Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on October 5, 2005
Human Molecular Genetics 2005 14(22):3493-3498; doi:10.1093/hmg/ddi374

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 14/22/3493    most recent ddi374v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (6) Request Permissions Google Scholar Articles by Sheridan, M. B. Articles by Cutting, G. R. Search for Related Content PubMed PubMed Citation Articles by Sheridan, M. B. Articles by Cutting, G. R. Social Bookmarking          What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mutations in the beta-subunit of the epithelial Na⁺ channel in patients with a cystic fibrosis-like syndrome

Molly B. Sheridan¹, Peying Fong², Joshua D. Groman¹, Carol Conrad³, Patrick Flume⁴, Ruben Diaz⁵, Christopher Harris⁶, Michael Knowles⁷ and Garry R. Cutting^1,*

¹McKusick–Nathans Institute of Genetic Medicine, ²Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, ³Stanford University, Palo Alto, CA 94304, USA, ⁴Medical University of South Carolina, Charleston, SC 29425, USA, ⁵University of Nevada School of Medicine, Las Vegas, NV 89107, USA, ⁶Vanderbilt University, Nashville, TN 37232, USA and ⁷University of North Carolina, Chapel Hill, NC 27599, USA

^* To whom correspondence should be addressed at: Johns Hopkins Medical Institutions, BRB 559; 733 North Broadway, Baltimore, MD 21205, USA. Tel: +1 4109551773; Fax +1 4106140213; Email: gcutting{at}jhmi.edu

Received August 25, 2005; Accepted September 30, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cystic fibrosis (CF) is an autosomal recessive disorder of Cl^– and Na⁺ transport. The vast majority of CF patients have deleterious mutations in an epithelial Cl^– channel called the CF transmembrane conductance regulator (CFTR). In contrast, defects in the epithelial Na⁺ channel (SCNN1) have been associated with phenotypes dominated by renal disease (systemic pseudohypoaldosteronism type I and Liddle syndrome). We report two non-classic CF patients without CFTR mutations who have novel deleterious mutations in the ß-subunits of SCNN1 in the absence of overt renal disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cystic fibrosis (CF) is an autosomal recessive disorder that affects Cl^– and Na⁺ transport in the sweat gland, respiratory tract, pancreas and male reproductive system (1). Linkage and positional cloning identified the CF transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel as responsible for CF. The Na⁺ transport defect in CF patients is a consequence of the loss of functional interaction between the CFTR and the epithelial sodium channel (ENaC) (1). Several unusual patients with clinical features of CF, including an ion transport defect in the sweat gland, have been reported without mutations in the coding regions of CFTR (2–5). In familial cases, linkage analysis has excluded the involvement of CFTR (2,4). The presence of a Na⁺ transport defect in CF patients and the functional interaction between CFTR and ENaC (6,7) prompted us to explore whether defects in the genes encoding , ß or ENaC (SCNN1A, B or G) account for features of CF (sweat Cl^– greater than 60 mmol/l and CF-like pulmonary infections) in 20 non-classic CF patients without mutations in the coding regions of CFTR (4,8).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Sequencing of the exons and the flanking introns of the genes encoding the , ß and -subunits of ENaC in 20 non-classic CF patients without CFTR mutations identified six novel sequence changes. Five are predicted to cause amino acid changes: R181W in SCNN1A and S82C, P267L, G294S and E539K in SCNN1B, whereas the sixth changes the highly conserved penultimate A in the 3' splice site of SCNN1B intron 12 (1670–2 A to G). Four previously reported amino acid polymorphisms in SCNN1A at frequencies consistent with those reported for the general population were also found. Four of the five novel SCNN1B mutations were found in two patients (824 and 828). Pedigree analysis confirmed that each mutation was inherited on separate genes (Fig. 1). The three amino acids mutated in patients 824 and 828 are completely conserved in ENaC orthologues and paralogues in human, rabbit, mouse and rat (Fig. 2). These substitutions were not found in ethnically matched control alleles (P267L in 256 alleles, G294S in 324 alleles and E539K in 254 alleles) (data not shown). Together, these data indicate that the SCNN1B mutations discovered in these patients are likely to be deleterious.

