Resequencing Epithelial Sodium Channel Genes Identifies Rare Variants Associated With Blood Pressure Salt-Sensitivity: The GenSalt Study.
ABSTRACT: BACKGROUND:A resequencing study of renal epithelial sodium channel (ENaC) genes was conducted to identify rare variants associated with blood pressure (BP) salt-sensitivity. METHODS:The Genetic Epidemiology Network of Salt-Sensitivity (GenSalt) study was conducted among 1,906 participants who underwent a 7-day low-sodium followed by a 7-day high-sodium feeding-study. The 300 most salt-sensitive and 300 most salt-resistant GenSalt participants were selected for the resequencing study. Three ENaC genes (SCNN1A, SCNN1B, and SCNN1G) were resequenced using capillary-based sequencing methods. Traditional burden tests were utilized to examine association between rare variants and BP salt-sensitivity. Associations of low-frequency and common variants were tested using single-marker analyses. RESULTS:Carriers of SCNN1A rare variants had a 0.52 [95% confidence interval (CI): 0.32-0.85] decreased odds of BP salt-sensitivity compared with noncarriers. Neither SCNN1B nor SCNN1G associated with salt-sensitivity of BP in rare variant analyses (P = 0.65 and 0.48, respectively). In single-marker analyses, 3 independent common variants in SCNN1A, rs11614164, rs4764586, and rs3741914, associated with salt-sensitivity after Bonferroni correction (P = 4.4 × 10-4, 1.1 × 10-8, and 1.3 × 10-3). Each copy of the minor allele of rs4764586 was associated with a 1.36-fold (95% CI: 1.23-1.52) increased odds of salt-sensitivity, whereas each copy of the minor allele of rs11614164 and rs3741914 was associated with 0.68-fold (95% CI: 0.55-0.84) and 0.69-fold (95% CI: 0.54-0.86) decreased odds of salt-sensitivity, respectively. CONCLUSIONS:This study demonstrated for the first time a relationship between rare variants in the ENaC pathway and BP salt-sensitivity. Future replication and functional studies are needed to confirm the findings in this study. CLINICAL TRIAL REGISTRY:Trial Number NCT00721721.
Project description:Rare mutations of the epithelial sodium channel (ENaC) lead to mendelian forms of salt-sensitive hypertension or salt-wasting hypotension. We aimed to examine the association between common variants in the ENaC genes and salt sensitivity of blood pressure (BP).A total of 1906 Han Chinese participated in the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study, which includes a 7-day low-sodium intake (51.3 mmol sodium/d) followed by a 7-day high-sodium intake (307.8 mmol sodium/d). Nine BP measurements were obtained at baseline and each intervention period using a random-zero sphygmomanometer. Single-nucleotide polymorphisms, both tagging and functional, from the 3 ENaC subunits, ?, ?, and ? (SCNN1A, SCNN1B, and SCNN1G), were genotyped. Multiple common single-nucleotide polymorphisms in SCNN1G were significantly associated with BP response to low-sodium intervention (rs4073930, P=1.7×10(-5); rs4073291, P=1.1×10(-5); rs7404408, P=1.9×10(-5); rs5735, P=3.0×10(-4); rs4299163, P=0.004; and rs4499238, P=0.002) even after correcting for multiple testing. For example, under an additive model, the minor allele G of SNP rs4073291 was associated with 1.33 mm Hg lower systolic BP reduction during low-sodium intervention.This large dietary sodium intervention study indicates that common variants of ENaC subunits may contribute to the variation of BP response to dietary sodium intake. Future studies are warranted to confirm these findings in an independent population and to identify functional variants for salt sensitivity.URL: http://www.clinicaltrials.gov. Unique identifier: NCT00721721.
Project description:In order to investigate the associations of SCNN1A, SCNN1G and SCNN1B genes with blood pressure (BP) in the Han Chinese population, we included 2880 participants did not use antihypertensive medication in the month prior to the baseline survey in the current analysis. Forty-four tag-single-nucleotide polymorphisms (SNPs) in epithelial sodium channel (ENaC) genes were selected and genotyped, and nine BP measurements were obtained during the 3-day examination. In the single-marker analyses, we identified significant associations of SCNN1A marker rs13306613 with diastolic BP (DBP) and SCNN1B marker rs12447134 with systolic BP (SBP) under codominant model after Bonferroni's correction (P=2.82 × 10(-5) and 4.63 × 10(-4), respectively). In addition, five SNPs in SCNN1G and four SNPs in SCNN1B achieved nominal significance for SBP, DBP or mean arterial pressure (MAP) under the additive model. For example, the minor C allele of rs5735 in SCNN1G gene was associated with decreased SBP, DBP and MAP (P=0.016, 5.41 × 10(-3) and 4.36 × 10(-3), respectively). Gene-based results showed significant associations of SCNN1G and SCNN1B with BP levels. This study suggested that ENaC genes have important roles in BP regulation in the Han Chinese population. Future studies are warranted to replicate these findings, and functional studies are needed to identify true causal variants in ENaC genes.
