Molecular Genetics Analysis Of A Chinese Family With Idiopathic Brugada-like Electrocardiogram Pattern

Objective: To investigate the mutation in a family with Brugada-like electrocardiogram pattern. To analysis the gene mutation and possible mechanism of molecular genetics and electrophysiology if the mutation existed, as well as the connection between mutation and clinical manifestation including ECG features.Methods: Clinical data, ECGs, and blood samples of 14 family members were collected for later investigation. Genomic DNAs were extracted from peripheral blood of each member. Screening the candidate gene according the result of linkage analysis accompanying with information form documents and database of interenet. 32 pairs of primers were designed for all 28 exons of SCN5A gene which has the best chance to be the disease-causing gene. Amplifying all 28 exons of family members by polymerase chain reactions. Amplifying the recoveryed PCR products by BigDye single-stranded amplification after agarose gel electrophoresis. Direct DNA sequencings were performed after the purification of Single-stranded amplification products. Applying Restriction Fragment Length Polymorphism (RFLP) technology to validate the existence of mutation in all the family members and 50 normal Chinese. Analyzing the genetic features of the family, as well as the distribution of mutation and the relationship between mutation and ECG performances.Results: A type 2 Brugada ECG pattern (saddleback-like) was showed on ECG data of proband, other ECG results of 3 family members showed type 3 Brugada ECG pattern and elevated J-wave respectively. The result of linkage analysis showed the SCN5A gene could be the most possible candidate gene. Direct DNA sequencing revealed a"double peak"in the second nucleotide of codon 1001 in SCN5A gene of proband, which was identified to be a missense mutation (T3002A) after matching and searching in databases. The T→A replacement at the 3002 site of cDNA of SCN5A causing the substitution of leucine by glutamine(L1001P) in the P-loop of the pore-lining region between transmembrane segments S5-S6 of domain DII, which is one of key structures of Nav1.5 cardiac sodium channelα-subunit. This mutation were found in proband, 3 siblings of proband, the daughter of proband, the restriction enzyme digest analysis of other family members and 50 healthy individuals revealed no same mutation in normals were detected. 3 Single Nucleotide Polymorphisms were detected in family members with mutation at the same time.Conclusions: A novel mutation T3002A of SCN5A was detected in a Chinese Family with Brugada-like electrocardiogram pattern. 5 members of the family were comfirmed with this heterozygous mutation. The results of linkage analysis, direct sequencing and the restriction enzyme digest analysis unanimously support the characteristic of Brugada syndrome as an autosomal dominant inheritance. No history of syncope; nocturnal agonal respiration; ventricular fibrillation; polymorphic ventricular tachycardia; a family history of sudden death under 45 years old were recorded among family members, the only clinical evidence were atypical Brugada-like electrocardiogram pattern records of proband and her 3 siblings and her daughter. However, when undertook genetic detection, all the 5 family members mention above showed an heterozygous mutation T3002A in the second nucleotide of codon 1001 in SCN5A gene, which suggests the likelihood that the defects in sodium channel function repaired by environmental factors. It is also could be the contribution of negative weakening effect that single nucleotide polymorphism brought on the low expression of sodium channel protein caused by mutation. The third explanation is that despite the missense mutation T3002A led the replacement of leucine by glutamine in the P-loop between transmembrane segments S5-S6 of domain DII of protein encoded by SCN5A,the specific extent of the influence on function is unclear, which require the investigation of cell function, such as mutation induction, transfection, patch clamp, action protentials and other research. It's likely the mutation only partially affected the permeability and selectivity of sodium channel, which means the channel can still maintain its normal physiological function most of the time. This could explain why proband and other family members only have an asymptomatic and atypical Brugada-like electrocardiogram pattern.