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Liddle’s Syndrome Caused by a Novel Mutation in the Proline-Rich PY Motif of the Epithelial Sodium Channel ß-Subunit

Second Department of Internal Medicine, Sapporo Medical University School of Medicine (M.F., N.U., Y.Shin., K.S.), Sapporo, Japan; Second Department of Internal Medicine, Obihiro Kosei General Hospital (M.F., Y.Shik., K.-i.S., M.H., N.S., T.N.), Obihiro, Japan; and Department of Nephrology, Kumamoto University Graduate School of Medical Sciences (K.K., M.A., T.M., N.W., K.T.), Kumamoto, Japan

Address all correspondence and requests for reprints to: Dr. Masato Furuhashi, Second Department of Internal Medicine, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan. E-mail: furuhasi{at}sapmed.ac.jp.

	Abstract

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Liddle’s syndrome is an autosomal dominant form of salt-sensitive hypertension and has been shown to be caused by missense or frameshift mutations in the amiloride-sensitive epithelial sodium channel (ENaC), which is composed of three subunits: , ß, and . All disease mutations either remove or alter amino acids of the target proline-rich PPPxY sequence (PY motif) of ß- or -ENaC and result in increased channel activity. In this report, we present a family with Liddle’s syndrome whose abnormality is caused by a novel missense mutation, P616R, in the PY motif of the ßENaC. Functional studies using the P616R mutant expressed in Xenopus oocytes showed an approximately 6-fold increase in the amiloride-sensitive sodium channel activity compared with that of the wild type. These findings provide additional clinical evidence that a conserved PY motif is critically important for the regulation of ENaC activity.

	Introduction

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LIDDLE’S SYNDROME IS an autosomal dominant form of salt-sensitive hypertension characterized by increased plasma volume caused by excessive salt and water reabsorption in the distal nephron, resulting in low levels of plasma renin activity and aldosterone, and increased potassium excretion, resulting in low levels of serum potassium and metabolic alkalosis (1). The administration of an antagonist of the amiloride-sensitive epithelial sodium channel (ENaC), amiloride or triamterene, in combination with a low salt diet corrects these abnormalities, whereas an antagonist of the mineralocorticoid receptor, spironolactone, has no effect. Liddle’s syndrome has been shown to be caused by missense or frameshift mutations in the ENaC. The channel is composed of three subunits: , ß, and (2). The three subunits have similar structures; the N- and C-terminal parts are located in the cytoplasm, with two transmembrane spanning domains and a large extracellular loop. Mutations causing this disease are all clustered in very short segments of the cytoplasmic C termini of either the ß- or -subunit of the ENaC (Table 1). All disease mutations either remove or alter amino acids of the target proline-rich PPPxY sequence (PY motif) and result in increased channel activity (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16).

	Case Report

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The proband was a 41-yr-old female. When she underwent a health check-up at 26 yr of age, high blood pressure was found for the first time. Because of the continuance of high blood pressure (160–170/100–110 mm Hg), treatment with antihypertensive drugs was started. However, there was little blood pressure reduction. At 32 yr of age, she became easily fatigued during her work, and a routine examination showed that her serum potassium level was 2.9 mEq/liter, and blood pressure was 150/90 mm Hg. Supplementation with potassium chloride tablets (32 mEq/d) and administration of spironolactone had no effect on her hypopotassemia, hypertension, or other symptoms.

She was referred to and admitted to Obihiro Kosei General Hospital (Obihiro, Japan) for additional examination of her hypertension. Her weight was 49 kg, height was 160 cm, blood pressure was 160/90 mm Hg, and pulse rate was 88/min. Physical examination revealed no signs of virilization. She had normal genital organs and was not ingesting licorice. There were no abnormalities in her heart sounds or respiratory sounds. An electrocardiogram showed flat T waves consistent with hypopotassemia. No left ventricular hypertrophy was detected by ultrasonic cardiography. The hypertensive change in eye-grounds was Keith-Wagner I.

