Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders
Characterisation of renal chloride channel, CLCN5 , mutations in hypercalciuric nephrolithiasis (kidney stones) disordersSarah E. Lloyd1, Willy Günther2, Simon H.S. Pearce1, Amelia Thomson1, Maria L. Bianchi3, Maurizio Bosio4, Ian W. Craig5, Simon E. Fisher5, Steven J. Scheinman6, Oliver Wrong7, Thomas J. Jentsch2 and Rajesh V. Thakker1,*
1MRC Molecular Endocrinology Group, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK, 2Centre for Molecular Neurobiology Hamburg (ZMNH), Hamburg University, Martinistrasse 52, D-20246, Hamburg, Germany, 3Metabolic Bone Unit, Centro Auxologico Italiano IRCCS, Via Ariosto 13, 20145, Milano, Italy, 4Section of Paediatric Nephrology, Paediatric Department, Magenta Hospital, Milano, Italy, 5Genetics Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, 6Department of Medicine, SUNY Health Science Center, Syracuse, NY 13210, USA and 7Department of Nephrology, The Middlesex Hospital, Mortimer Street, London W1N 8AA, UK
Received March 3, 1997;Revised and Accepted May 16, 1997
Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has ~12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.
Four disorders of hereditary hypercalciuric nephrolithiasis (kidney stones), that have been referred to as Dent's disease, X-linked recessive nephrolithiasis (XRN), X-linked recessive hypophosphataemic rickets (XLRH) and the idiopathic low molecular weight proteinuria of Japanese children (JILP), have been reported to be due to mutations of the X-linked renal-specific chloride channel, CLCN5 (1 -8 ). All of these four diseases have features in common, and they represent renal tubular disorders that are characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and renal failure. In addition, other renal proximal tubular defects, which include aminoaciduria, phosphaturia, glycosuria, kaliureseis, uricosuria and an acquired impairment of urinary acidification, may also occur (1 -3 ,5 ,6 ). However, there are differences between these disorders; for example, rickets has been a particular feature of Dent's disease and XLRH, but not XRN and JILP, and severe renal failure has been a feature of Dent's disease and XRN (1 -6 ). Dent's disease, XRN and XLRH were mapped to Xp11.22 (2 ,4 ,5 ), and a microdeletion in one patient with Dent's disease facilitated the isolation and characterisation of a renal chloride channel gene, CLCN5 (2 ,9 ,10 ). DNA sequence analysis detected different CLCN5 mutations in patients with the four hypercalciuric nephrolithiasis disorders, thereby establishing its causal role in these diseases (7 ,8 ). The common genetic aetiology of CLCN5 mutations and the phenotypic similarities between all these syndromes indicates that they are variants of one disorder, which we propose to refer to as Dent's disease.
The human CLCN5 gene has a 2238 bp coding sequence, which consists of 12 exons that span 25-30 kb of genomic DNA, and encodes a 746 amino acid protein (9 ,10 ). CLCN5 belongs to the family of mammalian voltage-gated chloride channel genes (CLCN1-CLCN7, and CLCKa and CLCKb, respectively) that have ~12 transmembrane domains (9 ,11 ,12 ) (Fig. 1 ). These chloride channels have an important role in the control of membrane excitability, transepithelial transport and possibly cell volume (11 ,13 ). Heterologous expression of wild-type CLCN5 in Xenopus oocytes has revealed that the channel, CLC-5, conducts chloride currents that are outwardly rectifying and time-independent, and similar expression of disease-associated CLC-5 mutants demonstrated markedly reduced or absent currents (7 ,8 ,14 ). However, a correlation between the mutations, their in vitro functional consequences and the phenotypic variations could not be established. We have pursued further investigations to define the structural and functional relationships of this novel renal chloride channel and its role in hypercalciuric nephrolithiasis, by characterising additional CLCN5 mutations in patients with familial and non-familial forms of this disorder.
