Investigating the underlying genetic mechanisms of inherited cardiac disorders

• Main Author: Petropoulou, Evmorfia
• Published: St George's, University of London 2017
• Subjects: 616.1
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.753990

Sudden cardiac death (SCD) is the most common cause of death worldwide, responsible for more than 50% of all deaths. SCD is associated with inherited cardiac conditions (ICCs) such as channelopathies and cardiomyopathies, that are heterogeneous disorders affecting the function and the structure of the myocardium. ICCs are predominantly Mendelian conditions whieh often present with reduced penetrance and variable expressivity. However, the underlying genetic cause is often not explained by screening for the current known genes, supporting the utility of whole exome and whole genome sequencing in these cases. Molecular diagnosis will enhance the clinical diagnosis and will provide a better and more targeted management for diseases. QRS interval prolongation is one of the features observed on the electrocardiogram (ECG) in Brugada Syndrome (BrS) cases. Initially a candidate gene study based on genome-wide association studies (GWASs) findings for QRS duration on the ECG was performed in a cohort of 156 unrelated SCN5A-mutation negative Brugada Syndrome (BrS) cases. Novel putative pathogenic mutations in the QRS duration-associated loci were found in only 4.5% of the BrS cases carried, suggesting that rare coding variants in the seven genes screened are not a major cause of BrS. However, a common putative benign variant (c.3218T>C - V1073A - rs6795970) in SCN10A was found to be significantly associated with BrS [P=1.39e-18; OR (95% CI) 3.02 (2.35-3.87)]. Then, the potential association with BrS with genes implicated in ICCs other than BrS, such as cardiomyopathies, was investigated using a multi-gene (174 genes) panel in a cohort of 79 unrelated -mutation negative BrS cases. Seven BrS cases (8.9%) had mutations in non SCN5A BrS-associated genes; 43% of these cases had a second mutation in cardiomyopathy (CM)-related genes. Thirteen BrS cases (16.5%) were found to have only one putative pathogenic mutation in CM- or other ICC-related genes. I then went on to investigate the utility of whole exome sequencing (WES) to identify the underlying genetic cause in a series of rare familial cardiac arrhythmias and cardiomyopathies in six Middle Eastern families - five consanguineous and one non- consanguineous. The proband of the first consanguineous family presented with DCM; WES identified a homozygous rare variant in PNPLA2 as potentially causal. Indeed, further investigation revealed the proband suffering from neutral lipid storage disease with myopathy (NLSD-M). The proband of the second consanguineous family died 90 days post birth and was diagnosed with hypertrophic CM (HCM). WES identified a homozygous rare variant in GAA (the gene mutated in Pompe disease) which was suggested to be the most possible cause. The proband of the next consanguineous family as well as all his siblings were diagnosed with HCM. WES of the proband only did not identify any good candidates; combining data from additional WES of his sister identified a homozygous rare variant in KLHL24. The proband of the non- consanguineous family was diagnosed with HCM. WES identified two heterozygous missense rare variants in two potential candidate genes, XIRP2 and SCUBE3, however none of the variants co-segregated with the disease. Two siblings of a consanguineous family were diagnosed with dilated CM (DCM) and were both sent for WES, which revealed digenic heterozygosity; two novel missense variants were identified one in TNNT2 and one in MYH7. Finally, I tried to investigate the genetic mechanism of a consanguineous family whose affected members displayed symptoms of cardiac arrhythmia and cardiomyopathy with variable expressivity among the affected members of the family. WES of two affected family members identified a rare heterozygous missense variant in CNOT1 (a gene previously associated with increased QT interval duration in a GWAS and a meta-analysis study); the variant was absent from a healthy family member that was also sent for WES. Following on from its identification, I used a gene knockdown approach in zebrafish to obtain more support for its potentially causative nature. The CN0T1 knockdown groups seemed to display atrial fibrillation. ECG measurements showed statistically significant difference between the CN0T1 knockdown groups and the control group of the heart rate of the fish (UIC vs SPL: P=0.037; UIC vs ATG: P=0.005). Statistically significant differences were also observed for the QRS and QT interval between the ATG morphants and the control group (P=0.008 and P=0.038, respectively). In conclusion, the above data together provide an insight into the role of rare and common variants in rare diseases, the advantages and disadvantages of the various approaches when trying to investigate the genetic background of rare familial diseases, the importance of co-segregation analysis for genotype-phenotype correlation and the necessity of functional analysis in diseases where the frequency of reduced penetrance is high.