View larger version (18K): [in this window] [in a new window]   Figure 1. Pedigrees of patients with two novel SCNNIB mutations. Parentheses indicate the individual with an inferred genotype. Solid symbols denote individuals who presented with features of CF (elevated sweat Cl– concentration and recurrent lung infections). Blood pressure is the most recent reading obtained in a medical setting. NA, not available.

 

View larger version (59K): [in this window] [in a new window]   Figure 2. Amino acid sequence alignment of ENaC orthologues and paralogues. Human (NP_001029 [GenBank] , NP_000327 [GenBank] , NP_001030 [GenBank] and NP_002969 [GenBank] ), rabbit (O97741 [GenBank] , O97742 [GenBank] and Q28738 [GenBank] ), mouse (NP_035454 [GenBank] , NP_035455 [GenBank] and NP_035456 [GenBank] ) and rat (NP_113736 [GenBank] , NP_036780 [GenBank] and NP_058742 [GenBank] ) , ß, and ENaC sequences were obtained from GenBank. Residues that are conserved among all orthologues and paralogues are highlighted in black. Amino acids found mutated in this study are indicated in bold. The alignment was generated with Clustal W.

 
RT–PCR of nasal epithelial RNA from patient 824 revealed that the 1670–2 A to G mutation results in two stable SCNN1B transcripts (Fig. 3). The first retains 83 nucleotides from the 3' end of intron 12 and is predicted to alter the amino acid sequence following codon 514 and terminate after the addition of 188 novel residues. The second transcript lacks the first 33 nucleotides of SCNN1B exon 13 leading to a deletion of 11 amino acids that precede the second transmembrane domain. These results confirm that 1670–2 A to G causes aberrant splicing leading to mRNA transcripts with substantial alterations in sequence.

View larger version (27K): [in this window] [in a new window]   Figure 3. RT–PCR analysis of SCNN1B 1670–2 A to G. (A) To determine whether the transcript resulting from the 1670–2 A to G mutation was stable, RT–PCR was performed using M13 tagged primers designed to amplify the region surrounding the P267L mutation in patient 824. Sequencing of the RT–PCR product revealed biallelic expression at SCNN1B nucleotide 927 (indicated by arrow), as seen in genomic DNA from the same individual. This confirms that the transcript resulting from the 1670–2 A to G mutation is stable. (B) To analyse the exon 12/exon 13 junction in transcripts resulting from the 1670–2 A to G mutation, RT–PCR was performed with primers placed in SCNN1B exons 12 and 13. RT–PCR products were cloned into pCR 2.1 TOPO (Invitrogen) and sequenced. Two different mutant transcripts were present. The first contains an insertion of 83 nucleotides from the 3' end of SCNN1B intron 12. This is predicted to result in frameshift and insertion of 188 novel amino acids after SCNN1B exon 12. The second transcript maintains frame, but lacks the first 33 nucleotides of SCNN1B exon 13. This is predicted to cause deletion of 11 amino acids preceding the second transmembrane domain.

 
The effect of the three missense mutations on ENaC function was investigated in Xenopus laevis oocytes (Fig. 4). Oocytes injected with wild-type -, ß- and -ENaC subunit RNAs displayed inwardly rectifying Na⁺ currents [mean –0.89±0.145 µA (SEM) at –100 mV; n=40; nine batches] that were inhibited by amiloride, as described previously (9). Oocytes injected with E539K ßENaC instead of wild-type ßENaC generated Na⁺ currents (–0.26±0.076 µA; n=12; six batches) that were significantly lower (P<0.001) than those of wild-type. Similarly, co-expression of P267L ßENaC with wild-type - and -ENaC was associated with Na⁺ currents (–0.33±0.09 µA; n=14; four batches) that were significantly reduced (P=0.002) compared with wild-type. In contrast, the co-expression of G294S ßENaC produced Na⁺ currents (–2.08±0.383 µA; n=27; six batches) that were significantly higher (P=0.004) than those of wild-type. Thus, each of the missense SCNN1B mutations found in patients 824 and 828 is associated with abnormal function.