Treatment with antihypertensive drugs was discontinued, and she was maintained on a regular diet (2000 kcal/d) that included 310 g carbohydrate, 50 g fat, 80 g protein, 120 mEq sodium, and 75 mEq potassium for 2 wk. Her serum potassium level was 2.5–2.9 mEq/liter and urinary excretion of potassium was 50–60 mEq/d. Because her urinary excretion of potassium was sufficient, the possibility of a deficiency of potassium intake was ruled out. Levels of other electrolytes, serum creatinine level, complete blood counts, and results of urinalysis, liver function tests, and serological tests were all within normal ranges. The results of arterial blood gas analysis showed mild metabolic alkalosis. Additional examination revealed suppression of plasma renin activity (<0.1 ng/ml·h) and a low plasma level of aldosterone (43 pg/ml). Plasma renin activity was not elevated 1 h after the administration of 50 mg captopril. Plasma levels of ACTH, cortisol, deoxycorticosterone, and corticosterone and 24-h urinary levels of catecholamines were all within normal limits. A 1-wk trial of dexamethasone (0.5 mg/d) or spironolactone (300 mg/d) had no effect on the hypertension, serum potassium level, or urinary potassium clearance. In contrast, 1-wk treatment with triamterene (150 mg/d) significantly decreased blood pressure and potassium clearance and increased the serum potassium level. The possibilities of 17-hydroxylase deficiency, 11ß-hydroxylase deficiency, and apparent mineralocorticoid excess (type I) were ruled out. Based on the clinical course and laboratory data, she was diagnosed as having Liddle’s syndrome.

The pedigree of this family is shown in Fig. 1. Although the proband’s father (I-1) had already died due to prostate cancer, he had been treated with antihypertensive drugs before his death. The proband’s mother (I-2) had been found to have hypertension at the age of 60 yr. Her recent blood pressure was 140/80 mm Hg during treatment with 40 mg valsartan, an angiotensin II receptor blocker, and her serum potassium level was 3.6 mEq/liter. The younger daughter (III-2) had slight hypopotassemia (3.3 mEq/liter) and low levels of plasma renin activity (0.1 ng/ml·h) and plasma aldosterone (24 pg/ml).

View larger version (22K): [in this window] [in a new window]   FIG. 1. Pedigree of a family with Liddle’s syndrome. Squares and circles represent males and females, respectively. Individuals with the P616R mutation are shown as filled symbols. Individuals lacking the mutation are shown as open symbols. Unexamined subjects are shown by dotted symbols. The proband is shown by the arrow. A deceased individual is shown as a symbol with a diagonal line. Below the symbols, in descending order, are shown the age of subjects, potassium level, plasma renin activity, plasma aldosterone concentration, and the presence of hypertension.

	Materials and Methods

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Venous blood samples for gene analysis were drawn from the proband and her family members after informed consent had been obtained according to the Helsinki guidelines. This study was approved by the institutional review board of Obihiro Kosei General Hospital and Kumamoto University. Genomic DNA was extracted from peripheral blood lymphocytes using a GFX Genomic Blood DNA Purification Kit (Amersham Biosciences, Piscataway, NJ). Primers specific for ß- and -subunits were designed and used to generate PCR products. PCR was performed with Ex Taq Hot Start Version (TaKaRa Bio, Otsu, Japan). PCR products were resolved with electrophoresis in 1.5% agarose gel stained with ethidium bromide and were purified using a GeneClean Spin Kit (Qbiogene, Montréal, Canada). Purified PCR products were subjected to direct sequencing using a deoxy-GTP BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). A newly identified mutation in the ß-subunit of the ENaC was introduced into cDNA of the wild type by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). A mutation generated by site-directed mutagenesis was confirmed by DNA sequencing in both forward and reverse orientations, then the mutant was used for subsequent functional studies.

