Recent advances in understanding the clinical and genetic heterogeneity of Dent's disease

Michael Ludwig 2 Boris Utsch 1 Leo A. H. Monnens 0 0 Department of Pediatric Nephrology, University Medical Center Nijmegen , Nijmegen , The Netherlands 1 Department of Pediatrics, University of Erlangen-Nuremberg , Erlangen , Germany 2 Department of Clinical Biochemistry, University of Bonn , Bonn Albuminuria Aminoaciduria Glucosuriaa Hypercalciuriab Nephrocalcinosis Renal stones Rickets Hypokalaemia ( 3.5) in patients with normal GFR aNo transport maximum (Tm) performed, no high performance chromatography (HPLC) analysis. bExcluding one patient with terminal renal failure. Fig. 1. Tubular phosphate reabsorption (TPR) in Dents disease. Closed circle, normal values, obtained from [11]; open circle, patients with Dents disease. Dents disease 1 and the CLCN5 gene The CLCN5 gene, affected by mutations in Dents disease 1 patients, was first identified in a family carrying a microdeletion [15,16]. The CLCN5 gene, encoding the voltage-gated chloride channel and chloride/proton exchanger (ClC-5) [17,18], spans 170 kb on chromosome Xp11.23/p11.22, comprises 17 exons and transcription initiates from at least four different start sites (Figure 2). Transcripts (GenBank accession numbers X91906 and BK000969) including the untranslated exon 1a (start site 2) [19] or 1b (start site 4) [16] are spliced to exon 2 containing the startATG, whereas a third mRNA (arising from start site 3) comprises a larger exon 1b and retains intron 1 [20]. Two further transcripts (start site 1), due to alternative splicing of exon II, include exons I to IV [21]. Both these transcripts carry the start-ATG in exon III, thereby encoding a longer ClC-5 isoform consisting of 816 amino acids with an additional 70 amino acids at the intracellular amino terminus. Since these two mRNAs maintain the reading frame, the startmethionine of the shorter form (746 amino acids) resides at codon position 71. The longer variant, however, has only been detected at mRNA and not at the protein level [21]. To date, more than 80 distinct CLCN5 mutations, consisting of nonsense, missense, splice site and insertional and deletional mutations, have been reported in patients with Dents disease 1 [35,16,2245]. These data provide no evidence for a genotypephenotype correlation, since various mutations were found to be associated with quite different clinical phenotypes ranging from classic Dents disease 1 to very slight urinary abnormalities, not only in unrelated patients but even within the same family [7]. Also, various groups provided evidence for genetic heterogeneity, in that, patients with typical features of Dents disease 1, in whom no CLCN5 mutations could be detected, were encountered [21,24,29,39,43]. Dents disease 2 Recent investigations have revealed that defects in the OCRL1 gene (GenBank accession numbers NM001587 and NM000276) encoding a phospatidylinositol 4,5-bisphosphate 5-phosphatase [PtdIns(4,5)P2 5-phosphatase; EC 3.1.3.36] are also responsible for a phenotype resembling Dents disease 1 [46]. In the OMIM database, Dents disease associated with OCRL1 mutations is now termed Dents disease 2 (OMIM #300555). Mutations in the OCRL1 gene, located at Xq25 [47], were initially found to cause Lowe syndrome [48], a pleiotropic disease (OMIM 309000), affecting eyes, the nervous system and the kidney. OCRL1 mutations observed in Dents disease 2 patients comprise insertion/deletion mutations, splice defects and missense mutations, located in various exons (E; E5: 259262delTGTT, E7: 436-437insAA, E11: T901G, E14: A1385G) or intron 6 (intervening sequence 6: IVS6-2A->G). Due to frameshifts and premature stop codons, three of these defects were shown to cause absence of OCRL1 protein in Western blot analysis, whereas the two missense mutations give rise to reduced protein levels and diminished PtdIns(4,5)P2 5-phosphatase activity [46]. Interestingly, in Lowe patients, these mutations have not been found to date, whereas mutations leading to a frameshift and/or premature stop codon have frequently been detected [49]. Thus the question remains: why, in these Dents 2 cases, does the observed mutations not cause cataracts or metabolic acidosis. One obvious possibility is that this subset of patients carries variations in other gene(s) which provides a degree of cerebral and/or ocular protection. It is also worth mentioning that formal neuropsychological testing has not been reported for the majority of classic Dents 1 patients, and it remains to be determined whether some might have subclinical neuropsychological problems. CLCN5, ~170 Kb Xp11.23 - p11.22 mRNA type 1 I II III 117 Kb type 2 type 3 Dents diseasea polygenic disorder? Expression and localization of ClC-5 V L B SG LL AI I G L D A H R E I T N K S A S R D F D D Y T G V E G 210 K A T I T D SG GY I I H N G F D M A T A P I D I F R V F N T W E Q 480 H Y Y A L G MQE L G V G R L A I A A V G P S M L F I G I R L carrying an amino acid substitution, on the one hand, revealed complete loss of function, whereas other mutant constructs retained up to 50% wild-type activity [4,5,22,23,29,43,50], indicating that type and/or location of a mutation does not predict functional consequences. Defective trafficking of mutant ClC-5 was also demonstrated in a pig renal tubular cell line [71]. CLCN5 knock-out mice Generation of transgenic mice (RZ) with reduced ClC-5 expression [72] or targeted disruption of the CLCN5 gene in mouse models revealed phenotypic consequences displaying the characteristic renal tubular defects observed in Dents disease 1 [58,73,74]. ClC-5 knock-out (KO) mice exhibited LMWP, glycosuria, aminoaciduria, polyuria and renal phosphate wasting. ClC-5 KO mice generated by different groups also developed hypercalciuria and nephrocalcinosis with progressive renal failure [72, 73] but these features were absent in the KO mice established by Piwon et al. [58]. Moreover, some of these studies indicated that hypercalciuria in the ClC-5 KO mousedespite elevated levels of 1,25-dihydroxyvitamin D3is of bone and renal origin [74], rather than caused by increased intestinal calcium absorption [72]. Dysfunction of ClC-5 in endosomes of ClC-5 KO mice further improves our understanding of Dents disease 1. Here, the internalization of the sodium proton exchanger NHE3 and the sodiumphosphate cotransporter NaPi-2 was slowed down, indicating an impaired endocytosis from the apical membrane of proximal tubular cells [58]. At a steady state, both these proteins redistributed from the membrane to intracellular vesicles probably due to a rise in luminal PTH concentration. ClC-5 KO mice show greatly reduced abundance of two cell surface receptors, megalin and cubilin, which are involved in the uptake of proteins into the proximal tubular cells [75]. This constitutes a selective loss at the brush border reflecting a trafficking defect of megalin/cubilin. Consequences for the acidification of the lysosome Along the endocytotic pathway, a successively decreasing p (...truncated)