Molecular Autopsy in Young Individuals With Sudden Cardiac Death

Background: Sudden cardiac death (SCD) is defined as an unexpected, non-traumatic death from cardiovascular causes occurring within 1 hour of symptom onset in an apparently healthy individual or, if unwitnessed, within 24 hours of the individual last being seen alive and asymptomatic. In individuals younger than 35 years, inherited causes predominate, including primary arrhythmia syndromes and inherited cardiomyopathies. The concept of molecular autopsy, the post-mortem genetic analysis of victims of unexplained sudden death following conventional autopsy, was introduced in 2001. Data on molecular autopsy have been reported mainly from North American, European and Australian populations, and no data are available for the admixed Brazilian population. Study setting: This single-center prospective study involves two units of the University of São Paulo: the Death Verification Service of the City of São Paulo (SVOC-USP), where index cases are identified and samples are collected at autopsy, and the Heart Institute (InCor) of Hospital das Clínicas, University of São Paulo Medical School, where molecular and pathological analyses and cascade screening of relatives are performed. Study workflow: At SVOC-USP, eligible cases are identified, and informed consent is obtained digitally from the legally responsible family member through the Brazilian government platform (GOV.BR). A verbal autopsy questionnaire is applied to the family, the autopsy report and death certificate are reviewed, and biological samples are collected. Whole blood (EDTA tubes) or tissue samples are sent to the Laboratory of Genetics and Molecular Cardiology (LGCM) at InCor, and the heart is sent to the InCor Pathology Laboratory. Molecular analysis: Genomic DNA is extracted with the QIAamp DNA Blood Mini Kit and assessed for integrity (agarose gel electrophoresis), purity and concentration (NanoDrop). Whole exome sequencing is performed on the Illumina NextSeq platform, with an expected mean coverage of 100x over 95% of exonic regions. Reads are aligned to the reference genome, and variants are called with GATK HaplotypeCaller. Variants are annotated and prioritized in Franklin by Genoox, retaining variants with a minor allele frequency below 1% in gnomAD, ExAC, Bravo, Kaviar and ABraOM (a Brazilian genomic database), including intronic variants that may affect splicing. Copy number variants are also analyzed. Variants are classified according to the American College of Medical Genetics and Genomics (ACMG) criteria, and pathogenic, likely pathogenic and uncertain-significance variants are reported. Variants of uncertain significance are periodically reassessed. Secondary findings are reported only when included in the ACMG list of recommended secondary findings. Sanger sequencing is used to confirm variants with low coverage or quality and for targeted testing of relatives. Pathological analysis: The heart is weighed after washing the cavities, and a transmural ventricular myocardial sample is stored in liquid nitrogen. After formalin fixation, the heart is examined macroscopically for myocardial fibrosis, fatty infiltration, hypertrabeculation, wall thickness disproportion, valve and septal integrity, and coronary artery origin, course and patency. Five myocardial samples and the mitral and aortic valves are sampled for histology (hematoxylin-eosin), with C4d immunohistochemistry when acute ischemic injury is suspected. Cascade screening: When a pathogenic or likely pathogenic variant is identified in the index case, first-degree relatives are invited to participate at InCor, where they provide consent and a blood sample for targeted Sanger sequencing. Carriers are referred for clinical cardiovascular screening. Epidemiological analysis: Data on sudden death in individuals aged 5 to 50 years are obtained from the Brazilian Ministry of Health database (DATASUS). Data management and analysis: Data are collected and stored in REDCap, with participants identified only by a coded center and sequential number. Results are summarized using descriptive statistics.