Results

Overview of the forensic cohort and case selection

Between 2009 and 2023, a total of 343 medico-legal autopsies of SUD were performed at the Institute of Legal Medicine and Forensic Sciences of Murcia. All cases underwent comprehensive forensic investigation, including complete autopsy, standardized cardiac dissection, histopathological examination, and toxicological analysis. Following this evaluation, cardiomyopathies were established as cause of death in 156 cases (45.5%), with a mean age of 43.4 ± 13.6 years old; 135 (86.5%) males. Structurally normal hearts consistent with inherited channelopathies were identified in 72 cases (21.6%), with a mean age of 39.3 ± 17.1 years and 72.2% males decedents. Ischemic heart disease and other structural cardiac causes accounted for 61 cases (18.3%) (47.6 ± 9.9 years; 88.5% male), whereas 54 (15.7%) cases were attributed to non-cardiac causes (30.1 ± 19.4 years; 66.7% male). A personal or familial history suggestive of inherited cardiac disease was documented in 77 individuals (22.4%) prior to death.

Postmortem genetic testing was undertaken in 119 cases in which an inherited cardiac disorder was suspected. A genetic variant was identified in 93 cases (78.1%). Among these, 39 cases (41.9%) harbored pathogenic or likely pathogenic variants considered sufficient to explain death, while 54 cases carried variants of uncertain significance (VUS).

From this broader cohort, 12 cases were selected for detailed analysis in the present study. Selection was not based solely on the performance of genetic testing, but rather on the coexistence of sub-diagnostic or borderline cardiac findings that were insufficient in isolation to establish a definitive cause of death and/or the presence of circumstances with potential medico-legal implications, such as death during exertion, emotional stress, minor trauma, or occupational activity. In all selected cases, an underlying inherited cardiac disorder was suspected and clarification of the cause and manner of death carried potential legal consequences.

Pathological findings of case selection

The twelve selected cases demonstrated a heterogeneous spectrum of cardiac findings ranging from structurally normal hearts to definite or suggestive arrhythmogenic cardiomyopathy (ACM), as well as inflammatory and rare structural abnormalities. The main demographic, circumstantial, and pathological characteristics are summarized in Table 1, while the genetic findings are detailed in Table 2.

Case ID | Gender | Ethnicgroup | Age | Circumstances of death | History | Main cardiac pathological findings | Heart weight(g) | ECG findings | Autopsy interpretation
1 | F | E | 15 | Emotional situation | Recurrent syncopes | Structurally normal heart | 220 | Normal | Inconclusive
2 | M | E | 9 | Physical stress (fair attraction) | No risk factors | Structurally normal heart | 150 | N/A | Inconclusive
3 | F | E | 36 | Resting at home | No risk factors | Fibro-fatty replacement of RV myocardium | 210 | N/A | Suggestive of ACM
4 | F | E | 55 | Public space collapse | No risk factors | Extensive fibro-fatty replacement of RV myocardium | 480 | N/A | Suggestive of ACM
5 | M | E | 38 | Physical exertion (athlete) | Competitive athlete. Prior syncope | Fibro-fatty replacement of RV and LV myocardium | 620 | Normal | Suggestive of ACM
6 | M | E | 17 | Physical exertion (soccer) | Fatigue/tiredness | Fibro-fatty changes with lymphohistiocytic myocarditis | 320 | N/A | Suggestive of ACM
7 | M | E | 43 | Sudden collapse | Idiopathic DCM with severely depressed EF | Fibro-fatty degeneration of LV myocardium | 495 | Abnormal | Suggestive of ACM
8 | M | Mo | 10 | Resting at home | Fatigue/tiredness | LV fibrosis with cardiomyocyte hypertrophy and inflammation | 170 | N/A | Inconclusive
9 | M | Mo | 24 | Physical confrontation | No risk factors | Cardiac hypertrophy without definitive structural cause | 566 | N/A | Inconclusive
10 | M | E | 19 | Acute illness | Febrile illness | Severe lymphocytic myocarditis | 370 | N/A | Inconclusive
11 | M | E | 38 | Working activity | No risk factors | Giant intramural lipoma | 350 | N/A | Inconclusive
12 | M | E | 24 | Physical exertion (heat exposure) | Recurrent syncopes in stressful situations | Structurally normal heart | 390 | N/A | Inconclusive

