Dent disease is an X-linked disorder of proximal tubular function characterized by low molecular weight proteinuria, the most consistent feature, as well as hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure [1,2]. The condition presents in boys during early childhood with symptoms of renal stones (pain abdomen, hematuria), bone pains or deformities due to rickets or with incidentally detected low molecular weight proteinuria. Progression to end stage renal disease (ESRD) occurs between 3rd to 5th decades in 30-80% of affected males.

Clinical and biochemical details were recorded at diagnosis and at follow up. Standard deviation score (SDS) for weight and height were calculated using World Health Organization charts and AnthroPlus v.1.0.4 calculator (www.who.int/growthref/tools/en). Blood levels of calcium, phosphate, alkaline phosphatase, electrolytes, creatinine, pH, bicarbonate, 25-hydro-xyvitamin D and parathormone were measured and compared to age-appropriate cut-offs. Timed urinary excretion of phosphate and creatinine was used to estimate tubular maximum for phosphate reabsorption/glomerular filtration rate (TmP/GFR); estimated glomercular filteration rate (eGFR) by the modified Schwartz formula was used to classify stages of chronic kidney disease (CKD) [10]. Height SDS, blood creatinine, eGFR and calcium excretion were compared at presentation and follow-up by Wilcoxon signed rank test (SPSS version 15.0, SPSS Inc., Chicago).

Genomic DNA, isolated from peripheral blood leukocytes, was amplified by polymerase chain reaction (PCR) using primers for all exons of the CLCN5 gene [11], followed by Sanger sequencing. In case the CLCN5 sequence was negative for variations, the OCRL1 gene was screened. Pathogenecity prediction software, sorting intolerant from tolerant (SIFT, http://sift.jcvi.org/) and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) were used to predict the effect of novel exonic variations.

Genetic analysis: Sequence analysis of the CLCN5 gene in 15 patients revealed eight mutations in exons 3, 7, 9, 10, 11 and 12 in 13 patients (Web Table II, Fig. 1) [12-15]. Five were missense mutations (p.Ser244Leu, p.Met504Lys, p.Trp58Cys, p.Glu527Gln and p.Gly57Arg), and three were nonsense mutations (p.Leu729X, p.Arg648X and p.Arg637X). Two patients did not show mutations in the CLCN5 or OCRL1 genes. Both the novel missense mutations (p.Glu527Gln and p.Gly57Arg) were predicted to affect protein function by either SIFT or PolyPhen2 computer programs. All three nonsense mutations including one novel mutation (p.Leu729X) were expected to be pathogenic.

Fig. I Schematic representation of ClC-5 mutations in 13 patients with Dent disease. The predicted topology of the protein is drawn from Wu, et al. [14] and Dutzler, et al. [15]. ClC-5 consists of 18 á helices, A to R, which are indicated by the boxed areas. Mutations in ClC-5 are shown as solid black circles with the resulting amino acid change indicated besides.

The present report describes a severe phenotype of Dent disease. Most patients showed pathogenic variants involving the CLCN5 gene. The median age at diagnosis was similar to a cohort of 117 European patients and pooled data from 377 patients [16]. However, patients in this report showed relatively early onset of symptoms compared to other series (Table II) [5,16-18]. While rickets is reported in infants with Dent disease, early onset with polyuria, polydipsia and night blindness is not described [1,12-19]. We also report the occurrence of renal failure in the first decade of life in patients with a common mutation in CLCN5, p.Ser244Leu.

TABLE II  Phenotypic Findings in Patients with Dent Disease 1 (CLCN5 mutations) in the Present and Previous Reports

Most series report rickets in up to one-third patients with Dent disease 1 [5,16-18]. Wrong, et al. [2] reported rickets in 40% patients and hypophosphatemia in one-third patients, with satisfactory response to vitamin D therapy. All our patients showed refractory rickets with hypophosphatemia in 94.4 % cases. Similar to four of our patients, all nine affected boys from two European families with the CLCN5 mutation p.Ser244Leu, in whom the mutation was first described, showed rickets [20]. However, none of the patients with this mutation from a large pedigree in southern United States had features of rickets [21]. It is unclear whether these phenotypic variations reflect differences in severity of coexistent vitamin D deficiency, dietary and environmental factors, delayed diagnosis, or effect of modifier genes.

Polyuria, an important symptom in the our patients, is reported in Dent disease [2]. While formal water deprivation testing was not performed, a defect in urinary concentration is likely, similar to other inherited renal tubular disorders with secondary nephrogenic diabetes insipidus [22]. This may be mediated by downregulation of expression of aquaporin 2 via the calcium sensing receptor in apical membrane of medullary collecting duct [22,23]. The high incidence of polyuria in these patients might explain the low prevalence of nephrolithiasis, the former serving as a physiological mechanism preventing stone formation. Moreover, it is also reported that the degree of hypercalciuria may not relate to development of nephrocalcinosis or renal failure [24]. Despite high rates of hypercalciuria and nephrocalcinosis (97.6% and 87.8%, respectively), only 12% of 41 patients of Dent disease showed renal failure [18]. Additional genetic or environmental factors may contribute to the occurrence of nephrocalcinosis/nephrolithiasis and consequent renal dysfunction.

Vitamin A-responsive night blindness, first reported in Dent disease in patients from our center [9], was observed in two-thirds of the present patients, compared to 37.5% in another report [25]. The condition is attributed to high urinary losses of RBP [3,25] and reduced blood levels of retinol and RBP [25], but information on dietary intakes and blood retinol levels is lacking. While insufficient vitamin A intake might predispose patients with Dent disease to clinically overt vitamin A deficiency, none of the unaffected family members developed night blindness.

The study is limited by small sample size, retrospective design, and lack of genetic testing in asymptomatic family members. However, there are important phenotypic differences from previously reported cohorts, including low prevalence of nephro-calcinosis and occurrence of CKD within first decade of life. Apart from the usual features, Dent disease in this cohort of Indian boys has a relatively severe phenotype with early onset of symptoms, hypophosphatemic rickets and night blindness.

Contributions: SB, RT, AS, PH, AB: diagnosis and management of patients; SB, RT: data collection; AS, PH, AB: analytical inputs and review of literature; HC: performed the sequencing of the genes. All authors participated in preparation of the manuscript and approved the final version submitted for publication.

Funding: Korean Health Technology R&D Project (HI12C0014), Ministry of Health & Welfare, Republic of Korea.

Competing interest: None stated.