The long QT syndrome (LQT) is an inherited cardiac disorder that can cause sudden cardiac death among apparently healthy young individuals due to malignant ventricular arrhythmias. LQT was found to be caused by mutations in four genes LQT1, LQT2, LQT3 and LQT5, and linkage was reported for an additional locus, LQT4, on chromosome 4q25-27. We have studied a large (n=131) LQT-affected Jewish kindred and identified tight linkage between the LQT-affected status and LQT3 (lod score 6.13, with an estimated recombination fraction of zero). We identified a new point-mutation, A to G substitution at nucleotide 5519 of the SCN5A gene, changing the aspartate 1840 to glycine, D1840G. This is a non-conservative change of an amino acid completely conserved in sodium channels from Molusca to human. The mutation was identified in all affected individuals (n=23), and not identified in all the unaffected family members (n=40), and not in 200 chromosomes of healthy control individuals. The mutation was identified in 3/12 individuals with equivocal phenotype, thus, providing an accurate diagnostic tool for all family members. This mutation is currently being used in a cellular electrophysiological model, to characterize the function of the mutated sodium channel in this syndrome. Β©1997 Wiley-Liss, Inc.
Article
The long QT syndrome (LQT) is a familial, autosomal dominant cardiac disorder characterized by recurrent syncope and sudden cardiac death among apparently healthy young individuals. The phenotypic hallmark of this disorder is delayed ventricular repolarization depicted as a prolonged QT interval on 12-lead ECG. LQT is genetically heterogeneous, and is caused by mutations in four genes LQT1, LQT2, LQT3 and LQT5 (Wang et al. 1995; Curran et al. 1995;Wang et al., 1996, Splawski et al., 1997. Linkage was reported for an additional locus, LQT4 on chromosome 4q25-27 (Schott et al., 1995). The aim of this study was to identify the molecular basis for LQT in a large LQT-affected Jewish family that has been previously reported not to be linked to the LQT1 locus (Benhorin et al., 1993).
Linkage analysis (Hasstedt 1989) with the D3S1100 marker (Jiang et al., 1994) showed that the LQT-affected status in this family is tightly linked to the D allele of this LQT3 marker (Figure 1), with a lod score of 6.13, and an estimated recombination fraction of zero, allowing 0.95 penetrance. SSCP analyses of exons 19, 22 and region B of exon 24, in which mutations have been previously identified (Wang et al., 1995a; Wang et al., 1995b) showed same banding pattern in both affected and non-affected individuals, suggesting that the molecular basis of LQT in this family is different from that identified in other LQT3 associated families. Analyses of exons 7, 9, 16, 18, 21, and 23 also revealed an identical patterns in affected and unaffected family members (data not shown). However, SSCP analysis of region A of exon 24 revealed a different banding pattern. Direct sequencing of a PCR product of this region, from one of the affected individuals revealed two base substitutions. One is a C->T substitution at nucleotide 5607 of the cDNA which is a polymorphism (Wang et al., 1995b), and the other is an A->G substitution at nucleotide 5519 of the cDNA which is expected to cause a non-conservative change of the aspartate at position 1840 to glycine, D1840G (based on sequence data from GENEBANK accession number AF007781). All family members for whom DNA was available (40 unaffected, 23 affected and 12 with equivocal phenotype) were then analyzed for the D1840G mutation by a specifically designed ASO hybridization test using 5' GAGGACGACTTCGATA 3' for detection of the normal allele, and 5' GAGGACGGCTTCGATA 3' for detection of the mutant allele. All LQT-affected individuals carried the D1840G mutation, whereas, all unaffected individuals did not. Among the 12 individuals with an equivocal phenotype three carried the mutation. One female, phenotypically defined as unaffected (mean QTc. of 0.440, from 5 different ECG tracings, registered over 9 years), who did not have any other non-ECG criteria for LQT, inherited the affected LQT3 allele in this family (Figure 1, marked by an asterisk) and the D1840G mutation. Following the linkage analysis two additional ECGs showed QTc of 0.460 sec, which according to our criteria indicated an equivocal affected status (Benhorin et al., 1993). The mutation was not found in four additional LQT-affected Jewish families which were too small for linkage analysis and not in 200 chromosomes of healthy unrelated control individuals. The D1840G mutation results in a substitution of aspartate to glycine at position 1840. This aspartate residue is completely conserved in sodium channels from Mollusca to human (Table 1), further suggesting that D1840G is likely to be a mutation causing disease, and not a polymorphism.
The identification of a new mutation in the studied family is not surprising since the different Jewish subgroups tended to live as relatively isolated populations until recent times, thus, specific genetic defects are expected to be found in their gene-pool. Indeed, a variety of genetic diseases have a significantly higher prevalence among Jewish than non-Jewish populations. Moreover, a specific repertoire of mutations causing common diseases in the general population has been reported among Jews (Kerem et al.,).
Diagnosis of LQT is a complicated issue since symptoms are found in only a minority of affected individuals with prolonged QTc. In the studied family all living affected patients are asymptomatic but one. Thus, the phenotypic definition was based on a large electrocardiographic database (Merri et al., 1989;Benhorin et al., 1990;Benhorin et al., 1993) which also considers age and gender differences. The mutation analysis in the studied family shows that the phenotypic definition scheme was quite accurate. The individual whose phenotypic status was changed from unaffected to equivocal during the course of this study represents a highly unusual occurrence of a QTc change over time, the reason for which is unclear.
In the studied family there were 12 individuals with equivocal phenotypic diagnosis, QTc's 0.440-0.463 sec, three of whom were found to carry the mutation. Their QTc's (0.440, 0.450, 0.460 sec) were similar to those of the other nine individuals from this subgroup who did not carry the mutation. Thus, in these 12 individuals a definitive diagnosis could have been made only by the mutation analysis. The D1840G mutation is currently being used in a cellular electrophysiological model, to characterize the function of the mutated sodium channel in this syndrome.
The SCN5A gene encodes the alpha subunit of the cardiac sodium channel which is responsible for the initial upstroke of the action potential, and also contributes small depolarizing currents during the plateau phase. Therefore, improper inactivation of this channel during the plateau phase is expected to cause delayed repolarization which is the electrophysiological hallmark of LQT (Gellens et al., 1992). The location of the mutation is not near a region previously-shown to affect the inactivation gating of the channel (Stuhmer et al.,