Six Missense Mutations of the Epithelial Sodium Channel B and G Subunits in Japanese Hypertensives

333

Original Article

Six Missense Mutations of the Epithelial Sodium Channel b and g Subunits in Japanese Hypertensives

Kei KAMIDE, Chihiro TANAKA＊, Shin TAKIUCHI, Yoshikazu MIWA, Masayoshi YOSHII, Takeshi HORIO, Yuhei KAWANO, and Toshiyuki MIYATA＊

Liddle’s syndrome is an autosomal dominant disease characterized by sodium-sensitive early hypertension and mutations in either the β- or γ-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: 333Ð338)

Key Words: Liddle’s syndrome, SCNN1B, SCNN1G, gene variants, hypertension

Neither the β or the γ subunit, when expressed alone or to- ＋ Introduction gether, produces any measurable Na current. However, co- expression with the α-subunits greatly enhances the ampli- The amiloride-sensitive epithelial sodium channel (ENaC) is tudes of the Na＋ current (4, 5). responsible for the rate-limiting step of sodium reabsorption Liddle’s syndrome is an autosomal dominant disease char- in the distal renal tubules, and thus may play a key role in acterized by sodium-sensitive early hypertension associated the maintenance of sodium balance and blood pressure. with hypokalemic metabolic alkalosis, low plasma renin ac- ENaCs are composed of three homologous subunits, termed tivity, and suppressed aldosterone secretion. Several muta- α, β, and γ, with a subunit stoichiometry of 2α:1β:1γ (1, 2). tions have been described in Liddle’s kindreds, some in the β These three subunits show a similar topology, consisting of subunit and others in the γ subunit (5–7). All these mutations two transmembrane domains separated by a large extracellu- abolish or modify a highly conserved PY motif present in lar domain (3). The α-subunit appears to be the conducting their intracellular carboxyl terminal region, thereby altering unit, and the role of the other two subunits is less certain. the binding of ENaC to its partner, Nedd4, a ubiquitin-pro-

