Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias.

Genetic factors contribute to the risk of sudden death from cardiac arrhythmias. Here, positional cloning methods establish KVLQT1 as the chromosome 11-linked LQT1 gene responsible for the most common inherited cardiac arrhythmia. KVLQT1 is strongly expressed in the heart and encodes a protein with structural features of a voltage-gated potassium channel. KVLQT1 mutations are present in affected members of 16 arrhythmia families, including one intragenic deletion and ten different missense mutations. These data define KVLQT1 as a novel cardiac potassium channel gene and show that mutations in this gene cause susceptibility to ventricular tachyarrhythmias and sudden death.

Fast inactivation causes rectification of the IKr channel.

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current-voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.

Potassium ion channels and human disease: phenotypes to drug targets?

A significant difficulty faced by the pharmaceutical industry is the initial identification and selection of macromolecular targets upon which de novo drug discovery programs can be initiated. A drug target should have several characteristics: known biological function; robust assay systems for in vitro characterization and high-throughput screening; and be specifically modified by and accessible to small molecular weight compounds in vivo. Ion channels have many of these attributes and can be viewed as suitable targets for small molecule drugs. Potassium (K+) ion channels form a large and diverse gene family responsible for critical functions in numerous cell types, tissues and organs. Recent discoveries, facilitated by genomics technologies combined with advanced biophysical characterization methods, have identified novel K+ channels that are involved in important physiologic processes, or mutated in human inherited disease. These findings, coupled with a rapidly growing body of information regarding modulatory channel subunits and high resolution channel structures, are providing the critical information necessary for validation of K+ channels as drug targets.

Linkage of a QTL contributing to normal variation in bone mineral density to chromosome 11q12-13.

Osteoporosis is a leading public health problem that is responsible for substantial morbidity and mortality. A major determinant of the risk for osteoporosis in later life is bone mineral density (BMD) attained during early adulthood. BMD is a complex trait that presumably is influenced by multiple genes. Recent linkage of three Mendelian BMD-related phenotypes, autosomal dominant high bone mass, autosomal recessive osteoporosis-pseudoglioma, and autosomal recessive osteopetrosis to chromosome 11q12-13 led us to evaluate this region to determine if the underlying gene(s) could also contribute to variation in BMD in the normal population. We performed a linkage study in a sample of 835 premenopausal Caucasian and African-American sisters to identify genes underlying BMD variation. A maximum multipoint LOD score of 3.50 with femoral neck BMD was obtained near the marker D11S987, in the same chromosomal region as the three Mendelian traits mentioned above. Our results suggest that the gene(s) underlying these Mendelian phenotypes also play a role in determining peak BMD in the normal population and are the first using linkage methods to establish a chromosomal location for a gene important in determining peak BMD. These findings support the hypothesis that a gene responsible for one or more of the rare Mendelian BMD traits linked to chromosome 11q12-13 has an important role in osteoporosis in the general population.

Genome screen for QTLs contributing to normal variation in bone mineral density and osteoporosis.

A major determinant of the risk for osteoporosis is peak bone mineral density (BMD), which is largely determined by genetic factors. We recently reported linkage of peak BMD in a large sample of healthy sister pairs to chromosome 11q12-13. To identify additional loci underlying normal variations in peak BMD, we conducted an autosomal genome screen in 429 Caucasian sister pairs. Multipoint LOD scores were computed for BMD at four skeletal sites. Chromosomal regions with LOD scores above 1.85 were further pursued in an expanded sample of 595 sister pairs (464 Caucasians and 131 African-Americans). The highest LOD score attained in the expanded sample was 3.86 at chromosome 1q21-23 with lumbar spine BMD. Chromosome 5q33-35 gave a LOD score of 2.23 with femoral neck BMD. At chromosome 6p11-12, the 464 Caucasian pairs achieved a LOD score of 2.13 with lumbar spine BMD. Markers within the 11q12-13 region continued to support linkage to femoral neck BMD, although the peak LOD score was decreased to 2.16 in the sample of 595 sibling pairs. Our study is the largest genome screen to date for genes underlying variations in peak BMD and represents an important step toward identifying genes contributing to osteoporosis in the general population.

