths. GENETIC COUNSELLING The insights gained from molecular autopsies have the potential to revolutionise the understanding of sudden unexpected deaths. By identifying specific genetic mutations associated with the deceased, forensic pathologists can organise a family conference focusing on genetic counselling for at-risk families. ETHICAL CONSIDERATIONS AND PRIVACY CONCERNS While molecular autopsies promise to uncover hidden causes of sudden unexpected death, they also raise ethical questions. Balancing the benefits of genetic information with individuals' privacy concerns and potential stigmatisation is a challenge that researchers and policymakers must address. Nonetheless, a molecular autopsy might yield unfavourable or uncertain outcomes even using the advanced application for genetic sequencing techniques. Indeed, not all instances of SUD can be attributed to genetically pre-determined factors, and it is frequent to come across genetic variations with unknown significance. Establishing a definite connection between the gene variant found and the characteristics of the deceased individual, along with analysing how the variant is passed down within the family, could hold significance in confirming a clear relationship between the genetic make-up and the observed traits [Figure 2].[1718]Figure 2: Diagram showing an integrated approach to investigating sudden unexplained death. This figure has been drawn with the premium version of BioRender (https://biorender.com/Accessed on 2023 Aug 14 2023) with the license number XN25Q8O9JV. Image credit: Susmita SinhaCONCLUSIONS A molecular autopsy is a crucial tool in forensic investigation, intending to achieve a genetic diagnosis in cases where the conventional autopsy fails to provide definitive conclusions. The molecular autopsy has emerged as a powerful tool in deciphering the genetic enigmas behind sudden unexpected deaths. By exploring the genomic terrain, scientists can uncover the molecular culprits that may have triggered these tragic events. As technology advances and our knowledge deepens, we stand on the brink of a new era in forensic investigation, offering hope for understanding and preventing sudden unexpected deaths in the future, more importantly, with the final goal of directing sequential genetic screening of the deceased's family members. Consent for publication The author reviewed and approved the final version and has agreed to be accountable for all aspects of the work, including any accuracy or integrity issues. Disclosure The author declares that they do not have any financial involvement or affiliations with any organisation, association or entity directly or indirectly with the subject matter or materials presented in this article. This includes honoraria, expert testimony, employment, ownership of stocks or options, patents or grants received or pending royalties. Data availability Information is taken from freely available sources for this editorial. Authorship contribution All authors contributed significantly to the work, whether in the conception, design, utilisation, collection, analysis and interpretation of data or all these areas. They also participated in the article's drafting, revision or critical review, gave their final approval for the version that would be published, decided on the journal to which the article would be submitted and made the responsible decision to be held accountable for all aspects of the work.