Liddle's syndrome: Heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel

Cell, Vol. 79, 407-414,

November

4, 1994, Copyright

0 1994 by Cell Press

Liddle’s Syndrome: Heritable Human Hypertension Caused by Mutations in the p Subunit of the Epithelial Sodium Channel Richard A. Shimkets,’ David G. Warnock,2 Christopher M. Bositis,’ Carol Nelson-Williams,’ Joni H. Hansson,’ Morris Schambelan,3 John R. Gill, Jr.,“ Stanley Ulick,5 Robert V. Milora,6 James W. Findling,’ Cecilia M. Canessa,8 Bernard C. Rossier,* and Richard P. Lifton’ lHoward Hughes Medical Institute Departments of Medicine and Genetics Boyer Center for Molecular Medicine Yale University School of Medicine New Haven, Connecticut 06510 2Departments of Medicine and Physiology and Biophysics University of Alabama and Birmingham Veterans’ Affairs Medical Center Birmingham, Alabama 35294-0007 %epartment of Medicine San Francisco General Hospital and University of California, San Francisco San Francisco, California 94110 4National institutes of Health National Heart, Lung, and Blood Institute Hypertension-Endocrine Branch Bethesda, MD 20892 5Veterans’ Affairs Hospital Bronx, New York 10468 “Capital District Renal Physicians Albany, New York 12208 Saint Luke’s Medical Center Medical College of Wisconsin Miilwaulkee, Wisconsin 83215 %stitut de Pharmacologic et de Toxicologic Universite de Lausanne 7 005 Lausanne S~i~~erla~d

Summary Liddle’s syndrome (pseudoaldosteronism) is an autosomal dominant form of human hypertension characterized by a constellation of findings suggesting constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel. We demonstrate complete linkage of the gene encoding the p subunit of the epithelial sodium channel to Liddle’s syndrome in Liddle’s original kindred. Analysis of this gene reveals a premature stop codon that truncates the cytoplasmic carboxyl terminus of the encoded protein in affected subjects. Analysis of subjects with Liddle’s syndrome from four additional kindreds demonstrates either premature termination or frameshift mutations in this same carboxy-terminal domain in all four. These findings demonstrate that Liddle’s syndrome is caused by mutations in the fi subunit of the epithelial sodium channel and have implications for the regulation of this epithelial ion channel as well as blood pressure omeostasis.

Introduction Hypertension is a common disorder of largely unknown cause, imparting an increased risk of stroke, myocardial infarction, and renal failure. Concordant evidence from studies of twins, familial aggregation, and
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when expressed alone in Xenopus laevis oocytes (Canessa et al., 1993) and p and y, which support only extremely low sodium conductance when expressed alone, but which greatly augment sodium conductance when expressed in conjunction with the a subunit (Canessa et al., 1994a). These three subunits show sequence similarities to one another, indicating descent from a common ancestral gene. Each encoded protein contains two transmembrane domains, with intracellular amino and carboxyl termini (Renard et al., 1994; Canessaet al., 1994b). A family of related genes, which when mutated cause mechanosensory transduction defects, has been identified in Caenorhabditis elegans (Driscoll and Chalfie, 1991; Huang and Chalfie, 1994). We report here identification of complete linkage of Liddle’s syndrome to the locus encoding the b subunit of the epithelial sodium channel in Liddle’s original kindred as recently reevaluated and extended (Botero-Velez et al., 1994). Analysis of this gene reveals a premature stop codon within the cytoplasmic carboxyl terminus of the encoded protein in affected subjects. Affected subjects from four additional Liddle’s syndrome kindreds are all shown to have either premature stop codons or frameshift mutations in the carboxy-terminal domain of the f3subunit gene. These findings demonstrate the molecular basis of an inherited form of hypertension and strongly motivate further study of these genes in the hypertensive population. In addition, the nature of these mutations has implications for the mechanism by which activity of this epithelial ion channel is regulated. Results