View larger version (17K): [in this window] [in a new window]   Figure 4. Functional studies of SCNN1B mutants expressed in Xenopus oocytes. The I–V relationship (t=182 ms) of the amiloride-sensitive currents for oocytes expressing (A) wild-type ENaC (squares) and ßENaC P267L (triangles); (B) wild-type (squares) and ßENaC G294S (circles); (C) wild-type (squares) and ßENaC E539K (diamonds) is shown. Each graph compares the amiloride-sensitive currents from a representative oocyte injected with RNA encoding one of the three ßENaC mutants with wild-type - and -ENaC against the amiloride-sensitive currents obtained from a paired, wild-type -, ß- and -ENaC-injected oocyte from the same batch. Currents resulting from expression of the three SCNN1B mutants with wild-type - and -ENaC were studied in a masked fashion. These currents reverse at potentials more depolarized than +20 mV, distinguishing them from non-specific background currents. (D) Histogram of normalized mean currents (±SEM) at a membrane potential of –100 mV for oocytes injected with RNA encoding wild-type - and -ENaC plus wild-type ßENaC, n=40 from nine batches; ßENaC P267L, n=14 from four batches; ßENaC G294S, n=27 from six batches; or ßENaC E539K, n=12 from six batches. Asterisks indicate P-values less than 0.005 when compared with wild-type.

 
Mutations that cause loss of ENaC function have been associated with systemic pseudohypoaldosteronism type I (PHA I) (10), an autosomal recessive renal salt-wasting disorder, whereas mutations that cause gain of ENaC function occur in patients with Liddle syndrome, an autosomal dominant form of renal hypertension (11,12). Aldosterone and renin, hormones that regulate ENaC activity in the kidney, are elevated in PHA I patients and reduced in Liddle syndrome patients. In addition, urinary, salivary and sweat Na⁺ and Cl^– concentrations are elevated in PHA I patients (13). Patient 828 had an episode of dehydration at 6 months of age associated with transient elevation of aldosterone and renin levels; however, neither patient 828 nor patient 824 has abnormal renin or aldosterone levels (Table 1). Both patients have normal concentrations of Na⁺ and Cl^– in saliva, urine, and serum, and normal blood pressure measurements (Table 1 and Fig. 1). In contrast, they each have elevated sweat Cl^– concentrations and have had multiple pulmonary infections. Elevated sweat Cl^– concentrations and pulmonary infections with bacteria that commonly infect the lungs of CF patients (e.g. Pseudomonas) have been reported in PHA I patients early in life (<5 years) (14,15). However, neither patient has an excessive volume of airway surface liquid manifest as persistent watery rhinorrhea, which is characteristic of PHA I patients (13). Furthermore, PHA I and Liddle syndrome patients have abnormal amiloride-sensitive Na⁺ absorption across nasal epithelium (16). Measurement of ion transport across the nasal epithelium of patient 824 revealed normal amiloride-sensitive Na⁺ absorption [mean basal potential difference: –23.8±3.05 mV (SEM); n=2 and mean percent inhibition with amiloride: 60% ±7.6; n=4] and robust CFTR-mediated Cl^– conductance (mean isoproterenol response: –39.8±7.19 mV; n=4). The parents of patient 828 declined nasal potential difference (NPD) measurement testing.

View this table: [in this window] [in a new window]   Table 1. Clinical and laboratory findings in organ systems that are affected in CF, PHA I or Liddle syndrome

