A key challenge in using WES in molecular autopsy is finding the true causal variant among hundreds of rare variants. By filtering genes known to be associated with a particular HPO term, we shift the analysis focus from the entire exome to that part of the exome that is clinically interpretable in a diagnostic setting. Instead of using a fixed panel-based approach, we designed a HPO-driven virtual gene panel, with the advantage, that recently identified genes are automatically associated by HPO terms in the HPO database and developed an algorithm that prioritized 1.4% of the variants by several filtering steps.
The two likely pathogenic variants found in our study, were detected in children (< 12 months). Genetic studies in SIDS cohorts collectively suggest that up to 15–20% of SIDS cases might be explained by inherited cardiac diseases not detectable during conventional forensic autopsy investigations (15–17). However, our data further highlight, that interpretation of putative pathogenic variants in SIDS is challenging.
The homozygous variant in UPB1 was annotated by the HPO term “status epilepticus” and has been recently published to trigger seizures due to ß-ureidopropionase (UPB) deficiency in a recessive mode of inheritance (18). Assmann et al. reported the same variant also homozygous in a four months old boy with an acute life threatening event (ALTE) with febrile status epilepticus (19). The extent of the reduction in enzyme activity caused by a particular UPB1 variant, along with other genetic and environmental factors may determine whether people with UPB deficiency develop neurological problems and the severity of these problems. Therefore, in many affected individuals with absent or mild neurological problems, the condition may never be diagnosed, and may thus explain that the here identified variant has been found homozygous in one of 141,426 genomes from unrelated individuals. Importantly, epileptic seizures can induce malignant arrhythmias, possibly due to seizure-related effects on the autonomic nervous system (20). However, the homozygous likely pathogenic variant in UPB1, recently associated to status epilepticus, has not been linked to SD before. Thus, a fixed gene panel-based approach consisting well-known genes linked to SD would have missed the variant in UPB1. In comparison, the HPO-dirven virtual panel is a flexible system that does not have to be adjusted over time as new genes are added.
The second identified likely pathogenic variant was detected in DSG2 in an eight months old girl. Pathogenic variants in DSG2 are associated with arrhythmogenic right ventricle cardiomyopathy (ARVC), a disease that importantly predominantly affects adults in the 4th or 5th decade of life. If ARVC is diagnosed in the infantile stage, there should be clearly identifiable morphologic changes of the heart (fibrosis, dilation, fatty infiltration) before death occurs. Nevertheless, another study identified variants in DSG2 associated with SUD in infants (21), indicating that interpretation of variants in context with the age of the individual at the SUD event is challenging.
Beside the two likely pathogenic variants, we identified nine VUS. The majority of the VUS has been identified in genes previously having been reported to be associated with cardiac channelopathies (SCN5A, AKAP9, RYR2) and cardiomyopathies (RBM20, RAF1) (2, 22–24). One variant was identified in SCN4A in a four weeks old infant. SCN4A variants are described as cause of autosomal-dominant myotonia and periodic paralysis (25). Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth (26). Interestingly, variants in SCN4A have also been reported in patients with clinical diagnosis of Brugada syndrome, a primary arrhythmia syndrome (27). Another potentially causative variant was identified in SCN8A in a three months old infant. Pathogenic variants in SCN8A have been associated with a wide spectrum of epilepsy phenotypes, ranging from benign familial infantile seizures to epileptic encephalopathies with variable severity (28). Now, there are no forensic guidelines on the management and interpretation of VUS. Grassi et al recently discussed the main elements and issues that differentiate the forensic management of cases in which VUS are found (29). Our data highlight that HPO-based filtering could be used as complementary approach in particular to prioritize VUS by HPO-matches. Before one of the candidate variants can be defined as “causative variant” further investigations (f.e. co-segregation analyses, functional studies) are needed. To date, many studies that used HTS identified putatively pathogenic variants in molecular autopsy but only a small number performed co-segregation analysis. Due to complete anonymization, co-segregation analyses of the variants cannot be performed. Campuzano et al demonstrated the value of co-segregation in SUD (30). The presence of rare variants in asymptomatic family members aided the exclusion of some variants as being causative of the SUD. Glengarry and co-workers reported that co-segregation studies are challenging to perform especially if the proband is an infant, due to difficulties in tracking families once a pathogenic variant which explains SD is found (31).
Our data further highlight that phenotype and genotype data should be used in conjunction to prioritize variants for further evaluation and may thus increase the overall solve rate especially in cases without specific clinical phenotypes like SD. In particular, HPO provides a structured, comprehensive and an international standard that could be used for developing algorithms and computational tools for clinical differential diagnostic in SUD.