Figure 1. Characterization of the Human Locus Encoding the f3 Subunit of the Epithelial Sodium Channel (A) Maps of overlapping cosmid clones. Maps of the human genomic DNA inserts of eight independent cosmid clones that hybridize to the cDNA of the f3 subunit of the rat epithelial sodium channel are shown. The horizontal bar adjacent to each indicated clone represents the span of the cloned segment; the locations of BamHl sites on each insert are represented by vertical bars; the different cloned segments are drawn with their overlaps indicated. At the top of the figure, the segment that hybridizes to probe BENaCGT-1 is indicated. At the bottom of the figure, genomic fragments that hybridize to four contiguous segments spanning the rat 9 cDNA are indicated by thick horizontal bars, and the transcriptional orientation of the gene is shown. (6) Sequence of genomic DNA encoding the carboxyl terminus of hf3ENaC. The sense strand of the human genomic DNA sequence from the exon encoding the carboxy-terminal segment of the j3 subunit of the epithelial sodium channel and a portion of the proximal intron is shown; the intron-exon junction is indicated by the arrow. Nucleotides that are identical to or different from those found in the corresponding position of the rat b subunit cDNA (Canessa et al., 1994a) are shown in capital or lowercase letters, respectively. The deduced encoded amino acid sequence is shown below the DNA sequence in single letter code: encoded residues that are different in the rat are indicated below the human sequence. Amino acids are numbered according to the rat sequence. The residues comprising the second

Cloning of the Human Homolog of rpENaC The pathophysiology of Liddle’s syndrome suggests constitutive activation of the epithelial sodium channel in the absence of circulating mineralocorticoids, motivating the cloning and development of genetic markers in human homologs of the channel subunits and other genes whose products are involved in the regulation of this channel. Cosmid clones spanning the human [3 subunit locus (hf3ENaC) were identified by hybridization of the rat 6 subunit cDNA (rpENaC; Canessa et al., 1994a) to a human cosmid library. Eight independent clones were isolated; maps of these clones were constructed, indicating that all

transmembrane domain of the encoded protein (Canessa et al., 1994b) are underlined. This sequence has been deposited in GenBank. (C) Hybridization of the segment encoding the carboxyl terminus of hf3ENaC to human genomic DNA. Genomic DNA from one affected (A) and one unaffected subject (N) of KIOO was separately digested with BamHI, EcoRI, or Hindlll, fractionated via agarose gel electrophoresis, and transferred to nylon membranes. A segment of the carboxyterminal exon of hf3ENaC was labeled and hybridized to the filter. A single genomic fragment hybridizes to the probe after digestion with each enzyme; the size of each fragment was determined by comparison to the products of digestion of phage lambda DNA with Hindlll and is indicated to the left of the autoradiogram. These hybridizing fragments are indistinguishable in the two subjects; fragmentsof identical size that hybridize to this probe are produced by digestion of chf3ENaCll with these same enzymes.

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Basis of Liddle’s Syndrome

#Repeats

Figure 2. Alleles Cetected by Marker pZVaCGT-7 Primers defining the markerpENaCG%7 were used to direct PCR using genomic DNA of unrelated subjects as template; the products of PCR were fractionated on denaturing polyacrylamide gels and exposed to X-ray film as described in Experimental Procedures. The number of GT repeats present on specific alleles was determined by comparison to the sequenced cloned segment derived from cosmid chPEnaCl2 and are indicated lo the left of the autoradiogram. In subsequent linkage studies, alleles are numbered consecutively based on decreasing length.

eight clones overlap and define a single contig of 75 kb (Figure 1A). Distinct contigs were defined by use of the rat a and y subunit cDNAs as probes (data not shown). Confirmation that this locus encodes the human homolog of the p subunit of the epithelial sodium channel gene was obtained by subcloning cosmid Sau3Al fragments that hybridize to the rat cDNA and subjecting these clones to DNA sequencing. This analysis revealed multiple exon fragments all showing strong homology to the DNA and protein sequence of @ENaC. The peptide segment extending from the H2 hydrophobic domain (Canessa et al., 1994a) through the carboxyl terminus is encoded in a single exon that shows 79% identity with the rat cDNA sequence and 81% identity with the rat protein sequence (Figure IB). Hybridization of the carboxy-terminal exon segment to human genomic DNA digested with BarnHI, Hindlll, or EcoRl demonstrated homology to asinglegenomic fragment of a size identical to that derived from the cosmid in each digest, confirming the presence of a single locus encoding this gene in the human genome (Figure 1C). Development of a Genetic Marker in hPENaC The cosmid contig was screened for (CT), repeats by hybridization of a (GT)?,, oligonucleotide to Sau3Al digests

