BASIC RESEARCH www.jasn.org A Missense Mutation in the Extracellular Domain of aENaC Causes Liddle Syndrome † † † Mahdi Salih,* Ivan Gautschi, Miguel X. van Bemmelen, Michael Di Benedetto, ‡ † Alice S. Brooks, Dorien Lugtenberg,§ Laurent Schild, and Ewout J. Hoorn* Departments of *Internal Medicine and ‡Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; †Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Switzerland; and §Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands ABSTRACT Liddle syndrome is an autosomal dominant form of hypokalemic hypertension due to mutations in the b-or g-subunit of the epithelial sodium channel (ENaC). Here, we describe a family with Liddle syndrome due to a mutation in aENaC. The proband was referred because of resistant hypokalemic hypertension, sup- pressed renin and aldosterone, and no mutations in the genes encoding b-orgENaC. Exome sequencing revealed a heterozygous, nonconservative T.C single-nucleotide mutation in aENaC that substituted Cys479 with Arg (C479R). C479 is a highly conserved residue in the extracellular domain of ENaC and likely involved in a disulfide bridge with the partner cysteine C394. In oocytes, the C479R and C394S mutations resulted in similar twofold increases in amiloride-sensitive ENaC current. Quantification of mature cleaved aENaC in membrane fractions showed that the number of channels did not increase with these mutations. Trypsin, which increases open probability of the channel by proteolytic cleavage, resulted in significantly higher currents in the wild type than in C479R or C394S mutants. In summary, a mutation in the extra- cellular domain of aENaC causes Liddle syndrome by increasing intrinsic channel activity. This mecha- nism differs from that of the b-andg-mutations, which result in an increase in channel density at the cell surface. This mutation may explain other cases of patients with resistant hypertension and also provides novel insight into ENaC activation, which is relevant for kidney sodium reabsorption and salt-sensitive hypertension. J Am Soc Nephrol 28: 3291–3299, 2017. doi: https://doi.org/10.1681/ASN.2016111163 Hypertension is one of the most common noncom- sodium channel (ENaC).6 In 1963, Liddle et al.7 municable disorders worldwide and a major risk reported a “familial renal disorder simulating factor for stroke, myocardial infarction, heart fail- primary aldosteronism but with negligible aldosterone ure, and ESRD.1 Primary or essential hypertension secretion.” Liddle syndrome or pseudoaldosteron- is a complex genetic trait that is also influenced by ism (OMIM 177200) is now known as an autosomal other risk factors, such as dietary sodium and po- dominant form of salt-sensitive hypertension that is tassium intake, obesity, and diabetes.2,3 In contrast, further characterized by suppressed plasma renin monogenic forms of hypertension are very rare but and aldosterone, hypokalemia, and metabolic have been instrumental in revealing the molecular pathways contributing to primary hypertension.4 The majority of these pathways point toward a Received November 2, 2016. Accepted June 4, 2017. role for increased sodium reabsorption by the kid- Published online ahead of print. Publication date available at neys, especially in the aldosterone-sensitive distal www.jasn.org. 5 nephron. Indeed, several monogenic forms of hy- Correspondence: Dr. Ewout J. Hoorn, PO Box 2040, Room pertension are caused by mutations increasing so- H-438, 3000 CA Rotterdam, The Netherlands. Email: e.j.hoorn@ dium reabsorption in this segment through the erasmusmc.nl sodium chloride cotransporter or the epithelial Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 28: 3291–3299, 2017 ISSN : 1046-6673/2811-3291 3291 BASIC RESEARCH www.jasn.org alkalosis.8 The syndrome was linked to mutations in the exomes as proxy for variant allele frequencies in the general SCNN1B or SCNN1G gene, encoding the b-org-subunit of population (seven times heterozygously in .100,000 alleles; ENaC.9,10 Mutations in SCNN1B or SCNN1G delete or modify Exome Aggregation Consortium). The ENaC blocker triam- the intracellular PY motifs in ENaC in such a way that Nedd4–2 terene normalized BP and serum potassium in the proband. fails to ubiquitylate the channel, leading to a retention of active Genotyping of the five siblings also identified the novel C479R ENaC at the cell surface.11,12 Here, we report a family with mutation in subject II-4. The mutation segregated with sup- Liddle syndrome due to a gain of function mutation in the pressed plasma renin and aldosterone but not with hyperten- extracellular domain of the a-subunit of ENaC (SCNN1A) sion (Figure 1C). Whole-exome sequencing in the proband did that predominantly increases channel open probability (Po) not identify additional mutations to explain the hypertensive but not channel surface density. trait in this family. Subject II-4 had mild hypertension (average ambulatory BP of 138/88 mmHg) that was