Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models.

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca(2+) entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.

Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development.

BACKGROUND: Moyamoya disease is an idiopathic vascular disorder of intracranial arteries. Its susceptibility locus has been mapped to 17q25.3 in Japanese families, but the susceptibility gene is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide linkage analysis in eight three-generation families with moyamoya disease revealed linkage to 17q25.3 (P<10(-4)). Fine mapping demonstrated a 1.5-Mb disease locus bounded by D17S1806 and rs2280147. We conducted exome analysis of the eight index cases in these families, with results filtered through Ng criteria. There was a variant of p.N321S in PCMTD1 and p.R4810K in RNF213 in the 1.5-Mb locus of the eight index cases. The p.N321S variant in PCMTD1 could not be confirmed by the Sanger method. Sequencing RNF213 in 42 index cases confirmed p.R4810K and revealed it to be the only unregistered variant. Genotyping 39 SNPs around RNF213 revealed a founder haplotype transmitted in 42 families. Sequencing the 260-kb region covering the founder haplotype in one index case did not show any coding variants except p.R4810K. A case-control study demonstrated strong association of p.R4810K with moyamoya disease in East Asian populations (251 cases and 707 controls) with an odds ratio of 111.8 (P = 10(-119)). Sequencing of RNF213 in East Asian cases revealed additional novel variants: p.D4863N, p.E4950D, p.A5021V, p.D5160E, and p.E5176G. Among Caucasian cases, variants p.N3962D, p.D4013N, p.R4062Q and p.P4608S were identified. RNF213 encodes a 591-kDa cytosolic protein that possesses two functional domains: a Walker motif and a RING finger domain. These exhibit ATPase and ubiquitin ligase activities. Although the mutant alleles (p.R4810K or p.D4013N in the RING domain) did not affect transcription levels or ubiquitination activity, knockdown of RNF213 in zebrafish caused irregular wall formation in trunk arteries and abnormal sprouting vessels. CONCLUSIONS/SIGNIFICANCE: We provide evidence suggesting, for the first time, the involvement of RNF213 in genetic susceptibility to moyamoya disease.

Confirmation of an association of single-nucleotide polymorphism rs1333040 on 9p21 with familial and sporadic intracranial aneurysms in Japanese patients.

BACKGROUND AND PURPOSE: Genetic factors are important determinants of intracranial aneurysm (IA). Recently, a multinational, genome-wide association study identified 3 loci associated with IA, located on 2q (rs700651), 8q (rs10958409), and 9p (rs1333040 and rs10757278). The aim of this study was to evaluate these associations. METHODS: Familial and sporadic cases were investigated. Familial cases, consisting of 96 subjects with IA, and 46 subjects of unknown status from 31 pedigrees were analyzed with the transmission disequilibrium test and linkage analysis. Associations of single-nucleotide polymorphisms (SNPs) with IA were tested in 419 sporadic IA cases and in 408 control subjects. Sequencing of CDKN2A, CDKN2B, and CDKN2BAS revealed additional SNPs, and their associations with IA were also tested. RESULTS: The transmission disequilibrium test revealed associations of 2 SNPs, rs700651 (P=0.036) and rs1333040 (P=0.002), with familial IA. Analysis of SNPs in sporadic cases revealed an allelic association of rs1333040 with IA (odds ratio=1.28; 95% CI, 1.04-1.57; P=0.02) but failed to show associations of rs10757278 and rs496892 with IA. We sequenced 3 candidate genes; CDKN2A, CDKN2B, and CDKN2BAS. All 6 index cases from IA families had the rs1333040-T allele and SNPs (rs10965215, rs10120688, and rs7341791) in CDKN2BAS. None of these SNPs had linkage disequilibrium with rs1333040 and was associated with IA. CONCLUSIONS: A region between introns 7 and 15 of CDKN2BAS carrying the rs1333040-T allele may confer risk for IA.

A rare Asian founder polymorphism of Raptor may explain the high prevalence of Moyamoya disease among East Asians and its low prevalence among Caucasians.

BACKGROUND: In an earlier study, we identified a locus for Moyamoya disease (MMD) on 17q25.3. METHODS: Linkage analysis and fine mapping were conducted for two new families in additional to the previously studied 15 families. Three genes, CARD14, Raptor, and AATK, were selected based on key words, namely, "inflammation", "apoptosis", "proliferation", and "vascular system", for further sequencing. A segregation analysis of 34 pedigrees was performed, followed by a case-control study in Japanese (90 cases vs. 384 controls), Korean (41 cases vs. 223 controls), Chinese (23 cases and 100 controls), and Caucasian (25 cases and 164 controls) populations. RESULTS: Linkage analysis increased the LOD score from 8.07 to 9.67 on 17q25.3. Fine mapping narrowed the linkage signal to a 2.1-Mb region. Sequencing revealed that only one newly identified polymorphism, ss161110142, which was located at position -1480 from the transcription site of the Raptor gene, was common to all four unrelated sequenced familial affected individuals. ss161110142 was then shown to segregate in the 34 pedigrees studied, resulting in a two-point LOD score of 14.2 (P = 3.89 × 10(-8)). Its penetrance was estimated to be 74.0%. Among the Asian populations tested (Japanese, Korean, and Chinese), the rare allele was much more frequent in cases (26, 33, and 4%, respectively) than in controls (1, 1, and 0%, respectively) and was associated with an increased odds ratio of 52.2 (95% confidence interval 27.2-100.2) (P = 2.5 × 10(-49)). This allele was, however, not detected in the Caucasian samples. Its population attributable risk was estimated to be 49% in the Japanese population, 66% in the Korean population, and 9% in the Chinese population. CONCLUSION: ss161110142 may confer susceptibility to MMD among East Asian populations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12199-009-0116-7) contains supplementary material, which is available to authorized users.

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder.

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan.

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.