Sudden cardiac death, which affects people of all ages at a rate of 1 per 1000 people per year, can be caused by inherited genetic conditions especially in young people under 45. Next generation sequencing allows simultaneous screening of multiple patients for multiple genes associated with cardiac conditions, establishing molecular diagnoses more quickly and cost-effectively than previous methods. This document describes the development and validation of targeted next generation sequencing panels for genes associated with thoracic aortic aneurysm/dissection, primary electrical diseases of the heart like Brugada syndrome, and cardiomyopathies. These panels identified genetic variants in 27-96% of patients tested and provided molecular diagnoses to help guide treatment and family screening.

1. Summary
Sudden cardiac death (SCD) or sudden unexplaineddeath is a leading cause of mortality with
an annual incidence of one death per 1000 person-years, affecting all ages. The vast majority
of SCD in persons older than 45 years are due to advanced atherosclerotic coronary artery
disease, whereas in the young (<45 years), inherited causes of SCD are more frequently
observed. The four most common categories in the young include: thoracic aortic
aneurysm/dissection (TAAD), primary electrical disease (PED), cardiom yopathies (CM) and
premature atherosclerosis. DNA sequencing allows establishing a molecular diagnosis,
working out a gene-specific treatment and management scheme, identifying high-risk
relatives and providing preventive treatment or presymptomatic follow-up. Initially, a gene-
by-gene screening approach was used to establish a molecular diagnosis, whereby the most
likely candidate gene(s) was selected based on clinical signs and symptoms. However, the
existence of overlap syndromes and the large clinical and genetic heterogeneity, posed major
challenges.The emergence ofnext generation sequencing (NGS) now allows to simultaneously
screen multiple patients for all genes involved in a certain pathology, in a much faster and
more time and cost efficient way.
In the first part, we developed a targeted NGS panel initially comprising 14 genes involvedin
TAAD. After validation, the assay was applied to 100 Marfan syndrome patients and 55
syndromic and non-syndromic TAAD patients.The mutation yieldreached96% for the Marfan
syndrome cohort, whereas a yieldof 27% was achievedfor the syndromic and non-syndromic
TAAD patients. Next, we screened 40 Marfan syndrome patients from Ukraine, for which a
yield of 67.5% was obtained. Finally, the assay was implementedinto our routine diagnostic
setting and further improvements to the design were made. To date, the TAAD assay
comprises eight additional genes, extra probes were designedfor the (partially) failed regions
and probes targeting 22 SNPs for sample identification were added.
In the second part, we first developed a small NGS panel comprising eight known Brugada
syndrome (BrS) genes. BrS is part of the PED spectrum. Since a large clinical and genetic
heterogeneity and a significant overlap is observed within PED, we soon opted to develop a
larger panel, the PED MASTR Plus assay, comprising 51 genes involved in primary
arrhythmias. Next, we screened 114 PED patients. 46.5% of patients had a variant, either
pathogenic, likely pathogenic or of unknown significance. An important finding was the
identification of the SCN5A c.4813+6_4813+7dupGGGT founder mutation. Finally, the PED
MASTR Plus assay was introduced into our genetic diagnostic laboratory.
In the last part, we used a commercially available HaloPlex NGS panel for CM. The assay
comprised 34 CM genes,including the largest human gene, TTN. We screened67 CM patients
and for 41.8% of patients we could establish a possible genetic diagnosis.Currently,the assay
is being improved by the addition of two more genes andenhancing the coverage of (partially)
failed exons and implemented into our diagnostic laboratory.
In conclusion, all of our NGS panels demonstrated that the confirmation of a clinical
diagnosis by a molecular diagnosis is a prerequisite for most patie nts as this will allow the
clinical geneticist/cardiologist to adjust the follow-up and therapeutic scheme accordingly.