of cosmid DNA. A strongly hybridizing fragment was subcloned and subjected to DNA sequencing. Primers flanking the identified GT repeat were synthesized and used to direct polymerase chain reaction (PCR) with DNA from unrelated individuals as template. This marker, /3ENaCGT-7, is polymorphic, with five alleles and 63% heterozygosity found in analysis of 50 unrelated Caucasian subjects (Figure 2). The PCR product amplified by these primers was localized by hybridization to an intron within the gene in independent clones (see Figure lA), confirlming that this GT repeat is linked to the p subunit locus in gienomic DNA. Complete Linkage of hpENaC and Liddle!‘s Syndrome in KlOO KlOO is Liddle’s original kindred as recently reevaluated; patients were classified as reported previously (Liddle et al., 1963; Botero-Velez et al., 1994). Thirteen subjects in this kindred who were diagnosed as frankly hypertensive before the age of 21 were classified as affected. Four subjects who were known to be severely hypertensive and who died before age 40 from cerebrovascular or cardiovascular disease were classified as affected; three of tlhese subjects had affected offspring, further supporting the diagnosis. Three subjects under age 20 who had blood pressures greater than 98% of the population for age and sex and who had low urinary aldosterone:K+ ratios were classified as affected. Subjects who were not known lo be hypertensive and had normal blood pressure at evaluation as well as normal serum K+ levels and normal aldoeterone:K+ ratios were classified as unaffected. Three patients were classified as affection status unknown, owing to normotension or borderline hypertension with low ratios of urinary aldosterone:K+ (Botero-Velez et al., 1994). The marker PENaCGT-7 was genotyped in this kindred, and the segregation of the marker was compared with the segregation of Liddle’s syndrome (Figure 3). Two models of inheritance were prospectively defined: a conservative model, wit,h 95% penetrance and a 2% phenocopy rate, which will ,accomodate a low degree of phenotypic misclassification, and a stringent model, with 100% penetrance and zero phenocopies. Analysis demonstrates complete linkage of the marker with Liddle’s syndrome, regardless of the model, with peak lod aoores of 7.26 and Figure 3. Linkage of hPENaC: and Syndrome in K10O

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The family relationships of members of KlOO are shown. Individuals classified as affected and unaffected with Liddle’s synidrome are indicated by closed and open symbols, respectively. Individuals who were classified as affection status unknown are indicated by stippled symbols. Diagonal iines indicate deceased members of the kindred. Two spouses who were not availaple for evaluation are indicated by dotted symbols. Below each symbol is indicated, in descending order, thle generationspecific relative identification number, the genotype for BENaCGT-I, and the genotype for the SSCP variant identified in this kindred (see Figure5); thecommonSSCPvariant in thepopulation is denoted as allele 1 and the novel SSCP variant iri KlQO is denoted allele 2.

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Liddle'slocus

Table 1. Pairwise Lod Scores and Recombination Fractions for Linkage of Liddle’s Syndrome with @N&G%7 and Chromosome 16 Loci Stringent Model

Conservative Model

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of the Liddle’s Trait Locus on the Genetic Map

The segment of the genetic map of human chromosome 16p (Gyapay et al., 1994) showing linkage to /3ENaCGT-7 and Liddle’s syndrome is represented as the thin horizontal bar. The location of markers and the recombination fractions between them are indicated. The location of /3ENaCGT-7 determined by multipoint linkage analysis in CEPH kindreds and KlOO is indicated below the map; this marker shows zero recombination with 0765420. The maximum likelihood location of DENaCGT-7 is indicated by the arrow; the 95% confidence interval for the location of /3ENaCGT-7 within this interval spans a recombination fraction of 3% and is indicated by the short thick horizontal bar above the arrow. Above the map, the multipoint analysis of linkage of Liddle’s syndrome to this region in KIOO is shown; analysis was performed with the trait locus and markers D76S472, PEN&G%7, and D76S407. The conservative and stringent models of inheritance defined in the text are shown as the solid and dashed lines, respectively. Liddle’s syndrome shows a peak lod score at zero recombination with pfNaCGT-7 under both models (indicated by the arrow), with odds of greater than 1OOO:l favoring location of this gene in the interval flanked by D76S472 and D76S407; the 95% confidence interval for location spans a recombination fraction of 4%, indicated by the thick horizontal bar below the arrow.

7.62 at a recombination fraction of zero under the conservative and stringent models, respectively (odds ratios in favor of linkage 1.82 x 1 O7to 1 and 4.17 x lo7 to 1). The use of this single marker, nonetheless, leaves a relatively wide 95% confidence interval (recombination fraction of 20%) for the location of the trait locus. Further refinement of the location of the Liddle’s trait locus was sought by locating h8ENaC on the human genetic map in order to permit testing linkage with flanking markers. /3ENaCGT-7 was initially localized by use of a somatic cell hybrid panel, which demonstrated that only hybrids containing human chromosome 16 supported amplification of this marker (data not shown). Confirmation of this location and refinement of the position on chromosome 16 were obtained by linkage analysis in the Centre d’Etudes du Polymorphisme Humain (CEPH) reference pedigrees as well as Kl 00. jENaCGT-7 showed strong linkage to a cluster of pericentromeric markers on 16p, with a pairwise lod score of 15.3 with D76S420 at a recombination fraction of zero. Multipoint analysis demonstrated thatfiE/VaCGT-7 lies in the interval defined byflanking markers D76S472 and D76S407, with odds favoring location in this interval of greater than 1OOO:l (Figure 4). Pairwise analysis of linkage with chromosome 16 mark-

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Loci are shown in their chromosomal order on 16~ (Gyapay et al., 1994); conservative and stringent models of inheritance are as specified in the text. z,,, maximum lod score; 6, estimated recombination fraction at .zmax.

ers confirmed linkage of Liddle’s syndrome to this chromosomal region (Table 1). Multipoint analysis comparing the segregation of Liddle’s syndrome with the segregation of /3ENaCGT-7 and flanking markers demonstrates that these flanking markers show recombination with both PfNaCGT-7 and Liddle’s syndrome in KlOO (Figure 4); genotypes of additional flanking markers confirm the presence of these meiotic recombinants. This analysis places the Liddle’s locus in the interval flanked by D76S472 and D76S407, with odds of greater than 1OOO:l; the resulting 95% confidence interval localizes the Liddle’s gene to a 4 CM region spanning the 6 subunit locus, with peak lod scores of 8.6 and 9.0 at zero recombination with PENaCGT-7 under conservative and stringent models of inheritance, respectively (Figure 4). A Stop Codon in hPENaC in KlOO The finding of complete linkage of h8ENaC with Liddle’s syndrome strongly motivated a search for the functional mutation in this gene in affected patients. The finding that dominant gain-of-function mutations as well as recessive mutations affecting mechanosensory transduction lie in or near the second transmembrane domain of related genes in C. elegans suggests that this domain comprises the functional ion channel (Driscoll and Chalfie, 1991; Huang and Chalfie, 1994; Hong and Driscoll, 1994) and prompted us to target this portion of the human gene. Segments of the final protein-coding exon of the gene (see Figure 1 B) were screened for molecular variants by singlestrand conformational polymorphism (SSCP). A novel SSCP variant was identified in all affected patients of Kl 00 (Figure 5), but was absent in all unaffected members (lod score of 8.6 and 9.0 at 8 = 0 for linkage with Liddle’s syndrome under alternative models). This variant was not detected by SSCP using genomic DNA of 250 unrelated subjects not known to have a diagnosis of Liddle’s syndrome. Direct sequence analysis of this variant demonstrates a single base substitution, a C-T transition at the first nucleotide of codon Arg-564 of the gene (Figures 6A and 6E). This mutation introduces a stop codon at this position, thereby deleting the last 75 amino acids from the encoded

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Basis of Liddle’s Syndrome

Figure 5. identification Syndrome by SSCP

of Molecular Variants in Patients with Liddle’s

Specific primers were used to amplify a segment of the exon encoding the carboxyl terminus of hj3ENaC from genomic DNA and analyzed on MDE gels at 4% as described in Experimental Procedures. The product from two representative unrelated subjects who do not have Liddle’s syndrome are shown in lanes labeled Normal. The products from patients with Liddle’s syndrome in KlOO, KlOl, K172, K175, and K176 are shown; the samples labeled KlOO-A and KtOO-B are from subjects Ill-6 and IV-12 in Figure 3, respectively. Variant bands that are unique to Liddle’ssyndrome patients are indicated by arrows; these same variants were observed in three independent amplified products from each subject. The novel variant in Ki 75 was better separated from the wild-type conformer on MDE gels fractionated at room temperature (data not shown) and was used as the template for sequence analysis.

protein. This truncation leaves the second transmembrane domain intact, but removes virtually the entire cytoplasmic carboxyl tail of the protein. That this striking variant is completely linked to the trait locus and is not found in unaffected subjects strongly argues that this is the functional mutation causing Liddle’s syndrome. Stop Codons and Frameshifts in Additional Liddle’s Kindreds Samples from affected patients from four additional independently ascertained kindreds with Liddle’s syndrome were studied; two affected patients from both K101 and K172 and single affected subjects from K175 and K176 were studied. All of these subjects had undergone extensive clinical evaluation, resulting in a definitive prospective diagnosis of Liddle’s syndrome; clinical evaluation of one of these kindreds has been previously reported (Gardner et al., 1971). In each case, the diagnosis of Liddle’s syndrome was made on the basis of early onset of hypertension with hypokalemia, suppressed levels of circulating mineralocorticoids, improvement of hypertension and hypokalemia in response to triamterene or amiloride but not spironolactone, and occurre8nce in at least two first degree relatives Patient DNA samples were screened by SSCP of the carboxy-terminal exon. SSCP variants were identified in samples from all four kindreds (see Figure 5); in one of these (K172), the variant comigrated with the variant seen in K100, while the other three variants were unique. None of these additional variants were seen in 250 control subjects. Sequence analysis demonstrated that the mutation in K172 is indeed’ identical to the mutation seen in KlOO; this variant is also seen in the affected daughter of the index case. Analysis of five highly polymorphic flanking markers in these two patients suggests an identical haplo-

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Figure 6. Sequence of Mutations Identified in Liddle’s Syndrome dreds

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SSCP variants identified in Figure 5 were subjected to direct DNA sequence analysis as described in Experimental Procedures. The sequence of independent variants is contrasted with normal sequence in (A)-(D). In each case, the sequence of the wild-type allele is shown above the sequence of the mutant allele; in all cases, the 5’ to 3’ orientation of the sequence is left to right. The effects of the observed mutations on the encoded protein are shown in (E). (A) Variant in KIOO; the sequence of the antisense strand is shown, and the variant base is indicated by an asterisk. (B) Variant in Ki 75; the sequence of the antisense strand is shown; the variant base is indicated by an asterisk. (C)Variant in K101; thesequenceof thesensestrand isshown, demonstrating the deletion of a single cytosine. (D) Variant in Kl76; the sequence of the antisense strand is shown, demonstrating the insertion of a single guanosine. (E) A portion of the encoded carboxy-terminal amino acid sequence of the human b subunit of the epithelial sodium channel is shown; amino acid residues are numbered as in Figure i B. Codons altered by mutations in Liddle’s syndrome patients from indicated kindreds are shown; the normal DNA sequence as well as the mul!ant sequence is shown. The consequence of each mutation is indicated.

