| Cardiovascular disease is the leading cause of deaths and disability in the world. Cardiac arrhythmia is the most common cause of death for cardiovascular diseases. Cardiac arrhythmia results from the combined effects of multiple genes and environmental factors, and has become a significant burden for the national public health, social and economic development. Genome wide association studies (GWAS) have achieved great success in identifying common genetic variants associated with common human diseases, but theyexplain only a small portion of heritability, a phenomenon referred to as missing heritability.We hypothesize that rare variants with large effects may explain a large portion of the missing heritability. To investigate association of rare genomic variants in cardiac sodium channel subunit (34gene(SCN4B) with AF (atrial fibrillation) or VT (ventricular tachyarrhythmias),we carried out mutational screening with199VT patients and477AF patients to identify nonsynonymous variants in SCN4B.First, using DNA samples from a panel of AF patients, highly sensitive high-resolution melt (HRM) analysis was used for mutational analysis. After sequencing of samples showing abnormal HRM patterns, only one nonsynonymous variant in SCN4B, G8S, was identified in4of477AF patients.Variant G8S was located within the signal peptide sequence of Navβ4and occurred at a highly conserved residue across species during evolution.Then, to assess the association between the G8S variant and AF, we carried out case-control association studies with two independent populations (944AF patients versus981non-AF controls in the first population and732cases and1,291controls in the second replication population).Significant association between G8S and AF was identified in the two independent populations (P=0.017, OR=3.66in cohort1; P=0.006, OR=3.16in cohort2) and in the combined population (P=4.16x10-4, OR=3.14). These results demonstrate that SCN4B variant G8S confers a significant risk of AF.Second, we used the HRM analysis followed by direct DNA sequence analysis to screen for genomic variants in SCN4B in199VT patients. All five exons and exon-intron boundaries ofSCN4B were analyzed.We identified two novel nonsynonymous variants in SCN4B, including G8S at the signal peptide and present in four patients and A145S in one patient.Variant A145S was located within the extracellular region of Navβ4and occurred at a highly conserved residue among different species. Bioinformatic analysesshowed that G8S and A145S were demanging variants that mayreduce the stability of Navβ4.To assess the association between the G8S variant and VT, we used case-control association studies with two independent VT populationsSCN4B variant G8S was also significantly associated with VT (P=5.58x10-5, OR=9.17in population1with299cases vs.981controls; P=4.47x10-3, OR=4.31in population2with270vs.639controls; P=1.20x10-7, OR=6.42in the combined population569cases vs.1620controls). Theses results demonstrate that SCN4B variant G8S confers a significant risk of VT. The SCN4B variant A145S was identified in a single VT patient and case control studies could not be performed due to its low frequency.Whole cell patch-clamp for recording sodium current showed that both G8S and A145S did not change the sodium current densities. Western blot analysis showed thatG8S and A145Ssignificantly decrease the expression level of the Navβ4protein. It is possible that the difference in the expression level was too small to be detected by patch-clamping analysis. This study for the first time links rare functional variants with large effects to common VT and AF and strongly supports the "common disease, rare variant" theory for common sporadic VT and AF. |
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