J Am Soc Nephrol 11: 1460–1468, 2000 Characterization of Renal (CLCN5) in Dent’s Disease

KATSUSUKE YAMAMOTO,* JEREMY P. D. T. COX,* THOMAS FRIEDRICH,† PAUL T. CHRISTIE,*‡‡ MARTIN BALD,‡ PETER N. HOUTMAN,§ MARTA J. LAPSLEY,ʈ LUDWIG PATZER,¶ MICHEL TSIMARATOS,# WILLIAM G VAN’T HOFF,** KANJI YAMAOKA,†† THOMAS J. JENTSCH,† and RAJESH V. THAKKER*‡‡ *MRC Molecular Endocrinology Group, Hammersmith Hospital, London, United Kingdom; †ZMNH Centre for Molecular Neurobiology, University of Hamburg, Germany; ‡Department of Paediatric Nephrology, University of Essen, Germany; §Department of Paediatrics, Leicester Royal Infirmary, United Kingdom; ʈDepartment of Chemical Pathology and Metabolism, St Helier Hospital, Surrey, United Kingdom; ¶Children’s Hospital “Jussuf Ibrahim,” Friedrich-Schiller University, Jena, Germany; #Department of Paediatric Nephrology, Children’s Hospital of the Timone, Marseille, France; **Department of Paediatric Nephrology, Great Ormond Street Hospital, London, United Kingdom; ††Department of Paediatrics, Osaka Prefectural Hospital, Osaka, Japan; and ‡‡Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom.

Abstract. Dent’s disease is an X-linked renal tubular disorder endonuclease or sequence-specific oligonucleotide hybridiza- characterized by low molecular weight , hypercal- tion analysis and were not common polymorphisms. The ciuria, , nephrolithiasis, and renal failure. The frameshift deletions and nonsense predict truncated disease is caused by mutations in a renal chloride channel , and inactivated CLC-5. The effects of the putative missense CLCN5, which encodes a 746 (CLC-5), Asp601Val mutant CLC-5 were assessed by heterologous ex- with 12 to 13 transmembrane domains. In this study, an addi- pression in Xenopus , and this revealed a chloride tional six unrelated patients with Dent’s disease were identified conductance that was similar to that observed for wild-type and investigated for CLCN5 mutations by DNA sequence CLC-5. However, an analysis of the mutant CLCN5 transcripts analysis of the 11 coding of CLCN5. This revealed six revealed utilization of the novel donor splice site, resulting in mutations: four frameshift deletions involving codons 392, a truncated CLC-5. Thus, all of the six mutations are likely to 394, 658, and 728, one (Tyr617Stop), and result in truncated CLC-5 and a loss of function, and these an A to T transversion at codon 601 that would result in either findings expand the spectrum of CLCN5 mutations associated a (Asp601Val) or creation of a novel donor with Dent’s disease. splice site. These mutations were confirmed by restriction

Dent’s disease is a renal tubular disorder characterized by low and three other phenotypically similar disorders referred to as molecular weight proteinuria, hypercalciuria, nephrocalcinosis, X-linked recessive nephrolithiasis (4–6), X-linked recessive nephrolithiasis, and progressive renal failure (1,2). The disease hypophosphatemic rickets (7), and the idiopathic low molecu- also may be associated with aminoaciduria, phosphaturia, gly- lar weight proteinuria of Japanese children (8–12), has been cosuria, kaliuresis, uricosuria, and impaired urinary acidifica- established to be due to inactivating mutations of a renal tion, and is complicated by rickets or osteomalacia in some chloride channel gene designated CLCN5 (11,13,14). patients. Thus, Dent’s disease may be considered a form of the The human CLCN5 gene, which is located on renal Fanconi syndrome (2,3). The etiology of Dent’s disease, Xp11.22, has a 2238-bp coding sequence that consists of 11 exons that span 25 to 30 kb of genomic DNA and encode a 746 amino acid protein (Figure 1) (15,16). CLCN5 belongs to the Received September 24, 1999. Accepted January 12, 2000. family of voltage-gated chloride channel (CLCN1- Dr. Katsusuke Yamamoto and Dr. Jeremy P. D. T. Cox contributed equally to this work. CLCN7, and CLCKa and CLCKb) that have approximately 12 Correspondence to Dr. Rajesh V. Thakker, Molecular Endocrinology Group, transmembrane domains (17,18). These chloride channels have Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Ox- an important role in the control of membrane excitability, ϩ ϩ ford OX3 9DU, United Kingdom. Phone: 44 1865 222043; Fax: 44 1865 transepithelial transport, and possibly cell volume (17,18). 222049; E-mail: [email protected] Heterologous expression studies of wild-type CLCN5 in Xe- 1046-6673/1108-1460 Journal of the American Society of Nephrology nopus oocytes have revealed that the channel, CLC-5, conducts Copyright © 2000 by the American Society of Nephrology chloride currents that are outwardly rectifying and time-inde- J Am Soc Nephrol 11: 1460–1468, 2000 CLCN5 Mutations in Dent’s Disease 1461