Case ID | Gene(s) involved | Variant | Variant Classification(ACMG)* | Diseaseclassification | Genes analyzed** | Familyscreening
1 | RYR2 | NM_001035.3: c.14311G > T (p.Val4771Phe) | P (PS2, PM1, PM2, PM5, PP2, PP3) | CPVT | 1 gene associated with CPVT | Yes
2 | CALM2 | NM_001743.5: c.404G > T (p.Gly135Val) | P (PS2, PM1, PM2, PP2, PP3) | Long QT | 251 | Yes
3 | JUP | NM_002230: c.103 C > A (p.Pro35Thr) | VUS (PM2, PP3) | RV-ACM | 5 desmosomal genes | Yes
4 | TXNRD2 | NM_006440.4: c.1220G > A (p.Cys407Tyr) | VUS (PM2, PP3) | RV-ACM | 218 genes associated with SCD | No
5 | GJA5 | NM_005266.5: c.365 C > T (p.Ser122Phe) | VUS (PM2) | RV-ACM | 218 genes associated with SCD | Yes
6 | JUP | NM_021991.2: c.2105G > A (p.Arg702His) | VUS (PM2) | RV-ACM | 21 genes associated with ACM | Yes
7 | FLNC | NM_001458.4: c.5398G > T (p.Gly1800*) | P (PVS1, PM2, PP1) | LV-ACM | 21 genes associated with ACM | Yes
8 | PPP1R13L | NM_001142502.1:c.2035 C > T (p.Leu679Phe) (homozygous) | LP (PM3, PM2, PP3) | ACM/DCM | 251 genes associated with SCD | Yes
9 | LZTR1LZTR1 | NM_006767.3: c.410 C > A (p.Thr137Asn)NM_006767.3: c.614T > C (p.Ile205Thr) | VUS (PM2)VUS (PM2) | Inconclusive (Noonan syndrome) | 251 genes associated with SCD | No
10 | Negative |  |  |  | 21 genes associated with ACM | No
11 | Negative |  |  |  | 251 genes associated with SCD | No
12 | Negative |  |  |  | 288 genes associated with SCD | No

In three cases (Cases 1, 2 and 12), children and young individuals (27.3 ± 14.5 years old), the heart was structurally normal on macroscopic examination, and extensive histological sampling revealed no evidence of myocardial fibrosis, fatty infiltration, disarray, inflammatory cardiomyopathy, or significant coronary artery disease. These cases fulfilled criteria for autopsy-negative sudden death.

Six cases (Cases 3–8) showed pathological findings within the spectrum of ACM, including right ventricular, left ventricular, or biventricular involvement. The observed changes consisted of focal or diffuse fibrofatty myocardial replacement, sometimes associated with lymphohistiocytic inflammatory infiltrates. In several individuals, these alterations did not fully satisfy established diagnostic criteria for definite ACM and were therefore interpreted as suggestive or sub-diagnostic phenotypes within the ACM spectrum. HCM was excluded based on the absence of myocyte disarray, asymmetric septal hypertrophy, and characteristic architectural abnormalities. Ischaemic heart disease was ruled out by detailed coronary artery examination demonstrating no significant atherosclerotic stenosis or acute plaque complications. When inflammatory infiltrates were prominent, inflammatory cardiomyopathy was considered.

Case 9 showed marked cardiac hypertrophy with a heart weight of 566 g but lacked definitive histological criteria for hypertrophic cardiomyopathy or ACM, and coronary arteries were free of significant disease. Case 10 demonstrated acute, severe lymphocytic myocarditis involving the conduction system without additional structural abnormalities. Case 11 revealed a giant intramural lipoma measuring approximately 3 cm within the left ventricular wall, without associated fibrosis, inflammation, or coronary pathology. Representative pathological findings are illustrated in Fig. 1.