Electrophysiological studies

cDNAs of the human ENaC -, ß-, and -subunits in pcDNA3 were linearized with digestion of NotI. cRNAs were synthesized with an mMESSAGE mMACHINE T7 ULTRA Kit (Ambion, Inc., Austin, TX). Stage 5–6 Xenopus oocytes were treated with 1 µg/ml collagenase type I (Sigma-Aldrich Corp., St. Louis, MO) for 1 h to remove follicle cell layers and then injected with 1 ng cRNA for each subunit in 25 nl water. Injected oocytes were kept at 19 C in modified Barth’s saline [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 15 mM HEPES, 0.3 mM Ca(NO₃)₂, 0.41 mM CaCl₂, and 0.82 mM MgSO₄]. Electrophysiological studies were performed 16–24 h after cRNA injection using the two-electrode voltage clamp technique. The amiloride-sensitive sodium current was determined by subtracting the difference in current values before and after the application of 5 µM amiloride to the bath solution. The current was recorded at room temperature (22–25 C) and at a holding potential of –100 mV in a solution containing 96 mM sodium gluconate, 2 mM potassium gluconate, 1.8 mM CaCl₂, 10 mM HEPES (pH 7.2), 5 mM BaCl₂, and 10 mM tetraethylammonium chloride.

Statistical analysis

Numeric variables of the amiloride-sensitive sodium current in the electrophysiological studies are expressed as the mean ± SEM of eight experiments. The difference between two unpaired variables of the amiloride-sensitive sodium currents in Xenopus oocytes expressing wild-type or ßP616R mutant ENaC was analyzed by unpaired t test. P < 0.05 was considered statistically significant.

	Results

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Detection of mutation in ENaC genes

To identify a mutation in the ENaC gene in this family, genomic DNAs from each family member were obtained, and DNA sequencing of each exon in ß and ENaC genes was performed. As shown in Fig. 2, a novel point mutation in exon 13 of the ßENaC gene in which ⁶¹⁶Pro (CCC) was changed to ⁶¹⁶Arg (CGC), P616R, was detected. This mutation in the patient (II-1) was also found in her younger daughter (III-2). The proband’s mother (I-2), who was hypertensive, did not carry this mutation. Genomic DNA from her father (I-1), who had a past history of hypertension treated with antihypertensive drugs, could not be obtained because he had already died of prostate cancer.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Identification of mutation by direct sequencing of the ßENaC. The C terminus of ßENaC, amplified by PCR, was directly sequenced in a normal control subject (A) and in the proband (B). The sequence extending from codons 613–619 is shown. As indicated by underlining, transition from C to G in the second base of codon 616 was observed in the proband. This point mutation changes codon 616 from Pro to Arg (P616R) in an affected sequence.

Functional studies were performed to determine whether the mutation identified in this family increases ENaC activities. The P616R mutation was introduced in ßENaC, and amiloride-sensitive sodium currents were measured in a Xenopus oocytes expression system. As shown in Fig. 3, the P616R mutation caused an approximately 6-fold increase in sodium channel activity compared with that of the wild type.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Amiloride-sensitive sodium currents in Xenopus oocytes expressing wild-type or ßP616R mutant ENaC. cRNA encoding the - and -subunits and either normal or mutant ß-subunit of the human ENaC were coinjected into Xenopus oocytes. The activity of ENaC was measured as the amiloride-sensitive inward sodium current. *, P < 0.0001 vs. wild-type.