Figure 1. Schematic representation of CLCN5 mutations within the framework of the predicted topology of CLC-5, which consists of 746 amino acids (13,16). The correct topology of the CLC-5 putative transmembrane domains (D1 to D13) is not established, and this representation is based on a model reported previously (Inset) (13). The consensus phosphorylation and glycosylation sites are indicated by the asterisks and branch sites, respectively. The locations of the six mutations detected by the present study (Table 2) are indicated by the filled arrowheads, and the mutations are illustrated in bold and within boxes. These six mutations consist of four deletional frameshifts (392delGT, 394delT, 658delC, and 728del23bp), one nonsense (Tyr617Stop), and the loss of codons 601 to 645 (filled arrowheads, marked 601 and 645) due to the novel donor splice-site mutation (gacagt to gtcagt). Of the 52 CLCN5 mutations reported from previous studies (6,9–14,26,37–39), 31 are shown and eight of these have been observed to occur more than once; in addition, two large deletions encompassing the entire CLCN5 gene (10,13), two small intragenic deletions (12,13), and four splice-site mutations (two donor and two acceptor splice mutations) (11,13,39), which are not shown, have been reported. The two intragenic deletions would result in a loss of codons 132 to 241 (12,13) and codons 132 to 449 (12). The two donor splice sites would both result in a loss of codons 132 to 172 (13), and of the two acceptor splice sites, one was shown to result in a loss of codons 173 to 241 (39) and the effects of the other one were not characterized (11). pendent (19), and similar expression of disease-associated ciuria and the other features of Dent’s disease remain to be CLC-5 mutants has demonstrated markedly reduced or absent elucidated. The identification of additional CLCN5 mutations currents (9,11–14). The expression of CLC-5 in the human may help in these studies, and we have pursued such studies in nephron has been shown to be in the , the thick patients with Dent’s disease. ascending limb, and the intercalated cells of the collecting duct (20). Furthermore, CLC-5 has been localized intracellularly to the subapical and with the vacuolar H(ϩ)-ATPase, Materials and Methods thereby suggesting that it may have a role in the counterion Patients transport mechanism that facilitates acidification within endo- Six probands who suffered from Dent’s disease (Table 1) were investigated after giving informed consent. All six of the probands had somes (20,21). These endosomes form part of the - low molecular weight proteinuria, five had hypercalciuria, four had mediated endocytic pathway that transports such as nephrocalcinosis and/or nephrolithiasis, and three had renal impair- (20–22), and thus CLC-5 dysfunction in this pathway ment. None of the probands suffered from rickets. Five of the six may help provide an explanation for the observed low molec- probands were of Northern European origin and one proband (26/97, ular weight proteinuria in Dent’s disease (20,21). However, the Table 1) was of Japanese origin. A family history of Dent’s disease mechanisms by which CLC-5 dysfunction results in hypercal- could be established in four of the probands, while in the remaining 1462 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1460–1468, 2000

Table 1. Clinical and biochemical abnormalities in six male probands with Dent’s disease

