American Journal of Medical Genetics 128A:434–435 (2004)

Research LetterDent Disease-Like Phenotype and the Chloride ChannelClC-4 (CLCN4) Gene

To the Editor:

Dent disease (OMIM 300009) is a rare X-linked renalFanconi syndrome, first described by Dent and Friedman[1964]. The disease is characterized by tubular low-molecular-weight proteinuria (LMWP) and hypercalciuria; nephrocalci-nosis, kidney stone formation (nephrolithiasis), and progres-sive renal failure are frequently observed [Wrong et al., 1994].Additional symptoms are aminoaciduria, phosphaturia, glyco-suria, kaliuresis, uricosuria, and an impairment in urinaryacidification. Dent disease has been related tomutations in theX-linked (Xp11.22) chloride channel 5 gene (CLCN5), whichencodes the ClC-5 protein, a member of the family of voltage-gated chloride channels [Fisher et al., 1995].

Although CLCN5 mutations have been detected in most ofthe patients investigated, our group [Ludwig et al., 2003] andothers [Akuta et al., 1997; Morimoto et al., 1998] haveencountered subjects with signs and symptoms typical of Dentdisease, in whom no CLCN5 defects could be detected. Evenrevision of the genomic structure of the CLCN5 gene condi-tional on the discovery of four additional CLCN5 exonsprovided no clue for the evidence ofmutations in these patients[Ludwig et al., 2003].

In the search for a further candidate gene, we noted that asecond member of the ClC-family of chloride channels, ClC-4,gives rise to strongly outwardly rectifying anion currentsclosely resembling those of ClC-5 [Friedrich et al., 1999] and amost recent report revealed that ClC-4, alike ClC-5, contri-butes to endosomal acidification and trafficking by epithelialcells of the renal proximal tubule [Mohammad-Panah et al.,2003]. These authors also found that ClC-4 and ClC-5 could beco-immunoprecipitated and, therefore may interact in vivo.Finally, mutations in ClC-4 would also show an X-linkedmodeof inheritance, since the gene encoding this chloride channelprotein (CLCN4) is located on Xp22.3. These observations ledus to investigate whether Dent disease can also be associatedwith CLCN4mutations.

We studied seven male patients from five families with aphenotype resembling Dent disease. Diagnosis was based onthe presence of classical hallmarks (LMWP, hypercalciuria,nephrocalcinosis/nephrolithiasis) as given in detail by Ludwiget al. [2003]. After informed consent was obtained from thesesubjects or their guardian CLCN4 exons 1–13 (including theuntranslated exons 1 and 2) with exon–intron boundaries

(GenBank accession number: NM_001830) were amplifiedfrom genomic DNA by polymerase chain reaction (PCR). Thecorresponding PCR-products were subjected to single strandconformation polymorphism (SSCP) analysis and fragmentsdisplaying amigration pattern different from control samples,were investigated by direct automated sequencing (373A,Applied Biosystems, Foster City, CA).

Applying this method, we were able to identify only onesample showing a mobility shift. The sequence variationdetected in this PCR-product turned out to be a C-to-G-transversion in intron 10 affecting residue 43 upstreamof exon11 (IVS10, �43), which was not observed in a further 30 X-chromosomes (10 male and 10 female control samples). Thisnucleotide variation however, neither affects the acceptorsplice site of exon 11nor seems to abolish a consensus sequence(YNYYRAY; Y: pyrimidine, R: purine, N: any base [Krainerand Maniatis, 1988]) necessary for branch point formation,implying that it represents a rare neutral variant.

In conclusion, wewere not able to detect anymutation in ourpatient’sCLCN4 genes.Comparedwithnon-X-linkeddiseases,in CLCN4 the mutation could not have been missed due to thefailure to amplify the respective PCR product from one of thetwo alleles. Nevertheless, the failure of mutation detectioncould be due to (i) the limitations of resolution power of SSCPanalysis or (ii) that regulating elements of the CLCN4 gene(enhancer or other regulatory elements located in intronicregions or proximal or distal to the gene) were not investigatedand/or (ii) that inversions will not be identified by the ampli-fication technique. On the other hand, mutations in othergenes may provoke a disorder ‘‘phenocopying’’ Dent disease.

ACKNOWLEDGMENTS

We thank A. Bokenkamp, H. Crueger, M. Nuutinen, W.Rhede, T. Ring, L. Stapenhorst, and S.Waldegger for access topatients.

REFERENCES

Akuta N, Lloyd SE, Igarashi T, Shiraga H, Matsuyama T, Yokoro S, CoxJPD, Thakker RV. 1997.Mutations of CLCN5 in Japanese childrenwithidiopathic low molecular weight proteinuria, hypercalciuria, andnephrocalcinosis. Kidney Int 52:911–916.

Dent CE, Friedman M. 1964. Hypercalciuric rickets associated with renaltubular damage. Arch Dis Child 39:240–249.

Fisher SE, van Bakel I, Lloyd SE, Pearce SH, Thakker RV, Craig IW. 1995.Cloning and characterization of CLCN5, the human kidney chloridechannel gene implicated in Dent disease (an X-linked hereditarynephrolithiasis). Genomics 29:598–606.

Friedrich T, Breiderhoff T, Jentsch TJ. 1999. Mutational analysisdemonstrates that ClC-4 and ClC-5 directly mediate plasma membranecurrents. J Biol Chem 274:896–902.

Krainer AR, Maniatis T. 1988. RNA splicing. In: Hames BD, Glover DM,editors. Transcription and splicing. Oxford: IRL Press, pp 131–206.

*Correspondence to: Dr. Michael Ludwig, Department ofClinical Biochemistry, University of Bonn, Sigmund-Freud-Str.25, D-53105 Bonn, Germany. E-mail: mludwig@uni-bonn.de

Received 4 November 2003; Accepted 16 February 2004

DOI 10.1002/ajmg.a.30204

� 2004 Wiley-Liss, Inc.

Ludwig M, Waldegger S, Nuutinen M, Bokenkamp A, Reissinger A,Steckelbroeck S, Utsch B. 2003. Four additional CLCN5 exons encodea widely expressed novel long CLC-5 isoform but fail to explain Dent’sphenotype in patients without mutations in the short variant.

Mohammad-Panah R, Harrison R, Dhani S, Ackerley C, Huan LJ, Wang Y,Bear CE. 2003. The chloride channel ClC-4 contributes to endosomalacidification and trafficking. J Biol Chem 278:29267–29277.

Morimoto T, Uchida S, Sakamoto H, Kondo Y, Hanamizu H, Fukui M,Tomino Y, Nagano N, Sasaki S, Marumo F. 1998. Mutations in CLCN5chloride channel in Japanese patients with low molecular weightproteinuria. J Am Soc Nephrol 9:811–818.

Wrong OM,Norden AG, Feest TG. 1994. Dent’s disease: A familial proximalrenal tubular syndrome with low-molecular-weight proteinuria,hypercalciuria, nephrocalcinosis, metabolic bone disease, progressiverenal failure, and a marked male predominance. Quart J Med 87:473–493.

Michael Ludwig*Boris UtschDepartment of Clinical Biochemistry,University of Bonn, Bonn, Germany

Research Letter 435