 
These patients demonstrate that deleterious SCNN1B mutations can produce symptoms in the lungs and sweat gland without the renal features of PHA I and Liddle syndrome. Each mutation in patient 824 is predicted to reduce but not to eliminate ßENaC function. Preservation of some ENaC function may allow sufficient Na⁺ reabsorption in the kidney, so that the clinical features of PHA I are avoided when salt is abundant in the diet. Indeed, transgenic mice expressing low levels of ßENaC appear clinically normal, but when placed under salt restriction exhibit acute PHA I symptoms (17). Partial ßENaC function could also explain the normal basal ENaC function in the nasal respiratory epithelia and normal salivary electrolyte concentration. However, the presence of pulmonary infection and elevated sweat Cl^– suggest that the level of residual ßENaC function in patient 824 is not sufficient for proper sodium transport in the airway and sweat gland. The concept that decreased ENaC function can lead to lung infections is supported by the pulmonary phenotype of young PHA I patients. In contrast, the two mutations carried by patient 828 (G294S and E539K) each appear to affect ENaC function differently. E539K was associated with reduction of ENaC function in Xenopus oocytes, whereas G294S was associated with increased function. As noted earlier, gain of function in ENaC has been associated with hypertension in Liddle syndrome. The father of patient 828, who carries G294S, is hypertensive (Fig. 1). Unfortunately, further characterization of the father's phenotype has not been possible. Intriguingly, the combination of gain and loss of function SCNN1B mutations in patient 828 produces a phenotype similar to that of patient 824. Co-expression of the ßENaC mutants carried by patient 828 (G294S and E539K) produced Na⁺ current profiles in oocytes that were very similar to those exhibited oocytes expressing the gain of function mutant (G294S) alone (data not shown). Murine studies reveal that increased ßENaC function can lead to pulmonary disease. Lung-specific over-expression of the ß (but not or ) ENaC subunit increased Na⁺ absorption in the airway and produced CF-like lung disease in transgenic mice in the absence of a Cl^– transport abnormality (18). In summary, these patients illustrate that the spectrum of phenotypes associated with mutations in SCNN1B is broader than previously recognized. Overlap of this spectrum with features of CF indicates that genetic heterogeneity should be considered in non-classic CF patients without CFTR mutations.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Patient population
One hundred and fifty-eight patients with features of non-classic CF have been studied for mutations in coding regions of the CFTR gene (4,8). Of the 73 patients without CFTR mutations, 20 had elevated sweat chloride (>60 mmol/l) and pulmonary disease defined by one or more of the following: sputum cultures positive for Pseudomonas aeruginosa, chronic productive cough, documented pneumonia, reactive airway disease, abnormal pulmonary function tests, abnormal chest X-ray and/or CT indicating bronchiectasis, infiltrates or blebs, chronic sinusitis and/or nasal polyposis.

ENaC sequencing and genotyping
Exons and bordering introns of SCNN1A, SCNN1B and SCNN1G were amplified from genomic DNA using polymerase chain reactions with M13 labelled primers (primer sequences may be obtained from the corresponding author). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced using ABI PRISM BigDye Primer Sequencing kit (Applied Biosystems, Foster City, CA, USA). The resulting sequences were analysed bidirectionally by capillary electrophoresis (ABI3100 Genetic Analyzer, Applied Biosystems) and the Sequencher analysis program (Gene Codes, Ann Arbor, MI, USA). To determine whether SCNN1B missense mutations P267L, G294S and E539K were polymorphisms, ethnically matched control samples were analysed. Briefly, the regions containing these mutations were amplified from genomic DNA using PCR and screened by allele-specific oligonucleotide hybridization or unidirectional sequencing.

SCNN1B RNA analysis
Nasal epithelial cells were collected from patient 824. RNA was extracted with the use of RNAbee (TelTest, Friendswood, TX, USA) following the manufacturer's protocol. cDNA was synthesized using reverse transcriptase (StrataScript reverse transcriptase, Stratagene, La Jolla, CA, USA) according to manufacturer's protocol. To confirm the presence of transcript from each allele, SCNN1B-specific primers were designed to amplify the region containing the mutation P267L in patient 824 by RT–PCR. Resulting products were sequenced as described earlier. The exons 12 and 13 junction was studied in transcripts with the 1670–2 A to G mutation by RT–PCR followed by cloning into pCR2.1 TOPO (Invitrogen, Carlsbad, CA, USA) and dye terminator sequencing (ABI PRISM BigDye Terminator Sequencing Kit, Applied Biosystems).

Preparation of ENaC mRNA and injection into Xenopus oocytes
Plasmids containing wild-type human cDNA encoding the three subunits of the ENaC were obtained from Dr Dale Benos (University of Alabama). Mutations in the ß-subunit were generated using the Transformer Site-Directed Mutagenesis Kit (BD Clontech, Mountain View, CA, USA). RNA was synthesized using in vitro transcription and capping kits (mMessage mMachine; Ambion, Austin, TX, USA) according to manufacturer's instructions, aliquoted and stored at –80°C. Mature female X. laevis were anaesthetized in 0.2% ethyl m-amino benzoate (Tricaine; Sigma Chemical, St Louis, MO, USA) and then placed on ice prior to partial ovariectomy and manual isolation of Stage V and Stage VI oocytes as previously described (19). Oocytes were injected with cRNA encoding wild-type - and -ENaC, along with wild-type or a given mutant ß-ENaC. Oocytes injected with equal volumes of water (50 nl/oocyte) served as negative controls. Injected oocytes were incubated at 18°C in 50% L-15 media (Invitrogen) media containing 10 µM amiloride. Wild-type ENaC currents were detectable as early as 1 day after injection.