Fig. 1: Pathological Findings from Clinical Autopsies of selected cases. (a) Structurally normal heart. (b) Heart with arrhythmogenic cardiomyopathy predominantly affecting the left ventricle, showing areas of fibro-fatty infiltration. (c) Heart with arrhythmogenic cardiomyopathy predominantly affecting the right ventricle, showing myofibrillar degeneration and fibro-fatty replacement. (d) Heart with lipoma. (e) Histological image of myocarditis, with a lymphohistiocytic inflammatory infiltrate associated with areas of myofibrillar degeneration (H&E stain, 40x). (f) Extensive fibro-fatty infiltration in the left ventricle, showing multifocal degeneration (Masson’s trichrome stain, 40x). (g) Multifocal fibro-fatty degeneration in the right ventricle, characteristic of arrhythmogenic cardiomyopathy (H&E stain, 40x). (h) Excoriated skin lesion. Insufficient severity to cause death

Genetic findings in selected cases

Postmortem genetic testing identified pathogenic or likely pathogenic variants in 4 of the 12 selected cases (33.3%), involving genes associated with inherited arrhythmia syndromes or cardiomyopathies (Table 2). In the remaining cases, VUS or negative results were obtained.

In two pediatric cases with structurally normal hearts, genetic testing provided a definitive explanation for death. Case 1 carried a pathogenic de novo variant in RYR2, consistent with catecholaminergic polymorphic ventricular tachycardia, while Case 2 harbored a pathogenic de novo variant in CALM2, associated with severe calmodulinopathy and malignant arrhythmia syndromes. In both cases, the absence of structural abnormalities combined with the presence of well-established disease-causing variants supported classification as primary electrical diseases.

Among cases with pathological findings suggestive of ACM (Cases 3–8), a truncating pathogenic variant in FLNC (p.Gly1800*) was identified in Case 7, supporting a left-dominant ACM phenotype. Case 8 carried a homozygous likely pathogenic variant in PPP1R13L (p.Leu679Phe), compatible with early-onset cardiomyopathy within the ACM/dilated cardiomyopathy spectrum. In these cases, genetic findings reinforced interpretation of structural abnormalities as manifestations of inherited cardiomyopathy rather than isolated or incidental changes.

VUS were identified in five additional cases involving genes related to desmosomal integrity or myocardial structure, including JUP, TXNRD2, GJA5 and LZTR1. Although these variants could not be considered causative in isolation according to ACMG criteria, their identification contributed to the interpretation of borderline pathological findings and supported further clinical correlation and family screening, which was performed in several of these families (Table 2).

Postmortem genetic testing was negative in Cases 10, 11, and 12. In these individuals, death was attributed to non-genetic causes, including acute myocarditis, a rare structural cardiac abnormality in the form of a giant intramural lipoma, and exertional heatstroke, respectively. In these settings, negative genetic results supported exclusion of an inherited cardiac disorder and reinforced the conclusions derived from pathological examination.

Familial screening and medico-legal implications

Familial cascade screening was performed when feasible and provided clinically relevant information. In Cases 1 and 2, parental testing confirmed de novo status of the variants, allowing reassurance of first-degree relatives. In cases harboring variants within the ACM spectrum, additional carriers were identified among relatives, including clinically affected individuals who subsequently cardiological follow-up. In the family of Case 7, evaluation of 53 relatives identified five carriers of the FLNC truncating variant, two of whom fulfilled diagnostic criteria for cardiomyopathy and three who exhibited non-diagnostic abnormalities, while the remaining relatives were wild-type. In Case 8, heterozygous carriage of the PPP1R13L variant was identified in one sibling, whereas the other was non-carrier.

From a medico-legal standpoint, postmortem genetic testing was particularly relevant in deaths occurring under circumstances potentially suggestive of external responsibility. In Case 9, sudden death during a physical confrontation with only minor external injuries required exclusion of traumatic or homicidal mechanisms; integration of pathological and genetic findings supported a natural cardiac cause. In Case 2, death during a fairground attraction raised the possibility of third-party liability; demonstration of a primary arrhythmogenic disorder excluded external causation. In Case 12, death occurred during occupational heat exposure, and negative genetic findings reinforced attribution to environmental and metabolic factors rather than inherited disease.

In this selected subgroup of complex forensic cases, integration of detailed pathological assessment with postmortem genetic testing provided incremental diagnostic value beyond conventional autopsy alone and was critical for accurate determination of both cause and manner of death.