From the Division of Hypertension and Nephrology and ＊Research Institute, National Cardiovascular Center, Suita, Japan. This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan. Address for Reprints: Kei Kamide, M.D., Ph.D., Division of Hypertension and Nephrology, National Cardiovascular Center, 5–7–1 Fujishirodai, Suita 565–8565, Japan. E-mail: [email protected] Received September 5, 2003; Accepted in revised form January 24, 2004. 334 Hypertens Res Vol. 27, No. 5 (2004) tein ligase (8–10). This leads to a low intracellular turnover Ninety-two percent of the study subjects (884 subjects) of the channel and to an increase in the number of active were diagnosed with essential hypertension, and the rest had channels exposed at the cell membrane. The underlying hy- secondary hypertension, including renal hypertension (37 pothesis is that dysregulation of ENaC activity causes inap- subjects), renovascular hypertension (23 subjects), primary propriate sodium retention, with consequent volume expan- aldosteronism (11 subjects) and hypothyroid-induced hyper- sion and suppression of peripheral renin activity in patients tension (1 subject). The hypertension criteria were blood with Liddle’s syndrome. pressure above 140 and/or 90 mmHg, or the use of antihy- Several groups have identified other molecular variants of pertensive agents. The study protocol was approved by the the β subunit of ENaC (SCNN1B) (11–16). These variants Ethical Review Committee of the National Cardiovascular are missense mutations, and none of them affects the PY mo- Center. tif. Among them, the T594M mutant, which showed a prevalence of 6% to 8% in hypertensives of African descent, was Screening of Mutations in the 381 bp-Coding Regions well characterized (17–20). Whole cell voltage clamp studies in Exon 13 of the SCNN1B Gene and Exon 12 of the indicated that this variant showed an increased response to SCNN1G Gene cAMP analog (11, 17). This mutation has been reported to be the common in black people with essential hypertension Blood samples were obtained from each subject and geno- in some studies (18, 19), but not another (20). Some studies mic DNA was isolated from peripheral blood leukocytes by an have reported that this mutation is common among black in- NA-3000 nucleic acid isolation system (KURABO, Osaka, dividuals with essential hypertension (18, 19), but another Japan) (22). The 381 bp-coding region in exon 13 of study found no such association (20). SCNN1B was amplified by PCR using a pair of specific Regarding the γ subunit of ENaC (SCNN1G), Iwai et al. primers: 5′-agatggtcaccccctcccgttc-3′and 5′-taccctcccgaccctt reported that a single nucleotide polymorphism, G(－173)A, tggagag-3′, which flank the 597-bp region. The 381 bp-cod- in the promoter region of the SCNN1G gene was associated ing region in exon 12 of SCNN1G was amplified by PCR us- with blood pressure regulation in a Japanese population (21). ing a pair of specific primers: 5′-tgcacagagtaagaggaaacagg- They also showed that the promoter activity of the G(－173) 3′and 5′-agcaggctttttggtcagagtat-3′, which flank the 677-bp allele was 2- to 3-fold higher than that of the A(－173) al- region. The PCR products were directly sequenced on an lele. This suggests that the γ subunit level is one of the deter- ABI PRISM 3700 DNA analyzer (Applied Biosystems, Fos- minants of the ENaC activity. In addition, it suggests that the ter City, USA) as described previously (23). The obtained ENaC activity may be regulated by a subtle change of each sequences were examined for the presence of a mutation us- subunit protein that could be induced by the missense muta- ing the Sequencher software package (Gene Codes Corpora- tions. As mentioned above, a highly conserved PY motif pres- tion, Ann Arbor, USA), followed by visual inspection. ent in the intracellular carboxyl terminal region of the β and γ subunits of ENaCs is highly important for their regulatory Results function. This suggests that the surrounding regions—encoded by exon 13 of SCNN1B and exon 12 of SCNN1G—of the In a total of 956 Japanese patients with hypertension, we PY motif are also important for the the function of ENaCs. screened exon 13 of the SCNN1B gene and exon 12 of the In the present study, we amplified the 381 bp-coding re- SCNN1G gene. The 381 bp-coding regions were amplified gions of both exon 13 of SCNN1B and exon 12 of SCNN1G from the genomic DNA and a total of 948 and 953 sample by polymerase chain reaction (PCR) in 956 Japanese patients DNAs were successfully sequenced for SCNN1B and with hypertension and successfully sequenced the regions in SCNN1G, respectively. Although we did not identify the 948 and 953 patients, respectively. We identified several mu- same mutations previously reported in Liddle’s syndrome tations, including missense mutations, both in SCNN1B and kindreds, we identified seven mutations in the SCNN1B gene SCNN1G. Based on our results, we also discuss the clinical and five in the SCNN1G gene. The seven mutations in the profiles of the patients carrying these mutations. SCNN1B gene consisted of three missense mutations, one of which has not previously been reported, and four synonymous mutations. The five mutations in the SCNN1G gene Methods consisted of three novel missense mutations and two synonymous mutations (Tables 1 and 2). Subjects In SCNN1B, three of the 948 individuals had a C-to-T sub- A total of 956 hypertensive subjects (525 men and 431 stitution at nucleotide 32053, leading to an amino acid sub- women; average age: 65.0±10.6 years old; body mass in- stitution from Pro to Ser at position 592 (P592S). Two of the dex: 24.2±3.4 kg/m2) were recruited from the Division of 948 individuals had a C-to-T substitution at nucleotide Hypertension and Nephrology at the National Cardiovascu- 32060, leading to an amino acid substitution from Thr to lar Center; all patients provided their written informed con- Met at position 594 (T594M). This T594M mutation was sent to participate in this study. previously identified only in black peoples (11, 14, 17, 18). Kamide et al: Novel Mutations in SCNN1B and SCNN1G in Hypertensive Patients 335

Table 1. Summary of Sequence Variations of Exon 13 in SCNN1B Identiﬁed in 948 Japanese Patients with Hypertension

Allele 1 Allele 2 SNP name Region Amino acid Total Allele 1 Allele 2 substitution Homo Hetero Homo number 31824C>T exon13 Ile515Ile 937 1 0 938 0.999 0.001 31839G>C exon13 Ser520Ser 919 19 0 938 0.990 0.010 31878T>C exon13 Ser533Ser 940 1 0 941 0.999 0.001 32053C>T exon13 Pro592Ser 945 3 0 948 0.998 0.002 32060C>T exon13 Thr594Met 946 2 0 948 0.999 0.001 32061G>A exon13 Thr594Thr 937 11 0 948 0.994 0.006 32173G>A exon13 Glu632Lys 947 1 0 948 0.999 0.001 The A of the ATG of the initiator Met codon is denoted nucleotide ＋1, as recommended by the Nomeclature Working Group (Hum Mut, Vol.11, pp.1–3, 1998). The nucleotide sequence (GenBank Accession ID: NT_010604) was used as a reference sequence. SCNN1B, β subunit of epithelial sodium channel gene; SNP, single nucleotide polymorphism.