Project description:We examined the associations of epithelial sodium channel (ENaC) genes with blood pressure (BP) changes and hypertension incidence in a longitudinal family study.A total of 2,755 Han Chinese participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) baseline examination were eligible for this study. The associations of 43 tag single nucleotide polymorphisms (SNPs) in ENaC genes with BP changes and hypertension incidence were assessed using mixed models to account for the correlations of repeated measures among individuals and within families. A genotype by time interaction term was used to model differences in longitudinal BP change according to genotype over time. Gene-based analyses were conducted using the truncated product method. The Bonferroni method was used to adjust for multiple testing in all analyses.During an average of 7.4 years follow-up, systolic BP (SBP) and diastolic BP (DBP) increased, and approximately 33% of participants developed hypertension. SCNN1A SNP rs11064153 and SCNN1G SNP rs4401050 were significantly associated with longitudinal changes in SBP after adjustment for multiple testing (P interaction = 5.8×10(-4) and 0.001, respectively). Similar but nonsignificant trends were observed for the associations between both rs11064153 and rs4401050 and DBP changes (P interaction = 0.024 and 0.005, respectively) and between rs11604153 and hypertension incidence (P = 0.02). Gene-based analyses also supported the overall association of SCNN1G with longitudinal changes in SBP (P = 2.0×10(-4)).Our findings indicated that SCNN1A and SCNN1G may contribute to BP changes over time in the Han Chinese population. Replication of these findings is warranted.
Project description:Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of aldosterone. Mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G) of the epithelial sodium channel (ENaC) and the NR3C2 gene encoding the mineralocorticoid receptor, result in systemic PHA1 and renal PHA1 respectively. Common clinical manifestations of PHA1 include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. In this study, we describe the clinical and biochemical manifestations in two Chinese patients with systemic PHA1. Sequence analysis of the SCNN1A gene revealed a compound heterozygous mutation (c.1311delG and c.1439+1G>C) in one patient and a homozygous mutation (c.814_815insG) in another patient, all three variants are novel. Further analysis of the splicing pattern in a minigene construct showed that the c.1439+1G>C mutation can lead to the retainment of intron 9 as the 5'-donor splice site disappears during post-transcriptional processing of mRNA. In conclusion, our study identified three novel SCNN1A gene mutations in two Chinese patients with systemic PHA1.
Project description:This study was performed to investigate whether genetic variation in the epithelial sodium channel (ENaC) is associated with 24-h urinary sodium excretion and blood pressure. A total of 3345 participants of the KoGES_Ansan and Ansung study were eligible for this study. Genomic DNA samples were isolated from peripheral blood and genotyped on the Affymetrix Genome-Wide Human SNP Array 5.0. Thirty-four single nucleotide polymorphisms (SNPs) were extracted for gene regions (SCNN1A, SCNN1B, and SCNN1G) as additive components by using Plink. Twenty-four-hour sodium excretions were estimated from spot urine samples using the Tanaka formula. The general linear model (GLM) was applied to assess the association between SNPs and urinary sodium excretion or blood pressure. In the SCNN1G gene, six SNPs (rs4073291, rs12934362, rs7404408, rs4494543, rs5735, and rs6497657) were significantly different in 24-h urinary sodium excretion according to gene variants. However, no difference was found in blood pressure among participants with gene variants of ENaC. Our finding indicated that 24-h urinary sodium excretions were different according to variants of the SCNN1G gene in large samples. Further studies to replicate these findings are warranted.
Project description:Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl- baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47 ?M). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing ?-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of ?-ENaC in cholinergic UBC-salt perception, homeostatic ion transport, mechanoreception-remains to be determined.
Project description:Liddle syndrome is an inherited form of low-renin hypertension, transmitted with an autosomal dominant pattern. The molecular basis of Liddle syndrome resides in germline mutations of the SCNN1A, SCNN1B and SCNN1G genes, encoding the ?, ?, and ?-subunits of the epithelial Na? channel (ENaC), respectively. To date, 31 different causative mutations have been reported in 72 families from four continents. The majority of the substitutions cause an increased expression of the channel at the distal nephron apical membrane, with subsequent enhanced renal sodium reabsorption. The most common clinical presentation of the disease is early onset hypertension, hypokalemia, metabolic alkalosis, suppressed plasma renin activity and low plasma aldosterone. Consequently, treatment of Liddle syndrome is based on the administration of ENaC blockers, amiloride and triamterene. Herein, we discuss the genetic basis, clinical presentation, diagnosis and treatment of Liddle syndrome. Finally, we report a new case in an Italian family, caused by a SCNN1B p.Pro618Leu substitution.