Proband’s Family Characteristica 6/97 8/97 9/97 12/97 15/97 26/97

Age (yr) at first symptoms or diagnosis 43 68614 Urinary abnormalities low molecular weight proteinuriab ϩϩϩϩϩϩ hypercalciuria ϩϩϩϩϩϪ Renal impairmentc ϩϩϪϪϪϪ Nephrocalcinosis and nephrolithiasisd ϩϩϪϩϩϪ

a ϩ, present; Ϫ, absent. b Low molecular weight proteinuria refers to excessive urinary loss of one or more of the following: ␣1 microglobulin, ␤2 microglobulin, retinol-binding protein, and lysozyme; and/or the presence of albuminuria (2). c Renal failure: creatinine clearance Ͻ80 ml/min per 1.73 m2 or serum creatinine Ͼ130 ␮mol/L. No patient had end-stage renal failure. d Nephrocalcinosis and nephrolithiasis detected by ultrasonography or radiology.

two probands family members were not available for study to estab- liculated Xenopus oocytes and incubated for2dat17°C. Currents lish an inherited basis for the disease. Venous blood samples were were measured by standard two-electrode voltage-clamp techniques, obtained from these six probands and from three affected and six using a Turbo TEC-05 amplifier (NPI, Tamm, Germany) and unaffected family members for mutational analysis of the CLCN5 pCLAMP software (Axon Instruments, Foster City, CA). Measure- gene. ments were carried out in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM

CaCl2, 1 mM MgCl2, and 5 mM Hepes, pH 7.4). The mem- DNA Sequence Analysis of the CLCN5 Gene brane was held at the and stepped for 500 ms to Ϫ ϩ Leukocyte DNA was extracted and used with CLCN5-specific potentials ranging from 100 to 80 mV in 20-mV steps. Currents ϩ primers (9) for PCR amplification, using methods described previ- were determined at a of 80 mV. Results were Ϯ ously (9). The DNA sequence of PCR products was determined by expressed as mean values SEM. Taq polymerase cycle sequencing, using a semiautomated detection system (ABI 373XL sequencer; Applied Biosystems, Foster City, CA) Determination of Protein Expression in Xenopus (23). DNA sequence abnormalities were confirmed by either restric- Oocytes tion endonuclease analysis or sequence-specific oligonucleotide Oocytes were pooled after measurement and stored at Ϫ20°C. (SSO) hybridization analysis of the appropriate genomic PCR prod- After homogenization in 5 mM ethylenediaminetetra-acetic acid, 10 ucts (23,24). The DNA sequence abnormalities were demonstrated to mM Tris-HCl, and a protease inhibitor mix (Complete; Boehringer be absent as common polymorphisms in the DNA obtained from 74 Mannheim, Mannheim, Germany) on ice, the yolk platelets were unrelated normal individuals (34 males, 40 females), and to cosegre- removed by three low-speed centrifugations. From the supernatant, gate with the disorder in the available members from three of the the equivalent of two oocytes was dissolved in sodium dodecyl sulfate families. (SDS)-Laemmli buffer and loaded onto a 6% SDS-polyacrylamide gel electrophoresis. After separation, the proteins were blotted on poly- CLCN5 mRNA Analysis vinylidene difluoride membranes, and CLC-5 protein was detected RNA was extracted from Epstein-Barr virus (EBV)-transformed using a rabbit polyclonal antiserum raised against a peptide encom- lymphoblastoid cell lines established from peripheral blood cells of passing the 13 carboxy-terminal amino acid residues of hCLC-5 proband 15/97 (Table 1), and from three unrelated normal individuals. (11,21). Detection was carried out using a Protein A-peroxidase-based Reverse (RT)-PCR was performed using pairs of nested kit (Renaissance; DuPont, Boston, MA). CLCN5-specific primers (outer primers: forward 5Ј-CTTGGAGGAGTC- CAGAAGGCC-3Ј, and reverse 5Ј- GGTACCAGTTAATACAA- Results CATATCC-3Ј; inner primers: forward 5Ј-AGTCAGGGAGCTGATTG- DNA sequence analysis of the entire 2238-bp coding region CATCAC-3Ј, and reverse 5Ј- CATATCCATGGTCTGTAATGTCC-3Ј), at and -intron boundaries of the CLCN5 gene from the six an annealing temperature of 65°C for both the first and second rounds, which probands (Table 1) with Dent’s disease revealed six novel consisted of 20 and 30 cycles, respectively. The PCR products were gel- mutations that consisted of four deletional frameshifts (Figure purified, and the DNA sequences of both strands were determined as de- 2), one nonsense mutation, and an A to T transversion in the scribed (23). second base of codon 601 (Figure 3, Table 2). The deletional Functional Expression in Xenopus Oocytes frameshifts occurred in exons 8, 11, and 12, and the nonsense mutation (Tyr617Stop) and A to T transversion both occurred Wild-type and mutant CLC-5 were expressed in Xenopus oocytes as described previously (9,11,13,14). A cDNA encoding the human in exon 10 (Table 2). Three of these mutations (Tyr617Stop, CLC-5 protein was inserted into the expression vector PTLN (25). 392delGT, and 728del23bp) resulted in an alteration of a Mutations were introduced by recombinant PCR. Capped cRNA was restriction enzyme site (Table 2) that facilitated their confir- synthesized using SP6-RNA polymerase after linearization of the mation (Figure 1). The other three mutations (394delT, construct. About 10 ng of cRNA was injected into manually defol- 658delC, and the A to T transversion in codon 601) were not J Am Soc Nephrol 11: 1460–1468, 2000 CLCN5 Mutations in Dent’s Disease 1463