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type linked to the disease locus in both K172 and KlOO (data not shown), strongly suggesting that the mutations in these kindreds are descendent from the same ancestral mutation. The three remaining kindreds show novel mutations. In K175, a C-+T transition mutates Gln-589 to a stop codon (Figures 8B and 6E), truncating the encoded protein by 50 amino acids. In K176, the insertion of a single cytosine residue introduces a frameshift mutation into the encoded protein at Thr-592 (Figures 6D and 6E), altering the encoded protein from codon 593 through a new termination at codon 605. In KlOl, the SSCP variant is due to deletion of a single cytosine residue from a sequence of six consecutive cytosines in codons 593-595 (Figures 6C and 6E). This frameshift mutation alters the encoded protein from codon 595 through the new stop codon at position 673. The presence of these two frameshift mutations in genomic DNA was confirmed by electrophoresis of end-labeled PCR products on denaturing polyacrylamide gels, demonstrating products either one base longer or one base shorter than the wild-type sequence in these kindreds (data not shown). Discussion We have demonstrated complete linkage of a premature stop codon in the 8 subunit of the epithelial sodium channel with Liddle’s syndrome and have shown that similar termination or frameshift mutations are present in four additional Liddle’s syndrome kindreds. None of these mutations have been observed in 250 unrelated subjects. These findings constitute proof that these mutations cause Liddle’s syndrome. The physiological evidence and the dominant mode of inheritance suggest that the observed mutations cause constitutive activity of the epithelial sodium channel. The finding that all four independent mutations remove overlapping segments of the normal carboxyl terminus of this protein by either deletion or frameshift consequently implicates this segment in the normal regulation of channel activity. The tight clustering of all the mutations identified to date to a 95 bp segment of the cytoplasmic carboxyl terminus of the 8 subunit gene suggests that the target for mutations that produce Liddle’s syndrome is very small, i.e., mutations far proximal or distal to the observed sites would not have the Liddle’s syndrome phenotype. The evidence for a constitutively active epithelial sodium channel suggests the necessity of an intact second transmembrane domain, which is thought to be part of the functional ion channel. This likely defines a proximal boundary for mutations that produce the Liddle’s phenotype. Given that the functional epithelial sodium channel is composed of at least three subunits of homologous StrUCture, it is of interest that all Liddle’s kindreds studied to date have mutations in the f3 subunit. This observation suggests that the cytoplasmic carboxyl termini of these proteins are not functionally equivalent, rather that the 8 subunit plays a unique role in the normal regulation of epithelial sodium channel activity. A role for mutation in

the a subunit gene in Liddle’s syndrome in KIOO has been directly excluded, both from the location of this gene on a different chromosome, 12p (Voilley et al., 1994), as well as by linkage analysis using polymorphic markers in this gene (lod score for linkage with Liddle’s syndrome -12; data not shown). Similarly, additional candidate genes including the mineralocorticoid receptor and the G protein Goi3 have been excluded by linkage (data not shown). How the observed mutations in the 8 subunit gene might lead to abnormal regulation of channel activity is at present a matter of speculation, since the normal mechanism(s) of regulation of this channel is thus far poorly understood. The most distal mutation truncating the protein removes only the last 50 amino acids from the encoded protein; the most distal frameshift eliminates only the last 43 normal amino acids from the encoded protein, narrowing the functional target of the mutations. A novel SH8binding domain, localized to the cytoplasmic carboxyl terminus of the a subunit of this channel, has recently been defined and shown to mediate binding to a-spectrin, potentially providing a means of anchoring this channel to the apical membrane of cells (Rotin et al., 1994). The segment mediating this binding is a short proline-rich domain (PPLALTAPPPA; Rotin et al., 1994). A similarly proline-rich domain is present in the region affected by all the Liddle’s mutations; this domain, extending from codon 609 to codon 620 (sequence PIPGTPPPNYDS), is completely conserved between rat and human and contains potential sites for protein phosphorylation (Figure 1 B). Thisobservation raises the possibility that this site in the 8 subunit is involved in some aspect of the regulation of trafficking of the complex to and/or from the apical plasma membrane. It is noteworthy that regulated interaction of this channel with the cytoskeleton has been proposed as a mechanism of regulation of channel activity (Smith et al., 1991; Prat et al., 1993). Alternative, though not mutually exclusive, models could invoke one (or more) of the following: a site in the truncated domain that is normally modified (e.g., by phosphorylation) to inactivate the channel, a segment to which another protein that directly inactivates the channel normally binds, or a domain that is normally directly involved in maintaining the channel in a closed state. A host of additional models invoking effects on channel assembly, recruitment, or stability are tenable at present. Functional studies of mutant channel complexes, as well as biochemical studies to identify proteins that interact with this carboxy-terminal domain will clearly be of value in evaluating these possibilities. In this regard, it is of interest that amiloride-sensitive sodium channels can be detected in immortalized B cells (Bubien and Warnock, 1993; BUbien et al., 1994); in Liddle’s patients, these channels appear to be constitutively activated, suggesting that these cells may serve as a model for the disease (Bubien et al., unpublished data). Liddle’s syndrome has been thought to be a rare disorder, with a very small number of cases reported in the worlds literature (Botero-Velez et al., 1994). It is likely, however, that the disorder is underdiagnosed, since neither severe hypertension nor hypokalemia are obligatory,

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and there are no pathognomonic features to assist in the diagnosis. Consequently, the disease is most commonly recognized when hypertension and hypokalemia are found in multiple family members and other disorders are excluded. Nonetheless, the present study suggests that the genetic target for mutations causing Liddle’s syndrome is very small, implying that de novo mutations will be very rare. It will be of interest to determine the spectrum of mutations causing this disorder in additional kindreds. Moreover, given that the mutations causing this disorder appear to cluster within a small region of the f3 subunit, a rapid genetic screening test for this disorder among hypertensive subjects can be envisioned, permitting identification of previously unrecognized kindreds. In addition, screening for specific mutations in at-risk relatives of affected subjects can be used to identify previously unrecognized cases within families, permitting assessment of the natural history and phenotypic spectrum of the disorder. The identification of mutations in this channel gene family that cause increased sodium reabsorption in the distal nephron firmly establishes that this channel is indeed the functional mineralocorticoid-sensitive epithelial sodium channel in humans. This finding suggests that mutations in subunits of this channel will prove in some cases to be the cause of another disorder of salt and water metabolism, pseudohypoaldosteronism, a salt-wasting disorder of infancy that behaves as though this channel is nonfunctional or unresponsive to mineralocorticoids (Kuhnle et al., 1990). Finally, it is noteworthy that increased sodium reabsorption via this channel appears to play a role in the pathogenesis of many of the known secondary causes of hypertension in humans. It might consequently prove surprising if variants in the genes encoding subunits of this channel do not contribute to the pathogenesis of some more common forms of hypertension. Hypertensive patients with low plasma renin activities may be a particularly interesting group for further study. Knowledge that these cloned genes constitute the functional channel in vivo and that mutation in these genes can cause hypertension strongly motivates careful examination of these genes as candidates in patients with essential hypertension. Experimental

Procedures

Cloning and Characterization of the Human Genomic j3ENaC A human genomic cosmid library was prepared by ligating sizeselected human genomic DNA partially digested with Sau3Al into the Xhol site of vector pWeX, a derivative of pWel.5 following filling in of the first two bases ofvector and insert. An 8-haploid genome equivalent library was plated and transferred to nylon membranes, and the DNA was denatured in situ by standard methods. The rat 6 subunit cDNA (Canessa et al., 1994a) was radiolabeled by random priming (Feinberg and Vogelstein, 1983) denatured, hybridized to the filters at low stringency (5 x SSC, 40% formamide, 10 m M sodium phosphate [pH 7.01, 5 x Denhart’s solution, 200 ug/ml salmon sperm DNA, 5% dextran sulfate, at 4Z°C), washed at a final stringency of 0.1 x SSC, 0.1% SDSat42°CandexposedtoX-rayfilm. DNAfrom identifiedcloneswas characterized by separate digestion with BamHl and EcoRl followed by eledtrophoresis on agarose gels and hybridization with four contiguous segments of the rat 8ENaC cDNA prepared using specific oligonucleotide’primers and the @EN& cDNA (Canessa et al., 1994a) as a tem-

plate in PCR. Maps of the clones defining this contig were constructed from their overlapping fragments and by inter se hybridization of specific fragments; maps were confirmed by analysis of the products of partial digestion (Evans and Wahl, 1987). Human genomic Southern blots were performed by hybridization of the 3’ promin-coding exon of the human j3subunit gene to human genomic DNA at high stringency (the same conditions described above, except the hybridization buffer contained 50% formamide and the final wash temperature was 60°C). Exon fragments were subcloned by digestion of cosmid DNA to completion with SaudAl and ligation of products into 13amHI-digested pBluescript; resultant clones hybridizing to the rat [I subunit cDNA were isolated and subjected to DNA sequence analysils by the dideoxy terminator method using an ABI 373 instrument following a standard protocol. Marker Development, Genotyping, and Linkage Alnalysis Clones bearing Sau3Al products of the cosmid contig cloned in pBluescript were screened for GT repeat sequences by liybridization to (GT)ro oligonucleotide; labeling of the oligonucleotide was performed using ]Y-~‘P]ATP and polynucleotide kinase. Hybridization was performed in 7% polyethylene glycol, 10% SDS at 42OC followed bywashing in 6 x SSC, 0.1% SDS at 42’C. Clone p8ENaCGT-2 was subjected to DNA sequence analysis and demonstrated a continuous array of (GT),.,; primers flanking this simple sequence repeat were designed and used to direct PCR from unrelated subjects (primer FENaCGT-A: 5’-CATGCTGAAGATGGACACGTG-3’; BENaCGT-B: SVZxZAAATGCACAGGAGCTGTTC-37. Genomic DNA was prepared from venous blood of patients by standard methods (Bell et al., 1!381). PCR was conducted in 10 ul containing 50 m M KC!, 1.5 m M MgC&, 5 m M Tris (pH 8.3) 125 pM dNTPs, 0.01% gelatin, 0.5 U of Taq polymerase, and 0.5 pM of each primer, with one primer labeled at the 5’ end with 3*P, and 100 ng of human genomic DNA. After an initi,al denaturation step at 94OC for 5 min, PCR was conducted for 30 cycles with denaturation at 94OC for 45 s, annealing at 55OC for 45 s, and extension at 72OC for 45 s. Genotypes were analyzed by denaturabon of the products of PCR by addition of 10 ~1 of formamide and heating to 94OC, followed by electrophoresis on standard DNA sequencing gels and subsequent autoradiography. Additional markers from chromosome 16 (D16.9499, D16S412, D16S420, 0165401, D76S406) were genotyped by PCR using specific end-labeled primers (Gyaptay et at., 1994). All genotypes were scored independently by two investigators, with both readers blinded to affection status. Analysis of linkage was performed using the LINKAGE programs (Lathrop et al., 1984). SSCP and DNA Sequencing Single-strand conformational polymorphism (SSCP; Orita et al., 1989) was performed by amplifying segments of the final exon of h8ENaC from genomic DNA using specific primers (j3ENaCl746: 5”GCTGGTGGCCTTGGCCAAGAG-3’; flfNaC7940: 5’-GTCCAGCGTCTGCAGACGCAG-3’). PCR and subsequent denaturation of products were performed as described above with the exceptiorr that products were labeled by inclusion of 1 pCi of ]u-32P]dCTP in tha reaction mixture. Products were fractionated at room temerature or 4OC by electrophoresis at 50 W constant power for 8 hr on 0.5 x MDE gels (AT Biochem) prepared in 0.6 x TBE (1 x TBE = 90 m M Tlris-borate [pH 7.81). Following autoradiography, conformers were excised from the gel and eluted by incubation in 100 pl of HZ0 at 45OC for 1 hr. A portion of the resultant product was then used to direct PCR with the same primers. These products were then subjected to direct DNA sequence analysis (Hata et al., 1990) using an ABl 373 automated DNA sequencer employing either dye terminators or dye-labeled primers, following a standard protocol suppied by the manufacturer (Applied Biosystems). in all cases, DNA sequences were confirmed by sequencing both DNA strands. Acknowledgments Correspondence should be addressed to R. P. L. We thank the family members of the kindreds studied for their invaluable contribution to this work, Andy Pakstis, Ken Kidd, Jean-Marc Lalouel, Pseter Aronson, and Gerhard Giebisch for helpful discussions, and Janet Budzinack for assistance with preparation of the manuscript. This work was sup

Cell 414

ported in part bygrantsfrom the National Institutes of Health. C. M. B. is a Howard Hughes Medical Institute Medical Student Research Training Fellow. Fi. P. L. is an Investigator of the Howard Hughes Medical Institute. Received September

12, 1994; revised October 4, 1994.

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