Use of Constant Denaturant Capillary Electrophoresis of Pooled Blood Samples to Identify Single-Nucleotide Polymorphisms in the Genes (Scnn1a and Scnn1b) Encoding the α and β Subunits of the Epithelial Sodium Channel (original) (raw)
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Clinical chemistry, 2002
The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. The detection limit of CDCE for ENaC variants was 1%, indicating that all members o...
Hypertension Research, 2004
Liddle's syndrome is an autosomal dominant disease characterized by sodium-sensitive early hypertension and mutations in either the-or-subunit of the amiloride-sensitive epithelial sodium channel encoded by SCNN1B and SCNN1G. We sequenced the 381 bp-coding regions in exon 13 of SCNN1B and the 381 bp-coding regions in exon 12 of SCNN1G in 948 and 953 Japanese patients with hypertension, respectively. In the SCNN1B gene, we identified three missense mutations, P592S (n 3), T594M (n 2), and E632K (n 1) in a heterozygous state in addition to four synonymous ones, Ile515 (n 1), Ser520 (n 19), Ser533 (n 1), and Thr594 (n 11). In the SCNN1G gene, we identified three missense mutations, A578V (n 1), P603S (n 1), and L609F (n 1) in a heterozygous state in addition to two synonymous ones, Ile550 (n 1) and Leu649 (n 91, heterozygous; n 2, homozygous). We did not identify the same mutations previously reported in Liddle's syndrome kindreds. Two of the six hypertensive patients with missense mutation in the SCNN1B gene showed atypical renin and aldosterone levels, though one of them was diagnosed with renovascular hypertension. One patient with T594M in the SCNN1B gene was resistant to hypertension. The roles of these missense mutations in the SCNN1B or SCNN1G gene identified in hypertensive patients are not clear in the pathogenesis of hypertension and the regulation of electrolytes. Thus, further investigation of these mutations, including functional analyses, will be needed. (Hypertens Res 2004; 27: 333-338)
Polymorphisms of the γ subunit of the epithelial Na+ channel in essential hypertension
Journal of Hypertension, 1999
Objective The ã subunit of the epithelial Na channel (ãENaC) has been implicated in Liddle's syndrome. The objective of this study was to examine its status in essential hypertension. Design and methods The search for molecular variants was performed using the SSCP technique after determination of the intron±exon boundaries of the transcribed sequence. We found an additional 205 bp intron splitting the published exon 10 in two. The last exon of ãENaC was tested with samples from a series of 245 normotensive patients and 453 hypertensive subjects (383 Caucasians, 70 Afro-Caribbeans), all probands of hypertensive families in the HYPERGENE data set. The search was extended to the other 11 transcribed exons in a subset of 65 patients with low-renin pro®le. Results Four neutral polymorphisms were detected, three in the third exon of the gene (T387C, T474C, C549T) and one in the last exon (C1990G). These four variants were found with similar frequencies in hypertensive and normotensive Caucasian subjects as well as in patients with low-renin pro®le. Hypertensive Caucasians and hypertensive subjects of African ancestry also had similar frequencies. Lastly, we found two rare mutations, one the insertion of a proline residue at position 594 of the mature protein (594insP), the other an Arg-to-His substitution at position 631 (R631H). Compared to wild-type (1.00 6 0.42, n = 26), expression of the 594insP (1.10 6 0.43, n = 26) and R631H (0.97 6 0.43, n = 26) variants in Xenopus oocytes produced no signi®cant increase in Na current. Conclusions Screening of the entire coding sequence of ãENaC does not suggest that this subunit is frequently involved in essential hypertension.
Clinical and experimental nephrology, 2002
Mutations have been found only in exons 8 and 12 of the β-subunit of the epithelial sodium channel (βENaC), but the presence of other mutations in the remaining exons remains to be determined in the Japanese population. New cases with the V434M mutation should be identified because the identified individuals have high plasma sodium concentration Exons 1 to 7 and 9 to 11 were screened by using single-strand conformational polymorphism (SSCP) in 200 subjects (100 normotensive and 100 hypertensive) randomly selected from 1245 participants in a community-based cohort study (Ohasama study) in northern Japan Four novel mutations were detected in exons 5, 6, and 7, and one of them was the novel missense mutation, P369H in exon 6. Then extended investigation of this mutation, together with those of V434M and P592S, which were identified in our previous studies, was performed in 1245 subjects. The final frequency of these mutations was 1/1245 for P369H, 5/1245 for V434M, and 5/1245 for P592S...