Figure 2. Detection of mutation in exon 12 by restriction enzyme analysis. DNA sequence analysis of the proband 26/97 (Table 1) revealed a 23-bp commencing at codon 728. This led to a frameshift that resulted in five missense amino acids (Gly, Glu, Pro shown) followed by a termination signal (Stop) at codon 733 (A). This deletion also resulted in the loss of an MseI restriction enzyme site (T/TAAA). PCR amplification and MseI digestion (B) would result in four fragments of 116, 82, 46 (not shown), and 10 bp (not shown) from the normal sequence, but only three products of 175, 46 (not shown), and 10 bp (not shown) from the mutant sequence, as illustrated in the restriction map (C). This deletion was not present in 74 normal individuals (N1 to N3 shown), indicating that it is not a common DNA sequence polymorphism. The standard size marker (S in Panel B) in the form of a 1-kb ladder is indicated. Similar restriction enzyme analysis was used to confirm the 392delGT and Tyr617Stop mutations (Table 2). The symbols denoting the in the individual are as described in Figure 3.

associated with altered restriction enzyme sites, and the difficult to predict. This transversion may result in either a method of SSO hybridization analysis (Figure 3) was used missense mutation, Asp601Val, or a novel donor splice site. (Table 2). The absence of each of these six DNA sequence The functional effects of such missense mutations may be to abnormalities in 110 alleles from 74 unrelated normal individ- abolish or markedly reduce CLC-5 chloride conductance uals established that these abnormalities were not sequence (9,11–14). The heterologous expression, in Xenopus oocytes, polymorphisms that would be expected to occur in Ͼ1% of the of the Asp601Val CLC-5 mutant revealed that ClϪ conduc- population. tance was unaffected and remained similar to that of the The four deletional frameshift mutations and the nonsense wild-type CLC-5 (Figure 4); translation of both the wild-type mutation are predicted to result in truncated CLC-5 that lack and mutant CLC-5 proteins was confirmed by Western blot from 14 to 346 amino acids (Table 2). The functional effect of analysis (data not shown) of the Xenopus oocyte membranes, such truncated CLC-5, which has been assessed previously using an antibody directed against the carboxy terminus (21). (9,11,13,14), is likely to be a loss of function, and this was However, an analysis of CLCN5 transcripts resulting from this confirmed by the expression of the CLC-5 mutant due to the A to T transversion revealed that the mutation was associated 23-bp deletional frameshift (Figure 2) at codon 728 (Figure 4). with a novel donor splice site (Figure 5).This novel donor The effects of the A to T transversion in codon 601were more splice site led to skipping of the 3Ј portion of exon 10 that 1464 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1460–1468, 2000