Molecular cloning, characterization, and genomic localization of a human potassium channel gene.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of resting membrane potential, and development of memory and learning. In addition to their role in regulating myocyte excitability, cardiac K+ channels control heart rate and coronary vascular tone and are implicated in the development of arrhythmias. We report here the cloning and sequencing of a k+ channel gene, KCNA1, derived from a human cardiac cDNA library and the chromosomal localization of the corresponding genomic clone. Oligonucleotides based on a delayed rectifier K+ channel gene were used in PCR reactions with human genomic DNA to amplify the S4-S6 regions of several different K+ channel genes. These sequences were used to isolate clones from a human cardiac cDNA library. We sequenced one of these clones, HCK1. HCK1 contains putative S2-S6 domains and shares approximately 70% sequence homology with previously isolated Shaker homologues. HCK1 was used to screen human cosmid libraries and a genomic clone was isolated. By sequencing the genomic clones, a putative S1 domain and translation initiation sequences were identified. Genomic mapping using human-rodent somatic cell panels and in situ hybridization with human metaphase chromosomes have localized KCNA1 to the distal short arm of human chromosome 12. This work is an important step in the study of human cardiac K+ channel structure and function and will be of use in the study of human inherited disease.

Molecular characterization and refined genomic localization of three human potassium ion channel genes.

Potassium ion (K+) channels are essential for a variety of cellular functions in both excitable and non-excitable cells and are likely to be involved in the pathogenesis of some cardiovascular and neurological disorders. To be useful in candidate gene analysis of inherited diseases it is important to identify new K+ channel genes and localize these sequences on the human physical and genetic maps. Using fluorescence in situ hybridization (FISH), we mapped two new K+ channel gene containing cosmids, c2-3a and c9-2a, to chromosomes 1 and 19, respectively. Partial DNA sequencing (c2-3a) and restriction enzyme site analysis (c9-2a) established the uniqueness of each clone. We refined the localization of c2-3a, c9-2a and a previously described K+ channel gene KCNA5, (c7-2), by performing contour length measurements of hybridized metaphase chromosomes and determining the average FLpter% value (fractional length relative to the fixed reference point pter x 100%). When compared to ideograms of banded metaphase chromosomes, these FLpter% values correspond to 12p13.31-->p13.33, 1p13.1-->p21.1 and 19q13.32-->q13.33, respectively. Using FISH, each of these clones has been finely mapped to a different human chromosome indicating a significant dispersion of K+ channel sequences in the human genome.

Genomic localization of the human gene for KCNA10, a cGMP-activated K channel.

Potassium (K) channels are important components of virtually all cells, and they play critical roles in many cellular functions. KCNA10 represents a new class of K channel specifically regulated by cGMP and postulated to mediate the effects of substances that increase intracellular cGMP. Since KCNA10 has the potential to be useful in candidate gene analysis of inherited diseases, the human gene for KCNA10 was characterized. Fluorescence in situ hybridization indicates that human KCNA10 maps to chromosome 1 at p13.1-->p22.1. Finer mapping of the gene was achieved by PCR of a set of CEPH YAC clones that spanned the region of interest. We found that YAC 818b9 contains human KCNA10. These data indicate human KCNA10 maps to 1p13.1 and resides within the genetic interval defined by microsatellite loci D1S2809 and D1S2726. That region of chromosome 1 contains another K channel gene, KCNA3.

Single HERG delayed rectifier K+ channels expressed in Xenopus oocytes.

HERG is a K+ channel with properties similar to the rapidly activating component (I(Kr)) of delayed rectifier K+ current, which is important for repolarization of human cardiac myocytes. In this study, we have characterized the single-channel properties of HERG expressed in Xenopus oocytes. Currents were measured in cell-attached patches with an extracellular K concentration of 120 mM. The single HERG channel conductance, determined at test potentials between -50 and -110 mV, was 12.1 +/- 0.6 pS. At positive test potentials (+40 to +80 mV), the probability of channel opening was low and slope conductance was 5.1 +/- 0.6 pS. The mean channel open times at -90 mV were 2.9 +/- 0.5 and 11.8 +/- 1.0 ms, and the mean channel closed times were 0.54 +/- 0.02 and 14.5 +/- 5.3 ms. Single HERG channels were blocked by MK-499, a class III antiarrhythmic agent that blocks I(Kr) in cardiac myocytes. The development of block was more rapid in inside-out patches than in cell-attached patches or in whole cell recordings, indicating that block occurs from the cytoplasmic side of the membrane. The single-channel properties of HERG are similar to I(Kr) channels of isolated cardiac myocytes, which provides further evidence that HERG proteins coassemble to form I(Kr) channels.

Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides.

We recently reported that mutations in HERG, a potassium channel gene, cause long QT syndrome. Heterologous expression of HERG in Xenopus oocytes revealed that this channel had biophysical properties nearly identical to a cardiac delayed rectifier K+ current I(Kr), but had dissimilar pharmacological properties. Class III antiarrhythmic drugs such as E-4031 and MK-499 are potent and specific blockers of I (Kr) in cardiac myocytes. Our initial studies indicated that these compounds did not block HERG at a concentration of 1 micromol/L. In the present study, we used standard two-microelectrode voltage-clamp techniques to further characterize the effects of these drugs on HERG channels expressed in oocytes. Consistent with initial findings, 1 micromol/L MK-499 and E-4031 had not effect on HERG when oocytes were voltage clamped at a negative potential and not pulsed during equilibration with the drug. However, MK-499 did block HERG current if oocytes were repetitively pulsed, or clamped at a voltage positive to the threshold potential for channel activation. This finding is in contrast to previous studies that showed significant block of I(Kr) in isolated myocytes by similar drugs, even in the absence of pulsing. This apparent discrepancy may be due to differences in channel characteristics (HERG versus guinea pig and mouse I (Kr)), tissue (oocytes versus myocytes), or specific drugs. Under steady state conditions, block of HERG by MK-499 was half maximal at 123 +/- 12 nmol/L at a test potential of -20 mV. MK-499 (150 nmol/L) did not affect the voltage dependence of activation and rectification nor the kinetics of activation and deactivation of HERG. These data indicate that MK-499 preferentially blocks open HERG channels and further support the conclusion that HERG subunits form I(Kr) channels in cardiac myocytes.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel.

Mutations in HERG cause an inherited cardiac arrhythmia, long QT syndrome (LQT). To define the function of HERG, we expressed the protein in Xenopus oocytes. The biophysical properties of expressed HERG are nearly identical to the rapidly activating delayed rectifier K+ current (IKr) in cardiac myocytes. HERG current is K+ selective, declines with depolarizations above 0 mV, is activated by extracellular K+, and is blocked by lanthanum. Interestingly, HERG current is not blocked by drugs that specifically block IKr in cardiac myocytes. These data indicate that HERG proteins form IKr channels, but that an additional subunit may be required for drug sensitivity. Since block of IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provides a mechanistic link between certain forms of inherited and acquired LQT.

The elastin gene is disrupted by a translocation associated with supravalvular aortic stenosis.

To identify genes involved in vascular disease, we investigated patients with supravalvular aortic stenosis (SVAS), an inherited vascular disorder that causes hemodynamically significant narrowing of large elastic arteries. Pulsed-field gel and Southern analyses showed that a translocation near the elastin gene cosegregated with SVAS in one family. DNA sequence analyses demonstrated that the translocation disrupted the elastin gene and localized the breakpoint to exon 28. Taken together with our previous study linking SVAS to the elastin gene in two additional families and existing knowledge of vascular biology, these data suggest that mutations in the elastin gene can cause SVAS.

A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome.

To identify genes involved in cardiac arrhythmia, we investigated patients with long QT syndrome (LQT), an inherited disorder causing sudden death from a ventricular tachyarrythmia, torsade de pointes. We previously mapped LQT loci on chromosomes 11 (LQT1), 7 (LQT2), and 3 (LQT3). Here, linkage and physical mapping place LQT2 and a putative potassium channel gene, HERG, on chromosome 7q35-36. Single strand conformation polymorphism and DNA sequence analyses reveal HERG mutations in six LQT families, including two intragenic deletions, one splice-donor mutation, and three missense mutations. In one kindred, the mutation arose de novo. Northern blot analyses show that HERG is strongly expressed in the heart. These data indicate that HERG is LQT2 and suggest a likely cellular mechanism for torsade de pointes.

