Novel missense mutation in the cyclic nucleotide-binding domain of HERG causes long QT syndrome.

Autosomal-dominant long QT syndrome (LQT) is an inherited disorder, predisposing affected individuals to sudden death from tachyarrhythmias. To identify the gene(s) responsible for LQT, we identified and characterized an LQT family consisting of 48 individuals. DNA was screened with 150 microsatellite polymorphic markers encompassing approximately 70% of the genome. We found evidence for linkage of the LQT phenotype to chromosome 7(q35-36). Marker D7S636 yielded a maximum lod score of 6.93 at a recombination fraction (theta) of 0.00. Haplotype analysis further localized the LQT gene within a 6.2-cM interval. HERG encodes a potassium channel which has been mapped to this region. Single-strand conformational polymorphism analyses demonstrated aberrant bands that were unique to all affected individuals. DNA sequencing of the aberrant bands demonstrated a G to A substitution in all affected patients; this point mutation results in the substitution of a highly conserved valine residue with a methionine (V822M) in the cyclic nucleotide-binding domain of this potassium channel. The cosegregation of this distinct mutation with LQT demonstrates that HERG is the LQT gene in this pedigree. Furthermore, the location and character of this mutation suggests that the cyclic nucleotide-binding domain of the potassium channel encoded by HERG plays an important role in normal cardiac repolarization and may decrease susceptibility to ventricular tachyarrhythmias.

Recent advances in the Laboratory of Molecular and Cellular Cardiology.

This article highlights some of the research in cardiac molecular biology in progress in the Department of Cardiology at Children's Hospital. It provides a sampling of investigative approaches to key questions in cardiovascular development and function and, as such, is intended as an overview rather than a comprehensive treatment of these problems. The featured projects, encompassing four different "model" systems, include (1) genetic analysis of the mef2 gene required for fruit fly cardial cell differentiation, (2) cardiac-specific homeobox factors in zebrafish cardiovascular development, (3) mouse transgenic and gene knockout models of cardiac potassium ion channel function, and (4) mapping and identification of human gene mutations causing long QT syndrome.

RNA-stimulated ATPase activity of eukaryotic initiation factors.

Previously, we have described an ATP-dependent recognition and binding of mRNA by eukaryotic initiation factors (eIF)-4A, eIF-4B, and eIF-4F (Grifo, J. A., Tahara, S. M., Leis, J. P., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1982) J. Biol. Chem. 257, 5246-5252; Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810). This finding was consistent with other studies which implicated eIF-4A and eIF-4B in binding mRNA to the 40 S ribosomal subunit, an ATP-requiring process. As part of ongoing studies of this step, and, in particular its ATP requirement, we have examined ATPase activity of various initiation factors. In this communication we describe an RNA-dependent ATP hydrolysis catalyzed by eIF-4A and eIF-4F. Although eIF-4B has little or no ATPase activity it can stimulate the RNA-dependent ATPase activity of either eIF-4A or eIF-4F. Similar to the ATP-dependent mRNA binding assay, the RNA-dependent ATPase activity is inhibited by the cap analogue m7GDP when globin mRNA is used as the activator. In addition, a variety of polynucleotides stimulate the ATPase activity of these factors including rRNA, tRNA, poly(U), and poly(A) but not poly(dA). Finally, an attempt has been made to discern whether phosphorylation or ATP hydrolysis is responsible for the ATP-stimulated binding of mRNA by eIF-4A and eIF-4B. We present evidence which is consistent with the interpretation that ATP hydrolysis and not protein phosphorylation correlates with ATP-stimulated binding of mRNA.

Multiple different missense mutations in the pore region of HERG in patients with long QT syndrome.

Long QT syndrome (LQTS), is an inherited cardiac disorder in which ventricular tachyarrhythmias predispose affected individuals to syncope, seizures, and sudden death. Characteristic electrocardiographic findings include a prolonged QT interval, T wave alternans, and notched T waves. We have screened LQTS patients from 89 families for mutations in the pore region of HERG , the K+ channel gene previously associated with chromosome 7-linked LQT2. In six unrelated LQTS kindreds, single-strand conformation polymorphism analyses identified aberrant conformers in all affected family members. These conformers were not seen in over 100 unaffected, unrelated control individuals, suggesting that they represent pathogenic LQTS mutations. DNA sequence analyses of the aberrant conformers demonstrated that they reflect five different missense mutations: V612L, A614V, N629D, N629S, and N633S. The missense mutation A614V was found in two unrelated families. Further functional studies will be required to determine what effect each of these changes may have on HERG channel function.

Missense mutation in the pore region of HERG causes familial long QT syndrome.