Genetic Analysis of the b Subunit of the Epithelial Na1 Channel in Essential Hypertension
2000
Mutations of the last exon of the b subunit of the amiloride-sensitive epithelial Na 1 channel (bENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of bENaC could be implicated in essential hypertension. After determination of the bENaC coding gene organization (12 exons spanning 23.5
Genetic Analysis of the β Subunit of the Epithelial Na + Channel in Essential Hypertension
Hypertension, 1998
Mutations of the last exon of the  subunit of the amiloride-sensitive epithelial Na ϩ channel (ENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of ENaC could be implicated in essential hypertension. After determination of the ENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22 Na ϩ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the ENaC gene in essential hypertension.
American Journal of Hypertension, 2000
We evaluated the association of a common polymorphism in ␥ENaC, consisting in a C to G transversion in codon 649, with essential hypertension and to the pressor response to salt in whites. Two hundred fifteen essential hypertensive patients, and 137 normotensive controls were genotyped for the ␥649 ENaC polymorphism by polymerase chain reaction method and diagnostic restriction enzyme digestion. The genotype distribution of the ␥649 ENaC polymorphism in the hypertensives, 129 CC (60%) and 86 CG/GG (40%) was not significantly different from that of the control group, 84 CC (61%) and 53 CG/GG (39%) (P ؍ ؍ .81). Salt sensitivity was assessed in a group of 48 patients by 24-h mean blood pressure response to changes in salt intake. Nineteen patients were diagnosed as salt sensitive, whereas 29 had salt-resistant hypertension. The ␥649 ENaC genotype distribution in salt-sensitive patients was 12 CC (63%) and 7 CG/GG (37%), not significantly different from the distribution in the salt-resistant group, 19 CC (65%) and 10 CG/GG (35%), P ؍ ؍ .87. The changes in systolic, diastolic, and mean blood pressure as measured by ambulatory blood pressure monitoring, and in plasma renin activity and plasma aldosterone induced by high salt diet were not different among the ␥649 ENaC genotypes. In the present study we found no association between the ␥649 ENaC polymorphism and essential hypertension or salt sensitivity. Although these data do not support a major causative role for this polymorphism, we cannot exclude that a functional mutation elsewhere in ENaC might be associated with essential hypertension.
Association of Sodium Channel -Subunit Promoter Variant With Blood Pressure
Hypertension, 2001
The SCNN1G gene, located on human chromosome 16p12, encodes the γ subunit of the amiloride-sensitive epithelial sodium channel, and mutations in SCNN1G can result in Liddle’s syndrome or pseudohypoaldosteronism type I. We identified sequence variations in the promoter region of SCNN1G and examined the association between this polymorphism and blood pressure in a large cohort (n=4075) representing the general population in Japan. We found T( − 1290)C , T( − 501)G , G( − 173)A , and G( − 104)T polymorphisms in the promoter region of SCNN1G and confirmed the existence of T387C and T474C polymorphisms in exon 3 and the C1947G polymorphism in exon 13. Because the genotypes of the T( − 1290)C , T( − 501)G , G( − 104)T , and T474C polymorphisms were in tight linkage disequilibrium, we selected the T474C and G( − 173)A polymorphisms for an association study. The G( − 173)A polymorphism of SCNN1G had a significant effect on systolic pressure ( P =0.0050) and pulse pressure ( P =0.0050). The ...
A novel epithelial sodium channel ?-subunit mutation associated with hypertensive Liddle syndrome
Pediatric Nephrology, 2005
Low-renin hypertension responsive to amiloride-thiazide therapy in a 4-year-old Afro-Haitian girl suggested Liddle syndrome. Urine steroid profiling substantiated the diagnosis and DNA analysis of the epithelial sodium channel (ENaC) revealed a novel heterozygous bENaC mutation in the patient and in her hypertensive father. Liddle syndrome should be considered as a cause of hypertension in young children particularly with suppressed renin activity.