Five families with Dent's disease (Table 1 ) were identified from the UK, USA, Italy (15 ) and the Indian subcontinent (Table 2 ), and 22 members (nine affected males, seven affected females, two unaffected males and four unaffected females) were studied. DNA sequence analysis of the entire 2238 bp coding region of the CLCN5 gene from one affected member of each of the five families revealed the presence of five different mutations (Table 2 ) which consisted of three missense (G57V, G512R and E527D), one nonsense (R648X) and one in-frame insertion at codon 30 (30:Hins). The G57V mutation resulted from a G to T transversion (GGC -> GTC), the G512R mutation resulted from a G to C transversion (GGT -> CGT), the E527D mutation (Fig. 2 ) resulted from an A to T transversion (GAA -> GAT), the R648X mutation from a C to T transition (CGA -> TGA) and the in-frame insertion (30:H) resulted from an insertion of ACC at codons 30 and 31 (Fig. 3 ). The G57V, G512R, E527D and R648X mutations resulted in alterations of MspI, HphI, Sau3A1 and TaqI restriction enzyme sites, respectively, that facilitated their confirmation (Fig. 2 ). Interestingly, the R648X (CGA -> TGA) mutation has been observed previously (7 ) in an unrelated (confirmed by microsatellite polymorphism analysis, data not shown) British family with Dent's disease, and this may represent a possible mutational hot-spot (Fig. 1 ). The ACC in-frame insertion (30:H) was not associated with an alteration in a restriction enzyme site, and this mutation was confirmed by sequence-specific oligonucleotide (SSO) hybridisation analysis (Fig. 3 ). The G57V, G512R and R648X mutations were demonstrated to co-segregate with the disease in each of families 8/95, 13.3/95 and 3/95, respectively (Table 2 ). However, the 30:H insertional and E527D mutations were demonstrated to arise de novo (Figs 2 and 3 ), and interestingly the E527D mutation was found to co-segregate with the disease in subsequent generations. The absence of each of these five DNA sequence abnormalities in 110 alleles from 69 unrelated normal individuals (28 males, 41 females) established that these abnormalities were mutations and not functionally neutral polymorphisms which would be expected to occur in >1% of the population.
The two missense mutations, G57V and G512R, and the nonsense mutation R648X predict structurally significant alterations to CLC-5 and are thus likely to be of importance in the aetiology of the disease. The nonsense mutation R648X predicts a truncated CLC-5 channel that lacks the 100 amino acids from the C-terminal loop (Fig. 1 ), and the missense mutations G57V and G512R predict a disruption of the helix in the first transmembrane domain, D1, and a disruption of the charge distribution within D11, respectively (Table 2 ). The effects upon CLC-5 function of such nonsense and missense mutations, which have been previously assessed (7 ,8 ), are likely to be a loss of function. The effects of the missense mutation E527D, in which there is a minor change involving a loss of a methyl group from the glutamate (E) to convert it to an aspartate (D) (Fig. 1 ), are difficult to predict, although it is important to note that E527 is one of the most conserved amino acids and is present in all the known CLCs, including those from plants, yeast, Escherichia coli, cyanobacteria, fish and mammals (11 ,13 ). Similarly, the insertion of the positively charged histidine (H) at codon 30 in the N-terminus region, amidst the peptide sequence between codons 24 and 34 (REKSRDRDRHR) that is already rich in the positively charged amino acids arginine (R) and histidine (H), is also difficult to predict; interestingly, this sequence also has positively and negatively charged amino acids in alternating positions and if these were arranged in a [beta]-pleated sheet with one side positive and the other negative, then insertion of any extra amino acid would result in a disruption of the charge distribution (Table 2 ). The functional effects of the missense and insertion CLC-5 mutants were therefore assessed by heterologous expression in Xenopus oocytes and measurements of chloride currents as previously described (7 ,8 ). The results demonstrated that the insertional (30H) and missense (G57V) mutations markedly reduced the chloride channels to ~30 and 50% of the wild-type CLC-5 respectively, whilst the missense mutations G512R and E527D abolished the chloride currents (Fig. 4 ).
. Clinical and biochemical abnormalities in nine patients from five families with Dent's disease
Familya
2/95
8/95
13.3/95
7.3/95
3/95
Individual
II.1p
II.1p
II.2
II.1p
II.1p
III.1
III.2
II.1p
II.2
Age (years) at diagnosis
2
5
4
3
19
4
3
3
0.5
Sex
M
M
M
M
M
F
F
M
M
Urinary abnormalities
Low molecular weight proteinuria
+
+
+
+
+
-
+
+
+
Hypercalciuria
+
+
+
+
-
+
+
+
+
Renal failureb
-
-
-
+
++
-
-
+
+
Ricketsc
-
-
-
-
+
-
-
+
+
Nephrocalcinosisd
-
+
+
+
+
-
-
+
+
+ = present, - = absent. aFamily designation is shown, and each individual is indicated by a generation (I, II or III) and sibship number (1 or 2) (Figs 2 and 3); pindicates the proband. bRenal failure: + = moderate (<97 ml/min/1.73m2) (ref. 8); ++ = end stage, requiring dialysis and transplantation. cRickets detected by raised serum alkaline phosphatase activity or radiologically. dNephrocalcinosis detected by ultrasonography or radiology.
