KCNJ5 Gly387Arg Mutation Responsible For Familial Long QT Syndrome

Background Sudden cardiac death is a major life-threatening disorder most frequently encountered in clinical practice with an overall incidence in the general population estimated to be between 0.1%and 0.2%, resulting in approximately 300,000 to 400,000 deaths annually in the United States. Sudden cardiac death in an otherwise healthy young individuals and the lack of an apparent etiology in many of those victims initially led to the classification as"sudden unexplained death syndrome"or"sudden infant death syndrome". In most cases, idiopathic polymorphic ventricular tachycardia described as"Torsade de pointes"(TdP) which referred to the characteristic rotation of the electrical axis along an imaginary iso-electric line on the surface electrocardiogram or ventricular fibrillation (VF) was gradually recognized as the primary trigger, and the underlying pathogenic determinants resulting in TdP or VF were lengthened cardic repolarization characterized by abnormal QT-interval prolongation on the surface electrocardiogram, that is, long-QT syndrome. Long-QT syndrome predisposes subjects to syncope, seizures, and sudden death resulted from TdP or VF. Long-QT syndrome is generally classified into two categories, acquired or idiopathic. In addition to inheritable susceptiblity, acquired long-QT syndrome is often attributable to local myocardial ischemia, imbalanced electrolytes, or administered medications and may be easily cured by getting rid of such risk factors. However, idiopathic long-QT syndrome has long been focus of medical research because of more complex mechanism and no efficacious mesures. In 1957, Jervell and Lange–Nielsen made the first report on a familial disease characterized by a striking prolongation of the QT-interval, congenital deafness and a high incidence of sudden cardiac death at young age . Subsequently, an almost identical disorder whereas lacking the trait of sensorineural deafness was identified by Romano and Ward. At present, Hundreds of mutations in 10 distinct genes accountable for a hereditary form of long-QT syndrome has been identified: KCNQ1 (KvLQT1, type 1 long-QT syndrome, LQT1), KCNH2 (hERG, LQT2), SCN5A (Nav1.5, LQT3), ANK2 (Ankyrin-B, LQT4), KCNE1 (MinK, LQT5), KCNE2 (MiRP1, LQT6), KCNJ2 (Kir2.1, LQT7), CACNA1C (Cav1.2, LQT8), CAV3 (Caveolin-3, LQT9) and SCN4β(Navβ4, LQT10). Overall, 8 of these genes encode proteins that are specifically involved in cardiac action potential generation, whereas ANK2 and CAV3 encoding non-channel proteins are considered to be more general-purpose. The main mechanism by which these mutated genes give rise to long-QT syndrome is an excessive and heterogeneous lengthening of the repolarization phase of the ventricular action potential. Nevertheless, the identified genetic defects only account for approximately 80 percent of congenital long-QT syndrome and the long-QT-associated genes in about 20 percent of cases remain to be identified. Furthermore, the accumulating evidence reveals that long-QT syndrome is of remarkable both genetical and clinical heterogeneity ranging from diverse culprit genes to different mutations of an identical gene in various patients with long-QT syndrome or from asymptome to syncope, even to sudden death in patients with the same mutation of a gene responsible for long-QT syndrome. Therefore, These findings highlight the need for a comprehensive genetic screening of unrelated patients, especially of familial ones with long-QT syndrome, for a novel causal gene.Objectives The aims of the present investigation were to map a novel locus linked to long-QT syndrome to a certain region on chromosome, to identify a novel gene responsible for long-QT syndrome by performing a systematic screening of selected candidate genes in a located position, and further to explore the molecular basis for long-QT syndrome. These researches may pave the way for early diagnosis, prognostic appraisal, prophylactic consultation, and gene-specific therapy.Methods As many as possible unrelated families with congenital long-QT syndrome were identified in China according to a set of diagnostic criteria proposed by Schwartz and colleagues and a cohort of around 500 unrelated healthy subjects as control were recruited from the same Chinese population. The diagnostic criteria for long-QT syndrome principally consist of a positive family history, clinical history, and electrocardio- graphic findings, and exclusion of organic heart diseases and other identified causative factors. The clinical data including medical records, electrocardiograms and echocardiography reports were collected. The genomic DNA from all participants, including the probands and all consenting family members, and controls was extracted from peripheral venous blood lymphocytes by use of Wizard Genomic DNA Purification Kit. After ruling out the known 10 long-QT syndrome-associated genes by polymerse chain reaction (PCR)-sequencing or linkage analysis of 3 to 5 of microsatellite markers near a gene, incliding KCNQ1,KCNH2,SCN5A,Ankyrin B,KCNE1,KCNE2,KCNJ2,CACNA1C,CAV3, and SCN4B, we performed the whole-genome scanning of microsatellites mainly from ABI PRISM Linkage Mapping Sets v2.5, genotyping on MegaBACE 500, analysis of haplotypes, and linkage analysis with software SAGE in order to map a novel gene linked to long-QT syndrome to a small region on a chromosome. The preferred candidate genes in the located region were established by bioinformatical knowledge and the entire coding sequences of selected candidate genes were screened in probands for mutations by direct PCR-sequencing and sequence alignment in order to identify a novel mutated gene leading to long-QT syndrome. The resultant mutant genes identified were