Voltage-clamp measurements
Two-electrode voltage-clamp measurements were performed in ND-96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂ and 5 NaHEPES; pH 7.5) in the presence and absence of 10 µM amiloride. Data were acquired using an oocyte voltage-clamp amplifier (Model OC 725A; Warner Instruments, Hamden, CT, USA). The acquisition and analysis of currents were facilitated by pClamp 9.2 software (Axon Instruments, Foster City, CA, USA). Under unclamped conditions, cells expressing wild-type ENaC showed strong membrane depolarization (with V_m often as great as +60 mV) on removal of amiloride from the bath solution.

Nasal potential difference
The function of ENaC in the nasal epithelia of patient 824 was evaluated by performing NPD measurements as previously described (20,21).

Statistical analysis
Values are expressed as mean±SEM. Two-tailed Student's t-test with unequal variance was used to assess statistical significance. P-values less than 0.05 were assumed significant.

	ACKNOWLEDGEMENTS

 
Special thanks to Greg Germino, William Guggino, Pam Zeitlin and Beryl Rosenstein for critical review of the manuscript; Dale Benos for providing the ENaC plasmids; Karen Butterfield for collection of clinical information and Cecilia Canessa for helpful discussion. This work was supported by the Cystic Fibrosis Foundation (CUTTIN98AO and R025-CR02) and the NIH (DK44003 and HL068927 to G.R.C.; U54 RR019480 and RR00046 to M.K.).

Conflict of Interest statement. None declared.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Welsh, M.J., Ramsey, B.W., Accurso, F.J. and Cutting, G.R. (2001) In Scriver, C.R., Beaudet, A.L., Valle, D. and Sly, W.S. (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York, Vol. III, pp. 5121–5188.

2. Mekus, F., Ballmann, M., Bronsveld, I., Dörk, T., Bijman, J. and Tummler, B. (1998) Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator. Hum. Genet., 102, 582–586.[CrossRef][Web of Science][Medline]

3. Hughes, D., Dork, T., Stuhrmann, M. and Graham, C. (2001) Mutation and haplotype analysis of the CFTR gene in atypically mild cystic fibrosis patients from Northern Ireland. J. Med. Genet., 38, 136–139.[Free Full Text]

4. Groman, J.D., Meyer, M.E., Wilmott, R.W., Zeitlin, P.L. and Cutting, G.R. (2002) Variant cystic fibrosis phenotypes in the absence of CFTR mutations. N. Engl. J. Med., 347, 401–407.[Abstract/Free Full Text]

5. Wallis, C. (2003) Atypical cystic fibrosis—diagnostic and management dilemmas. J. R. Soc. Med., 96 (Suppl. 43), 2–10.

6. Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C. and Boucher, R.C. (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science, 269, 847–850.[Abstract/Free Full Text]

7. Reddy, M.M., Light, M.J. and Quinton, P.M. (1999) Activation of the epithelial Na⁺ channel (ENaC) requires CFTR Cl^– channel function. Nature, 402, 301–304.[CrossRef][Medline]

8. Groman, J.D., Karczeski, B., Sheridan, M., Robinson, T.E., Fallin, M.D. and Cutting, G.R. (2005) Phenotypic and genetic characterization of patients with features of ‘nonclassic’ forms of cystic fibrosis. J. Pediatr., 146, 675–680.[CrossRef][Web of Science][Medline]

9. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.-D. and Rossier, B.C. (1994) Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature, 367, 463–467.[CrossRef][Medline]

10. Chang, S.S., Grunder, S., Hanukoglu, A., Rosler, A., Mathew, P.M., Hanukoglu, I., Schild, L., Lu, Y., Shimkets, R.A., Nelson-Williams, C. et al. (1996) Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat. Genet., 12, 248–253.[CrossRef][Web of Science][Medline]