Table 2. Summary of Sequence Variations of Exon 12 in SCNN1G Identiﬁed in 953 Japanese Patients with Hypertension

Allele 1 Allele 2 SNP name Region Amino acid Total Allele 1 Allele 2 substitution Homo Hetero Homo number 28898C>T exon12 Ile550Ile 952 1 0 953 0.999 0.001 28981C>T exon12 Ala578Val 952 1 0 953 0.999 0.001 29055C>T exon12 Pro603Ser 952 1 0 953 0.999 0.001 29075G>C exon12 Leu609Phe 952 1 0 953 0.999 0.001 29195C>G exon12 Leu649Leu 860 91 2 953 0.950 0.050 The nucleotide sequence (GenBank Accession ID: NT_010393) was used as a reference sequence. SCNN1G, γ-subunit of the epithelial sodium channel gene; SNP, single nucleotide polymorphism.

One of the 948 individuals had a G-to-A substitution at nu- with missense mutation in SCNN1G. An essential hyperten- cleotide 32,173, leading to an amino acid substitution from sive patient with a missense mutation of P592S in the Glu to Lys at position 632 (E632K). These missense muta- SCNN1B gene (case 1, Table 3) showed high plasma renin tions were found in a heterozygous form. In addition, we activity (PRA; 4.8 ng/ml/h), low plasma aldosterone concen- identified four synonymous mutations (31824C>T: n＝1; tration (PAC; 1.8 ng/dl) and normokalemia (4.3 mEq/l) even 31839G>C: n＝19; 31878T>C: n＝1; 32061G>A: n＝11) while taking β blockade and spironolactone. Both the PRA that encoded for Ile515, Ser520, Ser533 and Thr594, respec- and PAC in this patient were outside the normal ranges for tively (Table 1). Among them, two synonymous mutations our institute (normal PRA: 0.2–2.7 ng/ml/h; normal PAC: (Ser520 and Thr594) were previously reported in Japanese 2–13 ng/dl) and significantly different from the average val- (13, 16, 24). ues (PRA: 2.1 ng/ml/h; PAC: 15.6 ng/dl) among subjects of In SCNN1G, one of the 953 individuals had a C-to-T sub- the present study who had no secondary hypertension and no stitution at nucleotide 28981, leading to an amino acid sub- missense mutation of the SCNN1B or SCNN1G genes. stitution from Ala to Val at position 578 (A578V). One indi- One of the two patients with missense mutation of T594M vidual had a C-to-T substitution at nucleotide 29055, lead- in the SCNN1B gene (case 4, Table 3) showed severe hyper- ing to an amino acid substitution from Pro to Ser at position tension, with blood pressure of 174/96 mmHg even while 603 (P603S). One individual had a a G-to-C substitution at taking three kinds of antihypertensive drugs (calcium chan- nucleotide 29075, leading to an amino acid substitution nel blockade, angiotensin II receptor antagonist and α1- from Leu to Phe at position 609 (L609F). These missense adrenergic blocker). mutations were found in a heterozygous form. In addition, One patient with missense mutation of E632K in the we identified two synonymous mutations (28898C>T: n＝ SCNN1B gene and renovascular hypertension (case 6, Table 1; 29195C>G: n＝91, heterozygous and n＝2, homozygous) 3) showed much greater levels of both PRA (22 ng/ml/h) and that encoded for Ile550 and Leu649, respectively (Table 2). PAC (102.9 ng/dl) compared to the other subjects with reno- We also identified two single nucleotide polymorphisms in vascular hypertension (average PRA: 6.9 ng/ml/h; average intron 11, 28732G>A (n＝1) and 28776G>A (n＝1). PAC: 18.7 ng/dl), as well as clear hypokalemia (2.7 mEq/l) Tables 3 and 4 show the characteristics of the six patients despite spironolactone therapy. with missense mutation in SCNN1B and of the three patients Other hypertensive patients with missense mutations in 336 Hypertens Res Vol. 27, No. 5 (2004)

Table 3. Clinical Proﬁles of Hypertensives with Missense Mutations in Exon13 of SCNN1B

