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Dent disease-like phenotype and the chloride channel ClC-4 (CLCN4) gene.

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American Journal of Medical Genetics 128A:434 –435 (2004)
Research Letter
Dent Disease-Like Phenotype and the Chloride Channel
ClC-4 (CLCN4) Gene
To the Editor:
Dent disease (OMIM 300009) is a rare X-linked renal
Fanconi syndrome, first described by Dent and Friedman
[1964]. The disease is characterized by tubular low-molecularweight proteinuria (LMWP) and hypercalciuria; nephrocalcinosis, kidney stone formation (nephrolithiasis), and progressive renal failure are frequently observed [Wrong et al., 1994].
Additional symptoms are aminoaciduria, phosphaturia, glycosuria, kaliuresis, uricosuria, and an impairment in urinary
acidification. Dent disease has been related to mutations in the
X-linked (Xp11.22) chloride channel 5 gene (CLCN5), which
encodes the ClC-5 protein, a member of the family of voltagegated chloride channels [Fisher et al., 1995].
Although CLCN5 mutations have been detected in most of
the patients investigated, our group [Ludwig et al., 2003] and
others [Akuta et al., 1997; Morimoto et al., 1998] have
encountered subjects with signs and symptoms typical of Dent
disease, in whom no CLCN5 defects could be detected. Even
revision of the genomic structure of the CLCN5 gene conditional on the discovery of four additional CLCN5 exons
provided no clue for the evidence of mutations in these patients
[Ludwig et al., 2003].
In the search for a further candidate gene, we noted that a
second member of the ClC-family of chloride channels, ClC-4,
gives rise to strongly outwardly rectifying anion currents
closely resembling those of ClC-5 [Friedrich et al., 1999] and a
most recent report revealed that ClC-4, alike ClC-5, contributes to endosomal acidification and trafficking by epithelial
cells of the renal proximal tubule [Mohammad-Panah et al.,
2003]. These authors also found that ClC-4 and ClC-5 could be
co-immunoprecipitated and, therefore may interact in vivo.
Finally, mutations in ClC-4 would also show an X-linked mode
of inheritance, since the gene encoding this chloride channel
protein (CLCN4) is located on Xp22.3. These observations led
us to investigate whether Dent disease can also be associated
with CLCN4 mutations.
We studied seven male patients from five families with a
phenotype resembling Dent disease. Diagnosis was based on
the presence of classical hallmarks (LMWP, hypercalciuria,
nephrocalcinosis/nephrolithiasis) as given in detail by Ludwig
et al. [2003]. After informed consent was obtained from these
subjects or their guardian CLCN4 exons 1–13 (including the
untranslated exons 1 and 2) with exon–intron boundaries
(GenBank accession number: NM_001830) were amplified
from genomic DNA by polymerase chain reaction (PCR). The
corresponding PCR-products were subjected to single strand
conformation polymorphism (SSCP) analysis and fragments
displaying a migration 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 one
sample showing a mobility shift. The sequence variation
detected in this PCR-product turned out to be a C-to-Gtransversion in intron 10 affecting residue 43 upstream of exon
11 (IVS10, 43), which was not observed in a further 30 Xchromosomes (10 male and 10 female control samples). This
nucleotide variation however, neither affects the acceptor
splice site of exon 11 nor seems to abolish a consensus sequence
(YNYYRAY; Y: pyrimidine, R: purine, N: any base [Krainer
and Maniatis, 1988]) necessary for branch point formation,
implying that it represents a rare neutral variant.
In conclusion, we were not able to detect any mutation in our
patient’s CLCN4 genes. Compared with non-X-linked diseases,
in CLCN4 the mutation could not have been missed due to the
failure to amplify the respective PCR product from one of the
two alleles. Nevertheless, the failure of mutation detection
could be due to (i) the limitations of resolution power of SSCP
analysis or (ii) that regulating elements of the CLCN4 gene
(enhancer or other regulatory elements located in intronic
regions or proximal or distal to the gene) were not investigated
and/or (ii) that inversions will not be identified by the amplification technique. On the other hand, mutations in other
genes may provoke a disorder ‘‘phenocopying’’ Dent disease.
We thank A. Bökenkamp, H. Crueger, M. Nuutinen, W.
Rhede, T. Ring, L. Stapenhorst, and S. Waldegger for access to
Akuta N, Lloyd SE, Igarashi T, Shiraga H, Matsuyama T, Yokoro S, Cox
JPD, Thakker RV. 1997. Mutations of CLCN5 in Japanese children with
idiopathic low molecular weight proteinuria, hypercalciuria, and
nephrocalcinosis. Kidney Int 52:911–916.
Dent CE, Friedman M. 1964. Hypercalciuric rickets associated with renal
tubular damage. Arch Dis Child 39:240–249.
*Correspondence to: Dr. Michael Ludwig, Department of
Clinical Biochemistry, University of Bonn, Sigmund-Freud-Str.
25, D-53105 Bonn, Germany. E-mail:
Received 4 November 2003; Accepted 16 February 2004
DOI 10.1002/ajmg.a.30204
ß 2004 Wiley-Liss, Inc.
Fisher SE, van Bakel I, Lloyd SE, Pearce SH, Thakker RV, Craig IW. 1995.
Cloning and characterization of CLCN5, the human kidney chloride
channel gene implicated in Dent disease (an X-linked hereditary
nephrolithiasis). Genomics 29:598–606.
Friedrich T, Breiderhoff T, Jentsch TJ. 1999. Mutational analysis
demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane
currents. 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.
Research Letter
Ludwig M, Waldegger S, Nuutinen M, Bökenkamp A, Reissinger A,
Steckelbroeck S, Utsch B. 2003. Four additional CLCN5 exons encode
a widely expressed novel long CLC-5 isoform but fail to explain Dent’s
phenotype 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 endosomal
acidification 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 CLCN5
chloride channel in Japanese patients with low molecular weight
proteinuria. J Am Soc Nephrol 9:811–818.
Wrong OM, Norden AG, Feest TG. 1994. Dent’s disease: A familial proximal
renal tubular syndrome with low-molecular-weight proteinuria,
hypercalciuria, nephrocalcinosis, metabolic bone disease, progressive
renal failure, and a marked male predominance. Quart J Med 87:
Michael Ludwig*
Boris Utsch
Department of Clinical Biochemistry,
University of Bonn, Bonn, Germany
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like, channel, phenotypic, disease, genes, dent, clc, chloride, clcn4
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