Project description:Hypertension affects ?30% of adults in industrialized countries and is the major risk factor for cardiovascular disease.We sought to study the genetic effect of coding and conserved noncoding variants in syndromic hypertension genes on systolic blood pressure (BP) and diastolic BP to assess their overall impact on essential hypertension.We resequenced 11 genes (AGT, CYP11B1, CYP17A1, HSD11B2, NR3C1, NR3C2, SCNN1A, SCNN1B, SCNN1G, WNK1, and WNK4) in 560 European American (EA) and African American ancestry GenNet participants with extreme systolic BP. We investigated genetic associations of 2535 variants with BP in 19997 EAs and in 6069 African Americans in 3 types of analyses. First, we studied the combined effects of all variants in GenNet. Second, we studied 1000 Genomes imputed polymorphic variants in 9747 EA and 3207 African American Atherosclerosis Risk in Communities subjects. Finally, we genotyped 37 missense and common noncoding variants in 6591 EAs and in 6521 individuals (3659 EA/2862 African American) from the CLUE and Family Blood Pressure Program studies, respectively. None of the variants individually reached significant false-discovery rates ?0.05 for systolic BP and diastolic BP. However, on pooling all coding and noncoding variants, we identified at least 5 loci (AGT, CYP11B1, NR3C2, SCNN1G, and WNK1) with higher association at evolutionary conserved sites.Both rare and common variants at these genes affect BP in the general population with modest effects sizes (<0.05 standard deviation units), and much larger sample sizes are required to assess the impact of individual genes. Collectively, conserved noncoding variants affect BP to a greater extent than missense mutations.
Project description:The role of rare variants in blood pressure (BP) salt-sensitivity is unknown. We conducted a resequencing study of the renin-angiotensin-aldosterone system (RAAS) to identify rare variants associated with BP salt-sensitivity among participants of the Genetic Epidemiology Network of Salt-Sensitivity (GenSalt) study.The GenSalt study was conducted among 1,906 participants who underwent a 7-day low-sodium (51.3 mmol sodium/day) followed by a 7-day high-sodium feeding study (307.8 mmol sodium/day). The 300 most salt-sensitive and 300 most salt-resistant GenSalt participants were selected for the resequencing study. Seven RAAS genes were resequenced using capillary-based sequencing methods. Rare variants were tested for association with BP salt-sensitivity using traditional burden tests. Single-marker analyses were employed to test associations of low-frequency and common variants.Aggregate rare variant analysis revealed an association of the RAAS pathway with BP salt-sensitivity. Carriers of rare RAAS variants had a 1.55-fold [95% confidence interval (CI): 1.15, 2.10] higher odds of salt-sensitivity compared to noncarriers (P = 0.004), a finding which was significant after Bonferroni correction. A nominal association of the APLN gene with salt-sensitivity was also identified, with rare APLN variants conferring a 2.22-fold (95% CI: 1.05, 6.58) higher odds of salt-sensitivity (P = 0.03). Single-marker analyses did not identify variant-BP salt-sensitivity associations after Bonferroni adjustment. A nominal association of a low-frequency, missense RENBP variant was identified. Each minor allele of rs78377269 conferred a 2.21-fold (95% CI: 1.10, 4.42) increased odds of salt-sensitivity (P = 0.03).This study presents of the first evidence of a contribution of rare RAAS variants to BP salt-sensitivity. Clinical Trial RegistryTrial Number: NCT00721721.
Project description:The epithelial sodium channel (ENaC) is composed of three homologous subunits and allows the flow of Na(+) ions across high resistance epithelia, maintaining body salt and water homeostasis. ENaC dependent reabsorption of Na(+) in the kidney tubules regulates extracellular fluid (ECF) volume and blood pressure by modulating osmolarity. In multi-ciliated cells, ENaC is located in cilia and plays an essential role in the regulation of epithelial surface liquid volume necessary for cilial transport of mucus and gametes in the respiratory and reproductive tracts respectively. The subunits that form ENaC (named as alpha, beta, gamma and delta, encoded by genes SCNN1A, SCNN1B, SCNN1G, and SCNN1D) are members of the ENaC/Degenerin superfamily. The earliest appearance of ENaC orthologs is in the genomes of the most ancient vertebrate taxon, Cyclostomata (jawless vertebrates) including lampreys, followed by earliest representatives of Gnathostomata (jawed vertebrates) including cartilaginous sharks. Among Euteleostomi (bony vertebrates), Actinopterygii (ray finned-fishes) branch has lost ENaC genes. Yet, most animals in the Sarcopterygii (lobe-finned fish) branch including Tetrapoda, amphibians and amniotes (lizards, crocodiles, birds, and mammals), have four ENaC paralogs. We compared the sequences of ENaC orthologs from 20 species and established criteria for the identification of ENaC orthologs and paralogs, and their distinction from other members of the ENaC/Degenerin superfamily, especially ASIC family. Differences between ENaCs and ASICs are summarized in view of their physiological functions and tissue distributions. Structural motifs that are conserved throughout vertebrate ENaCs are highlighted. We also present a comparative overview of the genotype-phenotype relationships in inherited diseases associated with ENaC mutations, including multisystem pseudohypoaldosteronism (PHA1B), Liddle syndrome, cystic fibrosis-like disease and essential hypertension.