BACKGROUND: Long QT syndrome (LQT) is an inherited cardiac disorder that results in syncope, seizures, and sudden death. In a family with LQT, we identified a novel mutation in human ether-a-go-go-related gene (HERG), a voltage-gated potassium channel. METHODS AND RESULTS: We used DNA sequence analysis, restriction enzyme digestion analysis, and allele-specific oligonucleotide hybridization to identify the HERG mutation. A single nucleotide substitution of thymidine to guanine (T1961G) changed the coding sense of HERG from isoleucine to arginine (Ile593Arg) in the channel pore region. The mutation was present in all affected family members; the mutation was not present in unaffected family members or in 100 normal, unrelated individuals. CONCLUSIONS: We conclude that the Ile593Arg missense mutation in HERG is the cause of LQT in this family because it segregates with disease, its presence was confirmed in three ways, and it is not found in normal individuals. The Ile593Arg mutation may result in a change in potassium selectivity and permeability leading to a loss of HERG function, thereby resulting in LQT.

Evidence of genetic heterogeneity in Romano-Ward long QT syndrome. Analysis of 23 families.

BACKGROUND: The Romano-Ward long-QT Syndrome (LQTS) is an autosomal dominant inherited trait characterized by prolonged QT interval on ECG, life-threatening arrhythmias, syncope, and sudden death in affected individuals. A gene responsible for this disorder has been shown to be linked to the Harvey ras-1 locus (H-ras-1) DNA marker on the short arm of chromosome 11 (11p) in 7 families. The purpose of this study was to determine, by analyzing 23 families with LQTS for linkage to chromosome 11p, whether evidence exists for more than one gene causing LQTS (ie, locus heterogeneity). METHODS AND RESULTS: Twenty-three families (262 family members) were clinically evaluated using medical histories, ECGs, and Holter recordings. Each corrected QT interval (QTc) were determined using Bazett's formula. Blood for DNA extraction and cell line immortalization was obtained after informed consent. Southern blotting and polymerase chain reaction were performed, and linkage analysis carried out using the LINKAGE computer program (v 5.03). Genetic heterogeneity was determined using the HOMOG 2 (v 2.51) computer program. Twenty-three families were studied for evidence of linkage to chromosome 11p using the H-ras-1 locus probe pTBB-2 and multiple flanking markers, including tyrosine hydroxylase (TH). Two-point linkage analysis using pTBB-2 and TH markers was consistent with linkage in 15 of 23 families, with the maximum single-family LOD score of +3.038 occurring at theta = 0. However, 8 of 23 families had negative LOD scores, with the values in 4 families being less than -2 at theta = 0, consistent with exclusion of linkage. Analysis with the HOMOG program was consistent with genetic heterogeneity (P < .0001). Multipoint linkage data using pTBB-2 and TH were also examined for evidence of heterogeneity. HOMOG analysis of multipoint LOD scores from 100 cM surrounding the H-ras-1 locus also supported heterogeneity (P < .001). CONCLUSIONS: In the 23 families with LQTS analyzed for linkage to the H-ras-1 locus on chromosome 11p15.5, 15 of 23 families had LOD scores consistent with linkage. The remaining 8 of 23 families had negative LOD scores, 4 of which were definitively excluded from linkage. Thus, genetic heterogeneity is definitively (P < .001) demonstrated for this disorder.

Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current.

HERG, the human ether-a-go-go-related gene, encodes a K(+)-selective channel with properties similar to the rapidly activating component of the delayed rectifier K+ current (IKr). Mutations of HERG cause the autosomal-dominant long-QT syndrome (LQTS), presumably by disrupting the normal function of IKr. The current produced by HERG is not identical to IKr, however, and the mechanism by which HERG mutations cause LQTS remains uncertain. To better define the role of Erg in the heart, we cloned Merg1 from mouse genomic and cardiac cDNA libraries. Merg1 has 16 exons and maps to mouse chromosome 5 in an area syntenic to human chromosome 7q, the map locus of HERG. We isolated three cardiac isoforms of Merg1: Merg1a is homologous to HERG and is expressed in heart, brain, and testes, Merg1a' lacks the first 59 amino acids of Merg1a and is not expressed abundantly, and Merg1b has a markedly shorter divergent N-terminal cytoplasmic domain and is expressed specifically in the heart. The Merg1 isoforms, like HERG, produce inwardly rectifying E-4031-sensitive currents when heterologously expressed in Xenopus oocytes. Merg1a and HERG produce currents with slow deactivation kinetics, whereas Merg1a' and Merg1b currents deactivate more rapidly. Merg1b coassembles with Merg1a to form channels with deactivation kinetics that are more rapid than those of Merg1a or HERG and nearly identical to IKr. In addition, a homologue of Merg1b is present in human cardiac and smooth muscle. Thus, we have identified a novel N-terminal Erg isoform that is expressed specifically in the heart, has rapid deactivation kinetics, and coassembles with the longer isoform in Xenopus oocytes. This N-terminal Erg isoform may determine the properties of IKr and contribute to the pathogenesis of LQTS.