Exome Sequencing and Systems Biology Converge to Identify Novel Mutations in the L-Type Calcium Channel, CACNA1C , Linked to Autosomal Dominant Long QT Syndrome

Abstract

Background Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and Results We combined whole exome sequencing (WES) and bioinformatic/systems biology to identify the pathogenic substrate responsible for non-syndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree and established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with “genotype-negative/phenotype-positive” LQTS. WES was utilized on three members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, co-segregated with the disease within the pedigree, was ranked by three disease-network algorithms as the most probable LQTS-susceptibility gene, and involves a conserved residue localizing to the PEST domain in the II–III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain-of-function with increased ICa,L and increased surface membrane expression of the channel compared to wildtype. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Cav1.2’s PEST domain. Conclusions This study provides evidence that coupling WES and bioinformatic/systems biology is an effective strategy for the identification of potential disease causing genes/mutations. The identification of a functional CACNA1C mutation co-segregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.

Genetics of Long QT Syndrome

“…23 the ltCC is important for excitation-contraction coupling in the heart and mediates an inward depolarizing current in cardiomyocytes. functional characterization of the mutation using a whole-cell patch-clamp technique in hek293 cells revealed a gain-of-function with increased iCa,l and increased cell surface expression of the mutant ion channel compared to wild type.…”

“…importantly, although CaCna1C perturbations had been implicated previously in timothy syndrome (ts) and incorrectly given the genotype place holder as lQt8, this was the first demonstration that CaCna1C was a bona fide lQts-susceptibility gene, extending the breadth of distinct CaCna1C-related arrhythmogenic phenotypes. subsequently, mutational analysis of 103 unrelated lQts genotype-negative/phenotype-positive patients identified three additional CaCna1C missense mutations, suggesting that CaCna1C mutations may explain as much as 4% to 5% of genetically elusive lQts 23 or approximately 1% of lQts altogether. interestingly, three of the four mutations (k834d, p857l, and p857r) localized to the same critical pest-domain in the ii-iii linker of the ltCC, an amino acid sequence motif that represents an important signal for rapid protein degradation and ltCC channel stability.…”

Mutations in voltage-gated L-type calcium channel: implications in cardiac arrhythmia

“…This mutation in Ca V 1.2 also revealed a complex channel phenotypes, including loss of current density and inactivation in combination with increased window and late currents [83]. By whole-exome sequencing and bioinformatics/systemic biology, Boczek et al further identified 4 novel mutations of CACNA1C in LQTS, including p.K834E, p.P857R, p.P857L, and p.R1906Q [84], 3 of which were located at the conserved proline, glutamic acid, serine and threonine rich (PEST) domain of Ca V α 1 II-III loop, this domain acts to proteolytic signaling through the cellular quality control system [85]. Functionally, p.P857R mutation, cosegregated with the disease within the pedigree, significantly increased calcium currents and surface membrane expression of the channel as compared with wild type (WT) Ca V 1.2 channel [84].…”

“…By whole-exome sequencing and bioinformatics/systemic biology, Boczek et al further identified 4 novel mutations of CACNA1C in LQTS, including p.K834E, p.P857R, p.P857L, and p.R1906Q [84], 3 of which were located at the conserved proline, glutamic acid, serine and threonine rich (PEST) domain of Ca V α 1 II-III loop, this domain acts to proteolytic signaling through the cellular quality control system [85]. Functionally, p.P857R mutation, cosegregated with the disease within the pedigree, significantly increased calcium currents and surface membrane expression of the channel as compared with wild type (WT) Ca V 1.2 channel [84]. Interestingly, p.R1906Q locates one amino acid away from α 1C C-terminal STIM1 (stromal-interacting molecule 1, a calcium store sensor) binding domain, which may affect STIM1-mediated Ca V 1.2 channel gating inhibition and channel internalization [86,87].…”

“…The glutamic acid at 850 position is located within the PEST domain of Ca V α 1 II-III loop. The mutations in the PEST domain are proved to affect the surface membrane expression of Ca V 1.2 channels [84]. Functionally, p.E850 deletion in CACNA1C is found to lead to an almost complete loss-of-function in I Ca [90].…”