Direct activation of an inwardly rectifying potassium channel by arachidonic acid.

Arachidonic acid (AA) is an important constituent of membrane phospholipids and can be liberated by activation of cellular phospholipases. AA modulates a variety of ion channels via diverse mechanisms, including both direct effects by AA itself and indirect actions through AA metabolites. Here, we report excitatory effects of AA on a cloned human inwardly rectifying K(+) channel, Kir2.3, which is highly expressed in the brain and heart and is critical in regulating cell excitability. AA potently and reversibly increased Kir2.3 current amplitudes in whole-cell and excised macro-patch recordings (maximal whole-cell response to AA was 258 +/- 21% of control, with an EC(50) value of 447 nM at -97 mV). This effect was apparently caused by an action of AA at an extracellular site and was not prevented by inhibitors of protein kinase C, free oxygen radicals, or AA metabolic pathways. Fatty acids that are not substrates for metabolism also potentiated Kir2.3 current. AA had no effect on the currents flowing through Kir2.1, Kir2.2, or Kir2.4 channels. Experiments with Kir2.1/2.3 chimeras suggested that, although AA may bind to both Kir2.1 and Kir2.3, the transmembrane and/or intracellular domains of Kir2.3 were essential for channel potentiation. These results argue for a direct mechanism of AA modulation of Kir2.3.

Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel.

The slowly activating delayed-rectifier K+ current, I(Ks), modulates the repolarization of cardiac action potentials. The molecular structure of the I(Ks) channel is not known, but physiological data indicate that one component of the I(Ks), channel is minK, a 130-amino-acid protein with a single putative transmembrane domain. The size and structure of this protein is such that it is unlikely that minK alone forms functional channels. We have previously used positional cloning techniques to define a new putative K+-channel gene, KVLQT1. Mutations in this gene cause long-QT syndrome, an inherited disorder that increases the risk of sudden death from cardiac arrhythmias. Here we show that KVLQT1 encodes a K+ channel with biophysical properties unlike other known cardiac currents. We considered that K(V)LQT1 might coassemble with another subunit to form functional channels in cardiac myocytes. Coexpression of K(V)LQT1 with minK induced a current that was almost identical to cardiac I(Ks). Therefore, K(V)LQT1 is the subunit that coassembles with minK to form I(Ks) channels and I(Ks) dysfunction is a cause of cardiac arrhythmia.

Fine mapping of the chromosome 3p susceptibility locus in inflammatory bowel disease.

BACKGROUND AND AIMS: Genetic predisposition for inflammatory bowel disease (IBD) has been demonstrated by epidemiological and genetic linkage studies. Genetic linkage of IBD to chromosome 3 has been observed previously. A high density analysis of chromosome 3p was performed to confirm prior linkages and elucidate potential genetic associations. METHODS: Forty three microsatellite markers on chromosome 3 were genotyped in 353 affected sibling pairs of North European Caucasian extraction (average marker density 2 cM in the linkage interval). Marker order was defined by genetic and radiation hybrid techniques. RESULTS: The maximum single point logarithm of odds (LOD) score was observed for Crohn's disease at D3S3591. Peak multipoint LOD scores of 1.65 and 1.40 for the IBD phenotype were observed near D3S1304 (distal 3p) and near D3S1283 in the linkage region previously reported. Crohn's disease contributed predominantly to the linkage. The transmission disequilibrium test showed significant evidence of association (p=0.009) between allele 4 of D3S1076 and the IBD phenotype (51 transmitted v 28 non-transmitted). Two known polymorphisms in the CCR2 and CCR5 genes were analysed, neither of which showed significant association with IBD. Additional haplotype associations were observed in the vicinity of D3S1076. CONCLUSIONS: This study provides confirmatory linkage evidence for an IBD susceptibility locus on chromosome 3p and suggests that CCR2 and CCR5 are unlikely to be major susceptibility loci for IBD. The association findings in this region warrant further investigation.

