Humans ; Mutation ; NAV1.5 Voltage-Gated Sodium Channel/genetics*

Cardiomyopathy, Dilated ; genetics ; Humans ; NAV1.5 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics

OBJECTIVETo investigate the association of desmoplakin with the distribution and function of Nav1.5 by RNA silencing technology in HL-1 cells.

METHODSHL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav1.5 protein expressions by Western blotting, and the distribution and co-location of desmoplakin and Nav1.5 protein were detected by immunofluorescence staining. Patch-clamp recording was applied to analyze the changes in whole-cell sodium current after desmoplakin silencing.

RESULTSCompared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav1.5 proteins. Co-localization of desmoplakin and Nav1.5 was detected at cell-cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav1.5 with decreased peak current density (156.3∓6.2 vs 41.8∓3.1, n=6, P<0.05), a shift in voltage dependence of steady-state inactivation (-42 mV vs -61 mV, n=5, P<0.05), and prolonged time of recovery from inactivation.

CONCLUSIONDesmoplakin silencing caused redistribution of Nav1.5 protein and also changes in its electrophysiological properties in HL-1 cells.

Animals ; Cell Line ; Desmoplakins ; genetics ; metabolism ; Gene Silencing ; Mice ; Mutation ; Myocytes, Cardiac ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism

OBJECTIVEThree long QT syndrome(LQTS) pedigrees were brought together for genetic diagnosis by using short tandem repeat(STR) markers.

METHODSGenomic DNA was extracted from blood samples. STR markers (D7S1824, D7S2439, D7S483, D3S1298, D3S1767, D3S3521) in or spanning the HERG and SCN5A gene were amplified; the haplotype analysis for LQTS was performed.

RESULTSClinical diagnosis showed that 15 are LQTS patients (3 died) and 11 are probable patients. Linkage analysis showed that LQTS patients are linked with the SCN5A gene in family 1, HERG is linked with the disease in family 2 and 3. Fourteen gene carriers were identified, 2 patients and 7 probable patients were excluded.

CONCLUSIONLinkage analysis using STR markers can serve as useful tool for presymptomatic diagnosis.

ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; Female ; Genetic Linkage ; Haplotypes ; Humans ; Long QT Syndrome ; genetics ; Male ; NAV1.5 Voltage-Gated Sodium Channel ; Pedigree ; Potassium Channels ; genetics ; Potassium Channels, Voltage-Gated ; Sodium Channels ; genetics ; Tandem Repeat Sequences

OBJECTIVEBrugada syndrome is an inherited channelopathy that characterized by ST-segment elevation in the right precordial lead (V(1)-V(3)) on the electrocardiogram with or without right bundle branch block and related with high risk of sudden cardiac death and structurally normal hearts. The first and only gene linked to this disease is SCN5A, a gene encodes for alpha subunit of the cardiac sodium channel. The objective of this study is to explore SCN5A gene mutations in Chinese patients with Brugada syndrome.

METHODSFour patients diagnosed as Brugada syndrome and nine patients with suspected Brugada syndrome were chosen for the study. The exons in the functional regions of SCN5A gene were amplified with polymerase chain reaction and the amplified products were sequenced with Sanger method. If a mutation was identified, patient's family members were also screened.

RESULTSTwo heterozygous mutations were found in one family diagnosed as Brugada syndrome. One missense mutation was a G-->A transition in the first nucleotide of codon 95 in SCN5A gene exon 3, which was predicted to result in substitution of Valine with Isoleucine (V95I). The other missense mutation was a C-->T transition in the second nucleotide of codon 1649 in SCN5A gene exon 28, which was predicted to result in substitution of Alanine with Valine (A1649V). A heterozygous mutation was identified in one family suspected to have the disease. The mutation was a three nucleotides (TCT) deletion that caused Phenylalanine deletion in codon 1617 in SCN5A gene exon 28. The three mutations were not detected in 100 control chromosomes.

CONCLUSIONSMutation in SCN5A gene is one of the causes of Brugada syndrome in Chinese. Three novel SCN5A gene mutations were identified in Chinese with Brugada syndrome, which expands the spectrum of SCN5A mutations associated with the disease.

Adolescent ; Adult ; Aged ; Brugada Syndrome ; genetics ; Case-Control Studies ; Exons ; genetics ; Humans ; Male ; Middle Aged ; Muscle Proteins ; genetics ; Mutation ; NAV1.5 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics

OBJECTIVEBrugada syndrome is linked to sodium channel mutations and could induce arrhythmias that even lead to sudden death. The purpose of this study was to detect if there was gene mutation of SCN5A in 7 patients with Brugada syndrome and explore the molecular genetic characteristics of this disease.

METHODGenomic DNA was extracted from peripheral blood of all 7 patients with Brugada syndrome and 41 pairs of PCR primers were designed to amplify all the 28 exons of SCN5A.

RESULTThere was no novel mutation in exons of Gene SCN5A in these patients with Brugada syndrome.

CONCLUSIONBrugada syndrome might associated gene mutation or other mechanisms independent of SCN5A gene mutation.

Adult ; Brugada Syndrome ; genetics ; DNA Mutational Analysis ; Exons ; Humans ; Male ; Middle Aged ; Muscle Proteins ; genetics ; Mutation ; NAV1.5 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics

BACKGROUNDMutations in the cardiac sodium channel gene (SCN5A) may lead to a broad spectrum of familial arrhythmias, including long QT syndrome (LQTS), idiopathic ventricular fibrillation (IVF), and isolated cardiac conduction diseases. Recent studies have shown that polymorphisms in the SCN5A gene also play an important role in the manifestation of disorders involving cardiac excitability. In this study, we investigated the polymorphisms of the SCN5A gene in Han Chinese and its relation to Brugada syndrome (BS).

