| Objective:To identify and characterize a novel variation A197V in SCN1B associated with Brugada syndrome (BrS). To study sodium channel change affected by A197V mutation using cell immunofluorescence and whole-cell patch clamp techniques. To establish patient-sepcific induced pluripotent stem cells(iPSCs) from skin fibroblasts.Methods:1. All known exons and intron borders of the BrS-susceptibility6genes were amplified and sequenced in both directions.2. The ion channel variants were cloned by site-directed mutagenesis, plasmids were confirmed by restriction enzyme digestion and direct sequencing.3. Wild type (WT) and mutant genes were expressed in HEK293cells and studied using cell immunofluorescence and whole-cell patchclamp techniques.4. Reprogramming was performed with retroviruses encoding the four human factors, OCT4, KLF4, SOX2,and cMYC, then cells were cultured in a defined condition to induce iPSCsResults:1. Patient6was a46-year-old man with an type1ST-segment elevation in V1and V2supporting the diagnosis of BrS. A A197V variant was detectedin exon4of SCN1B gene in this proband, which was a variant in BrS.2. Plasmids containing A197V variant were cloned and sequenced. Result comfirmed success of site-directed mutagenesis.3. Coexpression of SCN5A/WT+SCN1B/A197V resulted in peak sodium channel current (INa) smaller compared to SCN5A/WT+SCNlB/WT.Cell immunofluorescence indicated no significant distribution of Navl.5protein between SCNlB/A197V and SCNlB/WT.4. iPSCs colonies started to appear about14days after first infection, they were mechanically isolated30d after infection and expanded on MEF feeder-free in mTesR1medium..Conclusion:1. A197V variation in SCNlB is a functional polymorphism that may serve as a modifier of the substrate responsible for BrS phenotypes via loss of function of sodium channel current.2. iPSCs can be induced from patient’s skin fibroblasts and can be used for disease model in the future. |
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