	Discussion

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Mutations of the ⁶¹⁴PPPxY⁶¹⁸ sequence (PY motif) of the C-terminus of the ßENaC have been reported in four kindreds to date. Hansson et al. (11) reported a family with Liddle’s syndrome who had a missense mutation of ⁶¹⁶Pro (CCC) to ⁶¹⁶Leu (CTC), P616L, and they demonstrated that this single amino acid substitution resulted in an 8.8-fold increase in sodium channel activity. They concluded that a proline-rich segment is critical for the regulation of channel activity. Tamura et al. (15) reported a family with Liddle’s syndrome who had a missense mutation of ⁶¹⁸Tyr (TAT) to ⁶¹⁸His (CAT), Y618H, and demonstrated that increased ENaC activity was functionally equivalent to the mutation at Arg⁵⁶⁴, R564X, identified in the original Liddle’s kindred. Inoue et al. (10) described a missense mutation of ⁶¹⁵Pro (CCC) to ⁶¹⁵Ser (TCC), P615S, in four affected members of a Liddle’s kindred and showed that the mutation resulted in an approximately 3-fold increase in the amiloride-sensitive sodium current. Uehara et al. (12) reported a family with Liddle’s syndrome who had a missense mutation of ⁶¹⁶Pro (CCC) to ⁶¹⁶Ser (TCC), P616S. In the present study we showed that a novel missense mutation changing ⁶¹⁶Pro (CCC) to ⁶¹⁶Arg (CGC) of the ßENaC resulted in an approximately 6-fold increase in sodium channel activity when the mutant was expressed in Xenopus oocytes and that it caused Liddle’s syndrome.

Proline-rich motifs, such as the PY motif, are generally involved in protein-protein interactions. The PY motif of the ßENaC was shown to interact with a cytosolic protein called Nedd4 (17). In the Xenopus oocyte expression system, Nedd4 protein is an ubiquitin ligase that controls the number of active ENaC channels at the cell surface (18); ENaC ubiquitination, a posttranslational modification that adds small ubiquitin proteins on the N terminus of the ENaC, represents a signal for rapid endocytosis and degradation of the ENaC in the cell. Disruption of the PY motif impairs ENaC retrieval from the plasma membrane and causes retention of active ENaC channels at the cell surface. This is the likely mechanism responsible for the increased sodium reabsorption in the distal nephron in patients with Liddle’s syndrome (19).

Because the siblings on the paternal side and the proband’s brother (II-2) refused to undergo tests for genetic diagnosis, our genetic survey of this family was not complete. Because the proband’s mother (I-2), who was hypertensive, did not carry this mutation, it can be speculated that the proband’s hypertensive father (I-1) had the mutation, and that the proband (II-1) inherited this condition from her father. Because, however, we could not obtain genomic DNA from her father (he had already died due to prostate cancer), the possibility that the proband (II-1) is a sporadic case that occurred as a de novo mutation cannot be ruled out. Some sporadic cases of Liddle’s syndrome have been previously reported (8, 11, 12, 14).

Although the proband’s 6-yr-old younger daughter (III-2) with P616R had slight hypopotassemia and low levels of plasma renin activity and plasma aldosterone, she was not hypertensive at the time of this study. This is consistent with the finding in a Liddle’s original pedigree that hypopotassemia and hypertension are not universal in affected members (4). Normotensive family members were found in the kindred of the original Liddle’s syndrome index case. We speculate that the degrees of hypertension, hypopotassemia, and suppression of plasma renin and aldosterone are influenced not only by gene mutations, but also by aging and environmental factors, such as dietary salt intake. Young age and low salt intake may explain the lack of high blood pressure, even in the presence of an activating ENaC mutation.

In summary, we have described a family with Liddle’s syndrome caused by a novel missense mutation, P616R, in the proline-rich PY motif of the ßENaC. Functional studies using the P616R mutant expressed in Xenopus oocytes showed an approximately 6-fold increase in the amiloride-sensitive sodium channel activity compared with that of the wild type. These findings provide additional clinical evidence that a conserved PY motif is critically important for the regulation of ENaC activity.

	Footnotes

 
First Published Online October 13, 2004

Abbreviation: ENaC, Epithelial sodium channel.

Received May 31, 2004.

Accepted September 23, 2004.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 90/1/340    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Furuhashi, M. Articles by Shimamoto, K. Search for Related Content PubMed PubMed Citation Articles by Furuhashi, M. Articles by Shimamoto, K. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM Protein UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-PROLINE Medline Plus Health Information High Blood Pressure Related Collections Adrenal and Hypertension