Analysis of single-nucleotide polymorphisms in the interleukin-4 receptor gene for association with inflammatory bowel disease.

Genetic linkage analysis in families with multiple cases of inflammatory bowel disease (IBD) has mapped a gene which confers susceptibility to IBD to the pericentromeric region of chromosome 16 (IBD1). The linked region includes the interleukin(IL)-4 receptor gene (IL4R). Since IL-4 regulation and expression are abnormal in IBD, the IL4R gene is thus both a positional and functional candidate for IBD1. We screened the gene for single-nucleotide polymorphisms (SNPs) by fluorescent chemical cleavage analysis, and tested a subset of known and novel SNPs for allelic association with IBD in 355 families, which included 435 cases of Crohn's disease and 329 cases of ulcerative colitis. No association was observed between a haplotype of four SNPs (val50ile, gln576arg, A3044G, G3289A) and either the Crohn's disease or ulcerative colitis phenotypes using the transmission disequilibrium test. There was also no evidence for association when the four markers were analyzed individually. The results indicate that these variants are not significant genetic determinants of IBD, and that the IL4R gene is unlikely to be IBD1. Linkage disequilibrium analyses showed that the val50ile and gln576arg variants are in complete equilibrium with each other, although they are separated by only about 21 kilobases of genomic DNA. This suggests that a very dense SNP map may be required to exclude or detect disease associations with some candidate genes.

Linkage of inflammatory bowel disease to human chromosome 6p.

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation. IBD is subdivided into Crohn disease and ulcerative colitis phenotypes. Given the immunologic dysregulation in IBD, the human-leukocyte-antigen region on chromosome 6p is of significant interest. Previous association and linkage analysis has provided conflicting evidence as to the existence of an IBD-susceptibility locus in this region. Here we report on a two-stage linkage and association analysis of both a basic population of 353 affected sibling pairs (ASPs) and an extension of this population to 428 white ASPs of northern European extraction. Twenty-eight microsatellite markers on chromosome 6 were genotyped. A peak multipoint LOD score of 4.2 was observed, at D6S461, for the IBD phenotype. A transmission/disequilibrium test (TDT) result of P=.006 was detected for D6S426 in the basic population and was confirmed in the extended cohort (P=.004; 97 vs. 56 transmissions). The subphenotypes of Crohn disease, ulcerative colitis, and mixed IBD contributed equally to this linkage, suggesting a general role for the chromosome 6 locus in IBD. Analysis of five single-nucleotide polymorphisms in the TNFA and LTA genes did not reveal evidence for association of these important candidate genes with IBD. In summary, we provide firm linkage evidence for an IBD-susceptibility locus on chromosome 6p and demonstrate that TNFA and LTA are unlikely to be susceptibility loci for IBD.

Genetic analysis of inflammatory bowel disease in a large European cohort supports linkage to chromosomes 12 and 16.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a complex disorder of unknown etiology. Epidemiological investigations suggest a genetic basis for IBD. Recent genetic studies have identified several IBD linkages. The significance of these linkages will be determined by studies in large patient collections. The aim of this study was to replicate IBD linkages on chromosomes 12 and 16 in a large European cohort. METHODS: Three hundred fifty-nine affected sibling pairs from 274 kindreds were genotyped using microsatellite markers spanning chromosomes 12 and 16. Affection status of the sibling pairs was defined as Crohn's disease (CD) or ulcerative colitis (UC). RESULTS: Nonparametric statistical analyses showed linkage for both chromosomes. Two-point results for chromosome 12 peaked at D12S303 (logarithm of odds [LOD], 2.15; P = 0.003) for CD and at D12S75 (LOD, 0.92; P = 0.03) for UC. Multipoint analyses produced a peak LOD of 1.8 for CD. Chromosome 16 showed linkage for CD at marker D16S415 (LOD, 1.52; P = 0.007). Multipoint support peaked above markers D16S409 and D16S411 (LOD, 1.7). CONCLUSIONS: These data are consistent with linkage of IBD to chromosomes 12 and 16. The replication of genetic risk loci in a large independent family collection indicates important and common susceptibility genes in these regions and will facilitate identification of genes involved in IBD.