Sudden cardiac death due to ARVC in the young: molecular autopsy by whole exome sequencing of DNA from dried blood spots (DBS) collected at birth

Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Uppsala University Hospital (ALF) and Marcus Börgström Foundation Background The post-mortem investigation of cases with sudden cardiac death (SCD) is critical for risk stratification and adequate care of surviving relatives. Genetic testing is limited by access to DNA from the proband, as blood samples are not routinely collected at the time of death, or DNA of poor quality obtained from formalin-fixed paraffin-embedded (FFPE) tissues. Whole exome sequencing (WES) on minute amounts of DNA has opened up for the possibility to use DNA from Dried Blood Spots (DBS). Purpose To evaluate the feasibility of WES on DNA from archived DBS collected at the time of newborn screening in cases of SCD. Methods Through National Health Databases and Registries, we identified all cases of SCD in the young (<35 y.o.a.) in Sweden with a post-mortem diagnosis of ARVC between 2000 and 2010 (n = 22). Clinical records, medical and family histories, and autopsy findings were collected. Surviving parents were invited to participate. DNA was extracted from samples in National Biobanks such as DBS collected at birth (n = 19), formalin-fixed paraffin-embedded (FFPE) heart tissues (n = 8) from autopsies, and frozen blood samples (n = 3). Patient and parental DNA samples (when available) underwent WES. DNA- and sequencing-quality check (QC) metrics were compared between two different sequencing platforms and specimen types. We analysed 392 cardiomyopathy and arrhythmia genes and variants were classified as pathogenic or likely pathogenic according to the criteria from the American College of Medical Genetics. Results A higher quality but a lower yield of DNA (7.3 – 140 ng) was obtained from DBS when compared to FFPE samples (515 – 3065 ng). However, 100% of DBS vs. 62,5% of FFPE samples passed QC before sequencing. The average mean target depth for DBS samples (160X) was similar to that in blood samples (155X) and higher than FFPE (82X). Furthermore, >97% of target regions were covered with read depths >30X in DNA from DBS and blood compared to 88% for FFPE samples (Min 63%; Max 97%). Analysis of WES data uncovered clinically relevant variants in 12 out of 19 families (63%), of which four were located in ARVC genes. In six cases, the molecular autopsy identified another arrhythmogenic syndrome. Additionally, we identified one SCD with hemochromatosis and one with myotonic dystrophy as a possible contributing cause. In two additional families, variants of unknown significance fulfilled criteria for further segregation analysis. The concordance of positive results between different tissue samples was 100%. Conclusions DNA obtained from DBS has a quality comparable to that of blood and is a reliable source for WES-based molecular autopsy that may improve the diagnostic yield in cases of SCD. In a proportion of cases diagnosed post-mortem with ARVC, our molecular autopsy analysis pointed to a different diagnosis, highlighting its importance for the correct medical care of surviving relatives.