Molecular autopsy: Twenty years of post-mortem diagnosis in sudden cardiac death.

In the forensic medicine field, molecular autopsy is the post-mortem genetic analysis performed to attempt to unravel the cause of decease in cases remaining unexplained after a comprehensive forensic autopsy. This negative autopsy, classified as negative or non-conclusive, usually occurs in young population. In these cases, in which the cause of death is unascertained after a thorough autopsy, an underlying inherited arrhythmogenic syndrome is the main suspected cause of death. Next-generation sequencing allows a rapid and cost-effectives genetic analysis, identifying a rare variant classified as potentially pathogenic in up to 25% of sudden death cases in young population. The first symptom of an inherited arrhythmogenic disease may be a malignant arrhythmia, and even sudden death. Early identification 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.

Discerning the Ambiguous Role of Missense TTN Variants in Inherited Arrhythmogenic Syndromes.

The titin gene (TTN) is associated with several diseases, including inherited arrhythmias. Most of these diagnoses are attributed to rare TTN variants encoding truncated forms, but missense variants represent a diagnostic challenge for clinical genetics. The proper interpretation of genetic data is critical for translation into the clinical setting. Notably, many TTN variants were classified before 2015, when the American College of Medical Genetics and Genomics (ACMG) published recommendations to accurately classify genetic variants. Our aim was to perform an exhaustive reanalysis of rare missense TTN variants that were classified before 2015, and that have ambiguous roles in inherited arrhythmogenic syndromes. Rare missense TTN variants classified before 2015 were updated following the ACMG recommendations and according to all the currently available data. Our cohort included 193 individuals definitively diagnosed with an inherited arrhythmogenic syndrome before 2015. Our analysis resulted in the reclassification of 36.8% of the missense variants from unknown to benign/likely benign. Of all the remaining variants, currently classified as of unknown significance, 38.3% showed a potential, but not confirmed, deleterious role. Most of these rare missense TTN variants with a suspected deleterious role were identified in patients diagnosed with hypertrophic cardiomyopathy. More than 35% of the rare missense TTN variants previously classified as ambiguous were reclassified as not deleterious, mainly because of improved population frequencies. Despite being inconclusive, almost 40% of the variants showed a potentially deleterious role in inherited arrhythmogenic syndromes. Our results highlight the importance of the periodical reclassification of rare missense TTN variants to improve genetic diagnoses and help increase the accuracy of personalized medicine.

Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome.

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.