sensitive to sodium chloride supplementation (145/91 mmHg) and also improved RESULTS with triamterene (121/71 mmHg). In a standardized diuretic test, the natriuretic response to triamterene in the proband Clinical and Genetic Characteristics of a Novel ENaC and II-4 was in the high range or increased compared with the Mutation response in healthy volunteers (Figure 1D).13 Thus, two siblings The proband was referred because of resistant hypertension, (the proband and II-4) show a clinical picture compatible with hypokalemia, metabolic alkalosis, and suppressed levels of Liddle syndrome and carry the C479R missense mutation. plasma renin and aldosterone. Despite a positive family his- tory for hypertension (Figure 1A), no mutations in SCNN1B C479 Is Located in the Extracellular Domain of ENaC or SCNN1G were identified. Diagnostic exome sequencing The ultimate proof of Liddle syndrome, however, is the dem- revealed a novel heterozygous, nonconservative T.Csingle- onstration that the mutation results in a gain of function of nucleotide mutation that results in the substitution of cyste- ENaC. The DNAvariant that encodes the C479R mutant has so ine 479 to arginine (C479R) in aENaC [c.1435T.C(p. far never been described. The C479 is a highly conserved Cys (Cys479Arg))] (Figure 1B). The mutation is reported at a residue that belongs to the second cysteine-rich domain very low frequency in a large database collecting .60,000 (CRD2) of the extracellular domain of ENaC that is likely in- volved in disulfide bridges.14 The human aENaC subunit (haENaC) C479 is con- served among not only the ENaC subunits and ENaC homologs but also the Acid- Sensing Ion Channel 1 (ASIC1) orthologs (Figure 2A). The crystal structure of chickenASIC1revealsthatthecASIC1 C366 forms a disulfide bond with another highly conserved cysteine, C291 in CRD2, that corresponds in haENaC to a disulfide bond between C479 and the C394 (Figure 2B).15 Therefore, we analyzed not only the consequences of the C479R mutation on hE- NaC function in Xenopus laevis oocytes but also, the functional effects of the mutation of the partner Cys C394S involved in the disul- fide bond. In addition, because C479 is a highly conserved Cys, we performed a similar functional analysis of the corre- Figure 1. The novel aENaC mutation is characterized clinically by hypertension, sup- sponding Cys mutations C507S and pressed plasma renin and aldosterone, and an exaggerated natriuretic response to an C422S in rat aENaC. ENaC blocker. (A) Pedigree showing three generations of the family with Liddle syn- fi drome. Generation II was analyzed by genotyping and biochemical pro ling. The arrow C479R Increases ENaC Current in indicates the proband. (B) Sequence chromatogram. (C) The C479R mutation segregated Oocytes with suppressed plasma renin and aldosterone but not with hypertension. Renin and aldosterone were measured in the absence of interfering drugs. Dashed lines represent Both C479R and C394S result in a similar lower limits of normal. HT, hypertension; NT, normotension; WT, wild type. (D) Results of approximately twofold increase in amilor- a standardized diuretic test showing the natriuretic response to a single dose of the ENaC ide-sensitive ENaC current (Figure 3A). blocker triamterene in the proband and subject II-4 in comparison with healthy volun- These results strongly suggest that the teers.13 *Proband; #subject II-4. channel gain of function is due to the 3292 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 3291–3299, 2017 www.jasn.org BASIC RESEARCH extracellular domain of rat ENaC results in a channel gain of function.14 Furthermore both C479Arg and C507Ser substitutions in haENaC and rat aENaC subunit have comparable stimulatory effects on ENaC current, indicating that the effect does not depend on the substituting amino acid. To- gether, these observations support the idea that the disruption of the C479-C394 disul- fide bridge by the C479R substitution is likely the primary cause of the observed gain of function in ENaC. Surface Density of C479R Channel Mutant The increase in ENaC activity due to the C479R mutation can result from an in- crease in channel Po,single-channelcon- ductance, or the number of channels at the cell surface. To test the latter possibility, we analyzed on Western blot the cleaved (CL) forms of the wild type and C479R a- and gENaC subunits (Figure 4, A and B), expressed in the whole oocyte, at the cell sur- face (Figure 4, C and D, Supplemental Figure 1) and analyzed urinary extracellular vesicles (Supplemental Figure 2).16 It is now well es- tablished that the CL forms of a and g rep- resent the mature ENaC subunits that are incorporated in the functional channel com- plex present at the cell surface.17 The full length (FL) of aENaC (93 kD) was detected in oocytes expressing a-subunit alone or abgENaC wild type and C479R mutant Figure 2. C479 is a highly conserved Cys that forms a disulfide bond with C394. (A) (Figure 4A).