11. Shimkets, R.A., Warnock, D.G., Bositis, C.M., Nelson-Williams, C., Hansson, J.H., Schambelan, M., Gill, J.R., Jr, Ulick, S., Milora, R.B., Findling, J.W. et al. (1994) Liddle's syndrome: heritable human hypertension caused by mutations in the ß subunit of the epithelial sodium channel. Cell, 79, 407–414.[CrossRef][Web of Science][Medline]

12. Gharvi, A., Lifton, R.P. (2001) In Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York, pp. 5399–5417.

13. Kerem, E., Bistritzer, T., Hanukoglu, A., Hofmann, T., Zhou, Z., Bennett, W., MacLaughlin, E., Barker, P., Nash, M., Quittell, L. et al. (1999) Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N. Engl. J. Med., 341, 156–162.[Abstract/Free Full Text]

14. Marthinsen, L., Kornfalt, R., Aili, M., Andersson, D., Westgren, U. and Schaedel, C. (1998) Recurrent Pseudomonas bronchopneumonia and other symptoms as in cystic fibrosis in a child with type I pseudohypoaldosteronism. Acta Paediatr., 87, 472–474.[CrossRef][Web of Science][Medline]

15. Schaedel, C., Marthinsen, L., Kristoffersson, A.C., Kornfalt, R., Nilsson, K.O., Orlenius, B. and Holmberg, L. (1999) Lung symptoms in pseudohypoaldosteronism type 1 are associated with deficiency of the alpha-subunit of the epithelial sodium channel. J. Pediatr., 135, 739–745.[CrossRef][Web of Science][Medline]

16. Baker, E., Jeunemaitre, X., Portal, A.J., Grimbert, P., Markandu, N., Persu, A., Corvol, P. and MacGregor, G. (1998) Abnormalities of nasal potential difference measurement in Liddle's syndrome. J. Clin. Invest., 102, 10–14.[Web of Science][Medline]

17. Pradervand, S., Barker, P.M., Wang, Q., Ernst, S.A., Beermann, F., Grubb, B.R., Burnier, M., Schmidt, A., Bindels, R.J., Gatzy, J.T. et al. (1999) Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel. Proc. Natl Acad. Sci. USA., 96, 1732–1737.[Abstract/Free Full Text]

18. Mall, M., Grubb, B.R., Harkema, J.R., O'Neal, W.K. and Boucher, R.C. (2004) Increased airway epithelial Na⁺ absorption produces cystic fibrosis-like lung disease in mice. Nat. Med., 10, 487–493.[CrossRef][Web of Science][Medline]

19. Fong, P., Rehfeldt, A. and Jentsch, T.J. (1998) Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata. Am. J. Physiol., 274, C966–C973.

20. Knowles, M.R., Paradiso, A.M. and Boucher, R.C. (1995) In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum. Gene Ther., 6, 445–455.[Web of Science][Medline]

21. Standaert, T.A., Boitano, L., Emerson, J., Milgram, L.J., Konstan, M.W., Hunter, J., Berclaz, P.Y., Brass, L., Zeitlin, P.L., Hammond, K. et al. (2004) Standardized procedure for measurement of nasal potential difference: an outcome measure in multicenter cystic fibrosis clinical trials. Pediatr. Pulmonol., 37, 385–392.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				L. Mutesa, A. K. Azad, C. Verhaeghe, K. Segers, J.-F. Vanbellinghen, L. Ngendahayo, E. K. Rusingiza, P. R. Mutwa, S. Rulisa, L. Koulischer, et al. Genetic Analysis of Rwandan Patients With Cystic Fibrosis-Like Symptoms: Identification of Novel Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel Gene Variants Chest, May 1, 2009; 135(5): 1233 - 1242. [Abstract] [Full Text] [PDF]

				A. Bush Update in Pediatric Lung Disease 2006 Am. J. Respir. Crit. Care Med., March 15, 2007; 175(6): 532 - 540. [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 14/22/3493    most recent ddi374v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (6) Request Permissions Google Scholar Articles by Sheridan, M. B. Articles by Cutting, G. R. Search for Related Content PubMed PubMed Citation Articles by Sheridan, M. B. Articles by Cutting, G. R. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics