Exome analysis
Segregation analysis was performed on all three phenotypes of interest
in the Lebanese family, which may exhibit epistatic interaction in
development of cardiomyopathy, heart block and AF. A computer script was
designed for variants annotation based on the novelty, conservation,
tissue expression and their effect on protein function. They were
considered non-conservative if the substituted amino acid was conserved
in all species. Filtering criteria included frequency
(<1/1000), and splice site location (-2 to +2 intron exon
boundary). Synonymous, intronic variants, and variants with low quality
were filtered out. Variants that were predicted to be benign by two in
silico tools (SIFT, and Polyphen2) as well as by other by other
laboratories were filtered out early in the analysis. CADD score was
additionally used to predict pathogenicity and variants with a score of
less than 10 were excluded. Regions prone to sequencing errors were
screened then all the related duplications and deletions were filtered
out. Also, all variants in known AF-associated genes were screened for
allele frequencies <1% in the Gnomad database. Lastly,
intolerance score and phyloP 46-way score were used as a measure of
conservation. The GTEx portal was used to filter out genes without
cardiac tissue expression.