METHODSGenomic DNA was isolated from 120 unrelated healthy volunteers and 48 unrelated Brugada syndrome patients by means of standard procedures. All exons including the putative splicing sites of the SCN5A gene were amplified by PCR and sequenced directly or after subcloning using an ABI Prism 377 DNA sequencer.

RESULTSA total of 5 single nucleotide polymorphisms (SNPs) were identified in the Han Chinese population, including 3 novel ones: G87A(A29A), 4245 + 82A > G, and G6174A. The allele frequencies of each SNP in the Han Chinese population were as follows: G87A (A29A) 27.5%, A1673G (H558R) 10.4%, 4245 + 82A > G 32.8%, C5457T (D1819D) 41.3%, and G6174A 44.9%. S1102Y and 10 other SNPs identified in other ethnic populations were not detected in this study. There was no significant difference in the allele frequency of A1673G (H558R) between different ethnic populations (all P > 0.5). On the other hand, the allele frequency of C5457T (D1819D) among Han Chinese was similar to its frequency among Japanese (P > 0.5), but higher than that among Americans (P < 0.005). The allele G1673 (R558) was over-represented in BS patients compared to controls (P < 0.005), but there was no significant difference in genotype frequencies at this locus. There were also no differences in either the allele or genotype frequencies of the 4 other identified SNPs when comparing BS patients with healthy controls.

CONCLUSIONSThe distribution of SCN5A SNPs may vary between different ethnicities. The polymorphism of A1673G might be associated with BS and may contribute to a susceptibility to BS in Han Chinese.

Case-Control Studies ; China ; ethnology ; Gene Frequency ; Humans ; NAV1.5 Voltage-Gated Sodium Channel ; Polymorphism, Single Nucleotide ; Sodium Channels ; genetics ; Syndrome ; Ventricular Fibrillation ; genetics

OBJECTIVEIn this study we investigated the functional restoration of nonsense mutations in the SCN5A gene.

METHODSThe readthrough-enhancing reagents were introduced to HEK293 cells to suppress one nonsense mutation W822X in the SCN5A gene. Patch-clamp was used to record the whole-cell current and dynamics. Western blot and immunofluorescence staining were used to certify the expression and the location of the sodium channel.

RESULTSIn transfected HEK293 cells, the nonsense mutation in SCN5A inhibited the expression level of full-length protein, and the sodium currents from the mutant channels were less than 3% of the wild-type level. Readthrough enhancement by decreasing translation termination efficiency with a siRNA targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1), the sodium current from the mutant cDNAs was restored to as much as 30% of the wild-type. After the treatment by the readthrough-enhancing reagents, the channels from cDNA carrying W822X remained the features of wild-type phenotype, and Western blot and immunochemical staining also showed the expression of full-length channel proteins.

CONCLUSIONReadthrough-enhancing reagents could effectively suppress nonsense mutations in SCN5A and partially restore the function of sodium channel and the expression of full-length channels.

Codon, Nonsense ; HEK293 Cells ; Humans ; NAV1.5 Voltage-Gated Sodium Channel ; Patch-Clamp Techniques ; Plasmids ; RNA, Small Interfering ; Sodium Channels ; genetics ; metabolism ; Transfection

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.
Animals ; Calcium Channels, L-Type ; chemistry ; genetics ; metabolism ; Channelopathies ; genetics ; metabolism ; Humans ; Mutation ; NAV1.1 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; Protein Domains ; Rabbits ; Structure-Activity Relationship

OBJECTIVETo elucidate the molecular and electrophysiological mechanisms of Brugada syndrome through functional analysis of a novel SCN5A gene mutation G1712C.

METHODSA recombinant plasmid pRcsodium channel α subunit (hH1) cDNA was constructed using in vitro PCR-based site-directed mutagenesis technique. LipofectamineTM 3000 was used to transfect the plasmid DNA into HEK293 cell line to induce stable expression of Nachannel β1-subunit, and the positive colonies were selected by screening with G418.The standard liposome method was used to transiently transfect HEK293 cells with either the wild-type or mutant Nachannel subunits (hH1 and mhH1, respectively), and the macroscopic Nacurrents were recorded using whole-cell patch-clamp technique. Data acquisition and analysis, generation of voltage commands and curve fitting were accomplished with EPC-10, PatchMaster and IGOR Pro 6.0.

RESULTSAn HEK293 cell line that stably expressed Nachannel β1-subunit was successfully established. After transient transfection with the WT subunit, large Nacurrents were recorded from the stable β1-cell line. Transient transfection with the G1712C subunit, however, did not elicit a Nacurrent in the cells.

CONCLUSIONCompared with normal Nachannel, the wild-type channel exhibits a similar sodium current. The characteristic kinetics of sodium channel of WT-hH1 was identical to that in normal cardiac muscle cell, and the missense mutation (G1712C) in the P-loop region of the domain IV may have caused the failure of sodium channel expression.

Brugada Syndrome ; genetics ; Genotype ; HEK293 Cells ; Humans ; Mutagenesis, Site-Directed ; Mutation ; NAV1.5 Voltage-Gated Sodium Channel ; genetics ; Patch-Clamp Techniques ; Polymerase Chain Reaction ; Transfection