The families of five probands (Table 1 ), who suffered from multiple proximal renal tubular defects in association with nephrocalcinosis, nephrolithiasis and renal failure, were investigated after obtaining informed consent. The proximal tubular abnormalities included: a low molecular weight proteinuria, which consisted of [beta]2 microglobulin (normal <0.05 mg/mmol creatinine), [alpha]1 microglobulin (normal <0.7 mg/mmol creatinine) and retinol-binding protein (normal <0.02 mg/mmol creatinine) (1 ); an increased urinary excretion of calcium (calcium:creatinine ratio >0.71 mmol/mmol), phosphate, potassium, glucose, urate and amino acids; and an acquired impairment of urinary acidification (1 -3 ,5 ,15 ). Venous blood samples were obtained from 16 affected and six unaffected members of the five families, which were from different populations (Table 2 ), and used for mutational analysis of the CLCN5 gene (7 ,8 ).
Leukocyte DNA was extracted and used with CLCN5-specific primers for PCR amplification utilising conditions previously described (7 ,8 ). The PCR products were gel purified and the DNA sequence determined by Taq polymerase cycle sequencing using fluorochrome-labelled di-deoxy terminators, and resolved on a semi-automated detection system (ABI 373A sequence, Applied Biosystems) (7 ,8 ,18 ,19 ). In addition, DNA sequence abnormalities were confirmed either by restriction endonuclease analysis of genomic PCR products obtained by the use of the appropriate primers or by SSO hybridisation analysis (7 ,18 ,19 ); for the SSO hybridisation analysis, the wild-type oligonucleotide (19mer) was TCTCGAGACCGGGATAGGC and the mutant oligonucleotide (19mer) was TCGAGACCACCGGGATAGG (Fig. 3 ). The DNA sequence abnormalities were demonstrated to co-segregate with the disorder and to be absent as common polymorphisms in the DNA obtained from 69 unrelated normal individuals (28 males, 41 females). Microsatellite polymorphism analysis at DXS988 and at D11S533, and Southern blot hybridisation analysis were performed as described previously (2 ,20 ).
After engineering an NcoI site at the initiator methionine, a cDNA encoding human CLC-5 was inserted into the NcoI site of the expression vector pTLN (21 ), which contains Xenopus globin untranslated sequences, to boost expression in the oocyte. Mutations were introduced by recombinant PCR and verified by sequencing. Capped cRNA was prepared by in vitro transcription, using SP6 RNA polymerase, from the linearised construct. About 10-20 ng of cDNA was injected into Xenopus oocytes, which previously had been manually defolliculated and briefly collagenased (22 ). After incubating for 3 days at 17oC in modified Barth's solution [88 mM NaCl, 1 mM KCl, 1 mM CaCl2, 0.33 mM Ca(NO3)2, 0.82 mM MgSO4, 10 mM HEPES pH 7.6], oocyte conductance was measured at room temperature by a standard two-electrode voltage-clamp technique (7 ) using a Turbo-TEC-05.amplifier (NPI, Tamm Germany) and pCLAMP software (Axon Instruments, Inc); recordings were performed in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES pH 7.4). The currents were measured at the membrane potential of +80 mV and were normalised to the wild-type amplitude for each mutant. Four different batches of cRNAs and eight different batches of oocytes for the wild-type and mutant constructs were used, and similar results were obtained. The results were expressed as mean values +- SEM.
We are grateful to T.M. Barratt, T. Kennedy, D.J. Rainford and S. Schurman, for access to patients and to A.G.W. Norden for urinary analysis and helpful discussions. This work was supported by the Medical Research Council (UK) (S.E.L., S.H.S.P., A.T. and R.V.T.); the Deutsche Forschungsgemeinschaft (W.G. and T.J.J.); the Human Genome Mapping Project (I.W.C. and S.E.F.); the National Institutes of Health (USA), grant number DK46838 (S.J.S.); and the North Atlantic Treaty Organisation (NATO) Collaborative Research Grant number CRG950114 (S.J.S. and R.V.T.). SHS Pearce was an MRC Training Fellow.
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*To whom correspondence should be addressed. Tel: +44 181 383 3014; Fax: +44 181 383 8306; Email: rthakker@rpms.ac.uk
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