Figure 3. Detection of the mutation in exon 10 by sequence-specific oligonucleotide (SSO) hybridization analysis. DNA sequence analysis of the affected proband II.1 from family 15/97 (Table 1) revealed an A to T transversion in the second base of codon 601 (A). This transversion would alter the wild-type sequence GAC, which encodes an aspartic acid (Asp), to GTC, which encodes a valine (Val), or to a novel donor splice site (gacagt to gtcagt). This mutation, which did not result in an altered restriction enzyme site, was confirmed and demonstrated to cosegregate with Dent’s disease in the family by the use of SSO hybridization analysis (B). The affected male II.1 is hemizygous for the mutant allele, and the mother I.1 is heterozygous for the mutant (m) and wild-type (WT) alleles. The absence of this mutation in 74 unrelated normal individuals (N1 to N3 shown) indicated that it was not a common DNA sequence polymorphism. Squares, male; circles, female; open sections, unaffected; filled sections, affected. encoded codons 601 to 645. Thus, the mutant CLCN-5 tran- study, is 58, and these are scattered throughout the channel script consisted of the 5Ј portion of exon 10 (encoding codons (Figure 1) with no evidence for mutational hot spots. Further- 512 to 600) spliced to exon 11. If translated, this abnormal more, there appears to be no correlation between the mutations CLCN5 transcript would result in 32 missense amino acids and (13,26). Of the total 58 CLCN5 mutations, from codons 601 to 632, followed by a termination signal approximately 32% are nonsense mutations, 22% are frame- (Stop) (Figure 5, Table 2). Such a truncated CLC-5 is likely to shift deletions or insertions, 2% are in-frame insertions, 4% are result in a loss of function that is similar to those observed by donor splice-site mutations, 3% are acceptor splice-site muta- heterologous expression of the 23-bp deletion commencing at tions, 27% are missense mutations, 8% are intragenic dele- codon 728 (Figure 4). Thus, all of the six mutations detected in tions, and 2% are complete deletions of the gene. The majority this study of patients with Dent’s disease are likely to be (Ͼ70%) are predicted to result in truncated or absent CLC-5, associated with a loss of CLC-5 function. which would lead to a complete loss of channel function. Only four of the mutations (30:His , Gly57Val, Leu278Phe, Discussion and Arg280Pro), which are predicted not to result in truncated Our results, which have identified six novel CLCN5 muta- CLC-5 and which are all located outside the predicted trans- tions (Table 2), expand the spectrum of mutations that are membrane domains, have been reported to be associated with associated with Dent’s disease. All of the six CLCN5 muta- reduced, but not abolished, ClϪ currents (Figure 1). Indeed, the tions predict structural alterations of CLC-5 that are likely to clustering of such missense mutations (Leu278Phe, result in a loss of function. The total number of CLCN5 Arg280Pro), which are associated with residual channel activ- mutations now reported, including the results of our present ity (9,11) in the putative loop between D5 and D6 (Figure 1), J Am Soc Nephrol 11: 1460–1468, 2000 CLCN5 Mutations in Dent’s Disease 1465

Table 2. CLC-5 mutations found in patients with Dent’s disease

Restriction Number b Family Exon a Codon Amino Acid Change Enzyme Predicted Effect and Base Change Change/SSOb

Nonsense mutation 8/97 10 1851 CϾA 617 Tyr 3 Stop (X) MseI Loss of 130 aa Deletions 6/97 8 1175–1176delGT 392 fs, 9 missense aa, Stop (X) BsrI Missense peptide from aa 392–400 and loss of 346 aa 12/97 8 1182delT 394 fs, 38 missense aa, Stop (X) SSO Missense peptide from aa 395–432 and loss of 314 aa 9/97 11 1974delC 658 fs, 18 missense aa, Stop (X) SSO Missense peptide from aa 659–676 and loss of 70 aa 26/97 12 2183–2205del23bp 728 fs, 5 missense aa, Stop (X) MseI Missense peptide from aa 728–732 and loss of 14 aa Novel donor 15/97 10 1802 AϾT 601 fs, 32 missense aa, Stop (X) SSO Missense peptide from aa 601–632 and loss of 114 aa

a Nomenclature as recommended (40). b SSO, sequence-specific oligonucleotide; fs, frameshift; aa, amino acid.