consequently targeted to detect in all relevant family members available and 500 unrelated subjects as controls to evaluate their prevalence in different populations. The conservation of amino acid residues altered by the identified mutations was determined by aligning protein homologs and orthologs among species. The total mRNA was extracted from human myocardium with TRIzol. The full-length wild-type cDNA of human KCNJ5 identified was obtained by reverse transcription (RT) - PCR using pfuUltraTM high- fidelity DNA polymerase and inserted into the oocyte expression plasmid pGEM-T with T4 DNA ligase. Mutation was introduced into a wild-type KCNJ5 clone with the use of a Quick Change? II XL Site-Directed Mutagenesis Kit. The wild-type and mutant KCNJ5-pGEM-T constructs were corroborated by sequencing prior to subsequent experiments. The appropriate wild-type and mutant recombinant vector KCNJ5-pGEM-T were transfected and cloned to obtain sufficient KCNJ5-pGEM-T for physiological research. The wild-type and mutant KCNJ5 cRNA was synthesized by standard in vitro run-off transcription from plasmid KCNJ5-pGEM-T using the T7 mMessage Machine kit in terms of manufacturer's instructions. Oocytes were surgically removed from anaesthetized X. laevis frogs and defolliculated enzymatically as described previously. The wild-type and mutant KCNJ5 cRNA was mocroinjected respectively or together in equal molar ratios into the prepared oocytes (0.5-2 ng / oocyte). The injected oocytes were kept in a low K solution. Whole-cell currents were mesured 24-48 hours after KCNJ5 cRNAinjection by two-electrode voltage-clamp amplifier. Pipettes were pulled from borosilicate glass and had a final tip resistance of 0.5-2.5 MΩwhen filled with 3M KCl and submerged in low K solution. Currents were measured in a high K solution. Data were sampled with pulse and analyzed using the Igor software and GraphPad software. All data are presented as mean±standard error of the mean. For statistical analyses t tests as well as one way ANOVAcombined with Tukey's mutiple comparison test were used. P < 0.05 was considered to be significant.Results A large kindreds with familial long-QT syndrome were identified in China and a cohort of 500 unrelated healthy subjects as ethnically matched control were recruited from the same Chinese population. Peripheral venous blood specimens were prepared and genomic DNA was extracted from lymphocytes. The clinical data including medical records, electrocardiograms and echocardiography reports were collected. The known 10 long-QT syndrome-associated genes were rule out from the present famiy with idiopathic long-QT syndrome. The genotypes of these family members were determined and the haplotype was constructed. By linkage analysis a novel locus linked to long-QT syndrome was finely mapped to a small region on the 11th chromosome, i.e., 11q23-24, roughly 5.34cM between microsatellite markers D11S990 and D11S4123, with a maximal two-point LOD score of 5.4183 for D11S4123 marker atθ=0.00. Two genes of KCNJ1and KCNJ5 located at the mapped chromosomal region were preferred by bioinfor- matical knowledge. A novel heterozygous missense mutation was identified in KCNJ5 from the proband representative of studied pedigree with long-QT syndrome. Rather, a 1473 G C mutation (accession number, NM₀00890) at nucleic acid level, predicting the substitution of arginine for glycine at codon 387 (Gly387 Arg) at amino acid level, was identified in the index case of the family. The mutation was located at the functionally important C-terminus of the KCNJ5-encoded ion channel protein. The same mutation was present in all the affected family members but absent in 500 matched controls. Additionally, no mutation in KCNJ1 was detected in the representative of this pedigree. A cross-species alignment of KCNJ5 - encoded Kir3.4 ion channel protein sequence displayed the mutaed amino acid was highly conserved evolutionarily among comprehensive species, which indicated rudime- ntarily that the mutation Gly387 Arg might be a pathogenic component rather than a rare benign molecular polymorphism. The human KCNJ5 gene was cloned and the mutation was introduced into it successfully by site directed PCR. The wild-type and mutant KCNJ5-pGEM-T recombine- ant constructs were constructed and substan- tiated by enzymatic analysis and direct PCR- sequencing. The appropriate recombinant constructs were amplified and sufficient cRNA of both wild-type and mutant KCNJ5 was synthesized in vitro. Enough oocytes were surgically acquired from anaesthetized toads. Electrophysiological analysis of the ion channels expressed in the oocytes surviving microinjection of cRNA revealed significant effect of the mutation on the the activity of inward rectifier potassium channel (Kir3.4) in contrast to its wild-type counterpart. At -50 mV, the average current was -23.2±1.2 pA/pF (n=11) and -8.5±0.8 pA/PF (n=8) respectively for the mutant and wild-type Kir3.4 (p<0.05); It was 15.2±1.1 pA/pF (n=11) for the mutant Kir3.4 and 5.1±0.5 pA/pF (n=8) for the wild-type Kir3.4 at -20mV (p<0.05). These experimental findings validate the pathogenic link between compromised Kir3.4 function and susceptibility to long-QT syndrome.Conclusions A novel locus linked to long-QT syndrome is mapped to 11q23-24, which is equivalent to a chromosomal region of about 5.34cM between microsatellite markers D11S990 and D11S4123. A novel gene, namely KCNJ5, responsible for long-QT syndrome was subsequently identified. KCNJ5 Gly387Arg mutation imposes remarkable effect on Kir3. 4 channel currents and may predispose individuals to long-QT syndrome. Our data establishes KCNJ5 as an 11th gene responsible for long-QT syndrome, indicating implications for genetic diagnosis, genetic counseling, and gene-specific therapy.