Case 123456 Polymorphism Pro592Ser Pro592Ser Pro592Ser Thr594Met Thr594Met Glu632Lys Age (years old) 62 73 67 58 91 66 Sex male male female female female female BMI (kg/m2) 28.2 26.3 22.8 28.5 25.6 20.5 Diagnosis EHT, NIDDM, EHT, HL, EHT, NIDDM, EHT, NIDDM, EHT, HL, RVHT, IGT, HL, HL, OMI EA HL HL OCI ASO, OCI HT duration (years) 30 20 11 23 50 11 HT family Hx father mother none brother mother, brother none SBP (mmHg) 140 138 150 174 122 150 DBP (mmHg) 88 70 80 96 62 70 Medication CCB, BB, CCB CCB CCB, ARB, CCB, BB CCB, ARB, spironolactone AB spironolactone Na (mEq/l) 137 138 139 140 142 142 K (mEq/l) 4.3 4.9 3.9 4.4 4.4 2.7 Cl (mEq/l) 101 109 100 102 108 98 Creatinine (mg/dl) 0.8 0.9 0.3 0.5 0.9 1.2 Overt proteinuria －－－＋－＋ PRA (ng/ml/h) 4.8 no data 1.1 0.8 1.4 22 PAC (ng/dl) 1.8 no data 8 22.6 9.3 102.9 FBS (mg/dl) 109 89 143 110 108 106 HbA1c (mg/dl) 7.2 5.3 8.0 5.3 5.6 5.7 SCNN1B, β-subunit of epithelial sodium channel gene; BMI, body mass index; EHT, essential hypertension; NIDDM, non-insulin dependent diabetes mellitus; HL, hyperlipidemia; OMI, old myocardial infarction; EA, effort angina; OCI, old cerebral infarction; RVHT, renovascular hypertension; IGT, impaired glucose tolerance; ASO, atherosclerotic obliterance; HT, hypertension; Hx, history; SBP, systolic blood pressure; DBP, diastolic blood pressure; CCB, calcium channel blocker; BB, β-adrenergic blocker; ARB, angiotensin II receptor blockade; AB, α1-adrenergic blocker; PRA, plasma renin activity; PAC, plasma aldosterone concentration; FBS, fasting blood sugar.

SCNN1B (cases 2, 3, 5, Table 3) and SCNN1G (cases 1–3, gion of exon 13 of the SCNN1B gene and exon 12 of the Table 4) did not show specific abnormalities of electrolytes, SCNN1G gene, i.e., in the regions where the causative muta- renin and aldosterone levels, or renal function. tions of Liddle’s syndrome are located. Among the three missense mutations we identified in exon 13 of the SCNN1B gene, the P592S mutation was previously Discussion identified in four out of 803 Japanese subjects by SSCP By the direct DNA sequence method, we determined the screening followed by DNA sequencing (13). It has been re- DNA sequence of the 381 bp-coding region in exon 13 of the ported that this missense mutation was not associated with SCNN1B gene and of the 381 bp-coding region in exon 12 of either home or casual blood pressure values. Although we the SCNN1G gene in 948 and 953 Japanese patients with hy- were not able to determine whether this P592S mutation in pertension and identified seven and five mutations, respec- the SCNN1B gene was associated with blood pressure eleva- tively. Several previous studies have screened for mutation tion in the present study, since all our subjects were hyper- in exon 13 of the SCNN1B gene using the single-strand con- tensive, one essential hypertensive patient with the P592S formational polymorphism (SSCP) method (11–13, 15, 16). mutation showed high PRA, low PAC, and normokalemia, SSCP is highly sensitive for detecting polymorphism (25). even though he was taking β blockade and spironolactone. We adopted the direct DNA sequence method instead of the The T594M mutation we identified in the SCNN1B gene SSCP method, because it is more accurate than the SSCP was originally reported in 6.1% of African-American sub- method. A screening of mutations by sequencing was previ- jects but not seen in Caucasians (11). In other studies, this ously performed in a large Japanese cohort including 90 hy- mutation was not observed in Japanese individuals (13, 14). pertensives and 51 controls (24). In that study, however, the So far, therefore, the T594M mutation has been identified authors only sequenced a part of SCNN1B and none of only in individuals of African descent (11–15). However, SCNN1G. Therefore, our study is the first complete, large- our intensive sequence study clearly showed that this muta- scale screening effort to detect mutations in the coding re- tion is also present in the Japanese population, even though Kamide et al: Novel Mutations in SCNN1B and SCNN1G in Hypertensive Patients 337

Table 4. Clinical Proﬁles of Hypertensives with Missense Mutations in Exon12 of SCNN1G