Val residue would be expected to abolish or reduce ClϪ cur- rents, and yet our results revealed no alteration in ClϪ conduc- tance (Figure 4). This suggested that the A to T transversion was likely to be altering CLC-5 structure and function by a mechanism other than that of a missense mutation. An exam- ination of the DNA sequence of codons 600, 601, and 602 (Figure 3) indicated that the A to T transversion may have resulted in a novel donor splice site (gacagt to gtcagt) in exon 10. The first two bases (gt) of donor splice sites are invariant (31), and the effects of this mutation leading to a putative novel donor splice site were demonstrated by an analysis of RNA obtained from the patient’s EBV-transformed lymphoblastoids Figure 4. Electrophysiologic analysis of Xenopus oocytes expressing (Figure 5). This novel donor splice site, which has not been human wild-type (WT) CLC-5, the putative mutant Asp601Val (Fig- previously reported in Dent’s disease, led to skipping of the 3Ј Ϫ ure 3), and 728del23bp (Figure 2) channels. The Cl currents were portion of exon 10 and a truncated, and likely inactivated, measured as described previously (11,13), and the averaged (mean Ϯ CLC-5 (Table 2). The utilization of such novel donor splice ϩ SEM) whole cell currents measured at 80 mV in the Xenopus sites, which may be used preferentially to the wild-type, has Ϯ oocytes injected with water control (0.34 0.067), WT CLC-5 been previously observed to occur in association with the (2.54 Ϯ 0.45), the putative Asp601Val mutant (2.61 Ϯ 0.44), and the 728del23bp mutant (0.33 Ϯ 0.09) are shown. The Asp601Val mutant Laron and Lesch-Nyhan syndromes (32,33). channel had currents that were similar to that of the WT channel, The mechanisms by which a functional loss of this renal whereas the deletional mutant resulted in abolished ClϪ currents. chloride channel (CLC-5) leads to a generalized proximal renal Western blot analysis of the Xenopus oocyte membranes using an tubular defect with low molecular weight proteinuria and hy- antibody directed against a carboxy-terminal fragment (21) identified percalciuria remain to be elucidated. Studies of CLC-5 expres- translated CLC-5 proteins (data not shown). sion in the human nephron have suggested some possibilities, as CLC-5 is expressed at multiple sites, including the proximal tubule, thick ascending limb of Henle, and intercalated cells of is of interest and suggests that this loop may have a regulatory the collecting duct (20). CLC-5 is located intracellularly and in role in CLC-5 function (11). In relation to this, our finding of the early endosomes, which contain the vacuolar H(ϩ)- the A to T transversion in codon 601, which predicted a ATPase and which form part of the receptor-mediated endo- possible missense mutation Asp601Val, was intriguing. The cytic pathway (20). This suggests a role for CLC-5 as a Asp601 is evolutionarily conserved in mouse (27), rat (19), and counterion transport mechanism allowing the action of the Xenopus (28) and in other CLC members, e.g., CLC-3 and electrogenic H(ϩ)-ATPase in the acidification of the endo- CLC-4 (29,30). Thus, the replacement of this negatively somes (20,21). Indeed, CLC-5 has been shown to have a charged conserved Asp residue with the nonpolar uncharged suitable pH dependence for its activity (34), and endosomal 1466 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1460–1468, 2000

other features (e.g., glycosuria and aminoaciduria) may be due to defective endosomal recycling of plasma membrane trans- porters responsible for the sodium-coupled reabsorption of these solutes (20,21,35). The hypercalciuria, which is not a typical feature of proximal tubular disorders, may partly be of the absorptive type and may be due to the associated high normal to elevated serum concentrations of 1,25-dihydroxyvi-

tamin D3 that are observed in patients with Dent’s disease (36). However, the localization of CLC-5 to the thick ascending limb of Henle, where 60% of calcium reabsorption occurs, suggests another interesting mechanism: The possible changes in endosomal recycling stemming from CLC-5 dysfunction in this segment may lead to altered recycling and activities of the surface transporters (18), e.g., the sodium-potassium-chloride cotransporter (NKCCT), the chloride channel (CLC-Kb), and the renal outer medullary (ROMK), which are involved in generating the transepithelial potential differ- ence that is necessary for paracellular calcium uptake. The role of the Dent’s disease-associated CLC-5 mutants (Figure 1) in these physiologic mechanisms of calcium homeostasis still remains to be elucidated.

Acknowledgments We are grateful to the Medical Research Council (MRC) (United Kingdom) (K. Yamamoto, J. P. D. T. Cox, P. T. Christie, and R. V. Thakker) and the Deutsche Forschungsgemeinschaft (T. Friedrich and T. J. Jentsch) for support. J. P. D. T. Cox is an MRC Training Fellow.

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