A Novel SCN5A Mutation Associated With Early Repolarization Syndrome

BackgroundThe early repolarization pattern consisting of a J-point elevation, notching or slurring of the terminal portion of the R wave (J wave) and tall/symmetric T wave is a common finding on the electrocardiogram (ECG) in the young healthy men or athletes and has been considered to be a "benign" ECG manifestation for a long time. However, recent evidence demonstrated that the early repolarization may be associated with increased risk for ventricular fibrillation (VF), depending on the locations and magnitude of J wave and ST-segment elevation. Thus, Antzelevitch and Yan proposed a new conceptual framework, inherited J wave syndromes, to describe the arrhythmic phenotypes. Inherited J wave syndromes, as a continuous spectrum of arrhythmic phenotypes, has been divided into four different subtypes in terms of anatomical locations of J wave and ST-segment elevation and clinical consequences including early repolarization in the lateral precordial leads (early repolarization syndrome, ERS, type 1), in the inferior or inferolateral leads (ERS type 2), globally in the inferior, lateral, and right precordial leads (ERS type 3) and in the right precordial leads (Brugada syndrome, BrS). So far, mutations in CACNA1C, CACNB2 and CACNA2D1 genes and "gain-of-function" mutations in KCNJ8 gene have been identified in the patients with ERS.ObjectivesThe present study was designed to identify the novel mutation that would be responsible for the ERS in a Chinese pedigree and further to characterize the functional consequences of the mutation to explore the pathogenesis of ERS.MethodsThe family members of the pedigree and 500 unrelated healthy Chinese individuals were recruited in the present study. All the exons of SCN5A, KCND3, CACNA1C, CACNB2, KCNE3, SCN1B and KCNJ8 genes were amplified by polymerase chain reaction (PCR) and were analyzed by direct sequencing. Whenever the variant was found in the proband, it would be confirmed in her family members and 500 unrelated healthy Chinese individuals to make the distinction between mutation and polymorphism by restriction fragment length polymorphism. The mutations based on the genotypes of the proband and her family members were created on the wide-type (WT)-SCN5A background by site-directed mutagenesis. Sodium current (INa) was measured from the HEK293 cells transiently transfected with the WT or mutant plasmid. Additionally, real-time PCR was performed to test the mRNA levels of WT and mutant sodium channel proteins. Furthermore, immunofluorescence was applied to detect the distribution pattern of the WT and mutant sodium channel proteins under a eonfocal laser scanning microscope.ResultsA 19-year-old female proband with recurrent syncope had a documented electrocardiogram with ventricular fibrillation proceeded by the large J waves and ST-segment elevation in leads I, II, III, aVF, and V2-V6. The baseline ECG was within normal limits with the exception of tiny, hump-like J waves in the inferior leads. Genetic analysis revealed that the patient carried a heterozygous missense mutation of G4297C and a heterozygous synonymous polymorphism of T5457C on the same allele of the SCN5A gene. The G4297C mutation led to a substitution of an arginine for a glycine at position 1433 that was predicted to be in the extracellular loop between the pore region and the S6 transmembrane segment in the domain III. of a subunit of the sodium channels, while the T5457C polymorphism did not cause change of the protein sequence. Patch-clamp experiments showed that the G4297C mutation significantly reduced the INa density and altered the gating properties of the channels. Immunocytochemistry studies demonstrated that the mutation dramatically inhibited the expression of sodium channels in the cytoplasm and on the cell membrane, although the mRNA level, detected by quantitative real-time PCR, remained in the normal range. Interestingly, the reduction of lNa density could be partially restored by co-existence of T5457C polymorphism on the same allele by up-regulation of the mRNA levels; however, the altered channel kinetics could not be rescued.ConclusionOur study indicated that the G4297C mutation caused the“loss-of-function”of sodium channels that might account for the clinic phenotype of ERS. The reduction of lNa density was due to decreased number of sodium channels caused by abnormal translation processes. The T5457C polymorphism partially rescued the lNa density of the mutant channels by up-regulation of the mRNA levels. BackgroundThe Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessively inherited disease clinically characterized by the presence of congenital bilateral sensorineural hearing loss, a markedly prolonged correct QT interval (QTc) on a documented electrocardiogram and a high incidence of sudden death secondary to polymorphic ventricular arrhythmias (e.g. torsade de pointes, TdP) in childhood. Most of the arrhythmic events (95%) are triggered by emotions or exercise. JLNS is caused by homozygous or compound heterozygous mutations in KCNQ1 and KCNE1 genes, which are responsible for encoding the slowly activated delayed rectifier current (lks). Mutations in the KCNQ1 gene account for more than 90%of individuals with JLNS. The“loss-of-function”of lls is the mainly pathophysiologic mechanism for JLNS.ObjectivesThe present study was designed to identify the novel mutation that would be responsible for JLNS in a Chinese pedigree and to characterize the functional consequences of the mutation to explore the pathogenesis of JLNS.MethodsThe family members of the pedigree and 200 unrelated healthy Chinese individuals were recruited in the present study. All the exons of the KCNQ1 gene were amplified by PCR and were analyzed by direct sequencing. Whenever the variant was found in the proband, it would be confirmed in her family members and 200 unrelated healthy Chinese individuals to make the distinction between mutation and polymorphism. The mutations based on the genotypes of the proband and her family members were created on the WT-KCNQ1 background by site-directed mutagenesis.lkswas measured from the CHO cells transiently transfected with the WT or mutant plasmid. Additionally, immunofluorescence was applied to detect the distribution pattern of the WT and mutant KCNQ1 channel proteins under a confocal laser scanning microscope. Furthermore, Western blot analysis was performed to test the expression of the WT and mutant KCNQl channel proteins.ResultsA 6-year-old deaf girl suffering from recurrent syncope had a documented electrocardiogram with polymorphic ventricular fibrillation since the age of 4 years. The baseline electrocardiogram showed a significantly prolonged QTc (524 ms). Genetic analysis revealed that the proband carried two heterozygous mutations of T2C and 1149insT in the KCNQl gene on separate alleles. The T2C mutation caused a substitution of threonine (Thr) for initiation codon at position 1 of KCNQ1 protein. In contrast, the 1149insT mutation caused a frameshift at codon 384 resulting in introducing a premature termination codon (PTC) at codon 462 that was predicted to form a nonfunctional truncated protein (A384fs/79). Patch-clamp analysis demonstrated that the T2C mutation resulted in significant reduction in the lks.Furthermore, Western bolt analysis and confocal imaging revealed that the T2C mutation produced a truncated protein with trafficking defect. In contrast, the 1149insT mutation failed to generate any measurable current, consistent with no protein expression in both the cell membrane and cytoplasm. Moreover, co-expression of the T2C and 1149insT mutations significantly reduced the peak tail current density to 8.27%of the wide-type (WT) current value, while co-transfected WT channels with either T2C or 1149insT mutant channels produced comparable current and channel kinetics to that of WT channels.ConclusionsOur study demonstrates that the compound heterozygous mutations T2C and 1149insT cause the "loss-of-function" of the lks that may account for the clinical phenotype of the proband. Multiple mechanisms have been involved in the pathogenesis of“loss-of-function”of lks.