Case 123 Polymorphism Ala578Val Pro603Ser Leu609Phe Age (years old) 70 80 65 Sex female male male BMI (kg/m2) 21.5 25.5 24.4 Diagnosis EHT, NIDDM EHT, NIDDM, HL EHT, EA HT duration (years) 30 32 7 HT family Hx none mother none SBP (mmHg) 134 160 128 DBP (mmHg) 74 110 90 Medication CCB, ACEI, ARB CCB － Na (mEq/l) 143 140 140 K (mEq/l) 3.8 4.2 4.3 Cl (mEq/l) 106 105 106 Creatinine (mg/dl) 0.5 1.2 1.0 Overt proteinuria －＋－ PRA (ng/ml/h) 0.3 1.3 2.8 PAC (ng/dl) 12.2 9.8 15.6 FBS (mg/dl) 136 105 93 HbA1c (mg/dl) 8.6 6.1 4.6 SCNN1G, γ-subunit of epithelial sodium channel gene; BMI, body mass index; EHT, essential hypertension; NIDDM, non-insulin dependent diabetes mellitus; HL, hyperlipidemia; EA, effort angina; HT, hypertension; Hx, history; SBP, systolic blood pressure; DBP, diastolic blood pressure; CCB, calcium channel blocker; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blockade; PRA, plasma renin activity; PAC, plasma aldosterone concentration; FBS, fasting blood sugar.

the prevalence is quite low (2/956, 0.2%). In a study on analysis of the mutant ENaC β subunit with E632K will be black residents in London, a gender difference was observed, needed. with females tending to carry the M allele (18). Notably, two In previous studies, individuals with either one of two individuals having the M allele in our study were also fe- synonymous mutations, Ser520 and Thr594, in exon 13 of males. the SCNN1B gene showed no significant differences in home Whole-cell voltage clamp recording of lymphocytes from or casual blood pressure values, age, body mass index, bio- patients having the variant channel with the T594M mutation chemical profiles of electrolytes or PRA, gender, or use of showed a greater response following stimulation with cAMP antihypertensive medication compared to individuals without analog compared to wild-type lymphocytes (11). Further- mutation (13, 16). In the present study, we confirmed that more, the cells isolated from the homozygote showed that these two mutations did not influence body mass index, bio- phorbol 12-myristate 13-acetate had no effect on cAMP ana- chemical profiles, or use of antihypertensive medications. log-induced electrophysiological responses, indicating that However, we were not able to evaluate these mutations with the T594M mutation plays an important role in regulating respect to blood pressure elevation, since all our subjects ENaC (17). These functional analyses may explain the clini- were hypertensives. cal phenotype of patients with the T594M mutation. The Three hypertensive patients with novel missense muta- T594M mutation contributes to the elevation of blood pres- tions in exon 12 of the SCNN1G gene did not have specific sure and suggests that consideration should be given to the abnormalities in electrolytes or the renin-angiotensin aldos- use of amiloride in affected individuals (19). terone system. However, it was considered worthwhile to In the present study, we found a novel missense mutation, perform functional analysis of these missense mutations in E632K, in exon13 of the SCNN1B gene. As shown in Table the SCNN1G, because single nucleotide polymorphism in the 3, a patient with this missense mutation (case 6) was diag- promoter region has been associated with blood pressure nosed with renovascular hypertension, and showed specific regulation in Japanese (21). abnormalities in potassium level, PRA and PAC. This pa- In summary, we identified three missense mutations, in- tient had taken angiotensin II receptor antagonist and cluding one novel mutation, E632K, in the SCNN1B gene spironolactone, but still had marked hypokalemia. Therefore, and three novel missense mutations, A578V, P603S and to clarify the consequences of this mutation, functional L609F, in the SCNN1G gene, in Japanese patients with hy- 338 Hypertens Res Vol. 27, No. 5 (2004) pertension. in African Americans. J Am Soc Nephrol 1996; 7: 2543– It remains unclear what roles these missense mutations of 2549. the SCNN1B or SCNN1G gene play in the pathogenesis of 12. Persu A, Barbry P, Bassilana F, et al: Genetic analysis of hypertension and the regulation of electrolytes in hyperten- the β subunit of the epithelial Na＋ channel in essential hy- – sive patients. Thus, further investigations, including func- pertension. Hypertension 1998; 32: 129 137. tional analyses, of these mutations will be needed. 13. 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