Discussion

Across heterogeneous forensic scenarios, integration of detailed pathological examination with postmortem genetic testing provided incremental diagnostic value beyond conventional autopsy alone. In autopsy-negative cases, genetic findings were decisive in establishing a natural cardiac cause of death, particularly in pediatric deaths and in events occurring during emotional stress or physical exertion. In cases with sub-diagnostic structural abnormalities suggestive of ACM, genetic results increased diagnostic confidence and supported interpretation of borderline myocardial changes as manifestations of inherited disease rather than incidental findings. Conversely, in cases characterized by inflammatory processes or uncommon structural lesions, negative genetic results contributed to delimiting the role of inherited cardiac disease and strengthened alternative pathological explanations.

Diagnostic yield and concealed cardiomyopathy as a central challenge

In the present study, 12 cases were selected from a cohort of 343 medico-legal SUD autopsies performed over a 15-year period because conventional investigation was inconclusive or raised medico-legal concerns requiring clarification. This was therefore not a prevalence study but a focused analysis of complex forensic cases in which the integration of genetics had potential diagnostic and legal implications.

Within this selected subgroup, pathogenic or likely pathogenic variants were identified in 4 of 12 cases (33.3%), a yield consistent with previous reports showing actionable variants in 20–40% of unexplained sudden deaths and approximately 30% when cardiomyopathy is present at autopsy [12–15]. The observed yield should therefore be interpreted as reflecting case enrichment rather than overall population prevalence.

A central finding of our series is the distribution of clinically actionable variants across both channelopathy-associated and cardiomyopathy-associated genes. In two autopsy-negative pediatric cases, pathogenic variants in RYR2 and CALM2 provided a definitive diagnosis of primary electrical disease. These cases illustrate the well-established concept that structurally normal hearts do not exclude a lethal arrhythmogenic substrate and confirm that postmortem genetic testing is essential in pediatric and adolescent SUD [16–19].

Equally relevant, however, is the observation that in cases with sub-diagnostic structural findings, causal variants were predominantly located in cardiomyopathy-associated genes. The identification of a truncating FLNC variant and a homozygous PPP1R13L variant in cases with borderline or evolving structural phenotypes supports the concept of concealed cardiomyopathy, defined as malignant ventricular arrhythmias occurring before overt morphologic criteria are fully expressed [6, 8, 20]. This concept has gained increasing recognition in recent years and challenges the traditional dichotomy between “structurally normal” and “structurally abnormal” hearts. Subtle fibrofatty replacement, limited fibrosis, or mild hypertrophy may represent early or partial expression of inherited cardiomyopathy rather than incidental findings. Our data align with recent studies demonstrating that actionable variants in cardiomyopathy genes are frequently identified in autopsy-inconclusive cases and may be more common in the presence of subtle structural abnormalities than in completely normal hearts [6, 13, 15].

Traditionally, genetic testing in unexplained sudden death focused predominantly on inherited arrhythmia syndromes such as long QT syndrome or Brugada syndrome [15]. However, increasing evidence demonstrates that this approach underestimates the prevalence of cardiomyopathy-related variants in SCD. Genes such as FLNC,_ TTN_,_ LMNA_,_ PKP2_,_ DSP_, and NKX2.5 are now recognized in autopsy-negative cases, often with subtle or sub-diagnostic pathological features [6, 21]. In fact, recent studies have shown that up to 70% of clinically actionable variants in autopsy-inconclusive cases are found in cardiomyopathy genes rather than channelopathy genes, and that these variants are four times more frequent in cases with sub-diagnostic structural abnormalities compared to structurally normal hearts [6, 13, 15]. Our findings support the systematic inclusion of both arrhythmia- and cardiomyopathy-associated genes in postmortem panels for SUD. Restrictive panels focused exclusively on channelopathies risk missing clinically actionable diagnoses in cases with subtle structural abnormalities.

Variants of uncertain significance and ongoing challenges

Despite advances in sequencing technologies, a substantial proportion of SUD cases remain genetically unresolved, with 70–80% lacking definitive pathogenic variants even after broad testing [6, 12, 15]. VUS are particularly problematic in the post-mortem setting, where clinical correlation and segregation studies are often unavailable [11, 12]. In our experience, VUS were identified in 5 out of 12 cases (41.7%) in genes such as JUP, TXNRD2, GJA5, and LZTR1, which, although not definitively pathogenic, may represent candidates of emerging relevance [22–25]. For example, TXNRD2 is not currently considered a major ACM-associated gene; however, accumulating evidence suggests an emerging role in DCM [26, 27]. These findings illustrate both the potential of postmortem genetic investigation and the interpretative complexity it entails. For these reasons, hypothesis-free approaches (whole exome or genome sequencing without a clear suspicion) are not routinely recommended in forensic practice [6, 12, 15].

Clinical, Preventive, and Medico-Legal Implications

Establishing a molecular diagnosis extends beyond determining the cause of death in the proband. Cascade family screening following sudden death has been shown to identify disease in 25–47% of screened relatives, particularly when multiple family members are evaluated [6, 28]. In our cohort, familial screening identified additional carriers, including clinically affected individuals who subsequently entered cardiological surveillance programs.

Identification of at-risk relatives enables targeted preventive strategies, including exercise restriction in ACM, pharmacological treatment, and prophylactic implantable cardioverter-defibrillator implantation in selected high-risk phenotypes [28, 29]. Given the age-dependent penetrance and variable expressivity of inherited cardiac diseases, longitudinal follow-up remains essential even in initially asymptomatic carriers.

One of the most distinctive contributions of postmortem genetic testing lies in its medico-legal impact. Several deaths in this series occurred under circumstances potentially suggestive of external causation, including physical confrontation, recreational activity, and occupational heat exposure. In such contexts, identification of an inherited cardiac substrate can prevent misclassification of death and avoid inappropriate attribution of criminal or civil liability [30, 31]. Conversely, negative genetic findings may reinforce attribution to inflammatory, environmental, or metabolic causes.

The legal significance of postmortem genetic evidence has been illustrated in internationally recognized judicial cases in which genetic findings fundamentally altered legal determinations. These examples underscore that postmortem genetic testing is not merely an academic adjunct but may serve as a safeguard against miscarriage of justice [32–34].

Towards a multidisciplinary standard of care

The body of evidence supports the establishment of multidisciplinary teams including forensic pathologists, cardiologists, geneticists, pediatricians, psychologists, and genetic counselors [14]. Best practice requires systematic preservation of DNA samples (e.g., EDTA blood, spleen, liver, thymus, or fresh-frozen myocardium), use of broad next-generation sequencing panels, and careful interpretation of VUS following established international guidelines [4, 11]. This framework will facilitate the transition of post-mortem genetic testing from a research tool to a routine component of forensic and preventive medicine.