Approximately 60% of all DD patients (DD1 pts) have inactivating mutations in the CLCN5 gene encoding the electrogenic Cl-/H+ exchanger ClC-5, which is expressed on the early endosomes in renal proximal tubules. ClC-5 is involved in the receptor-mediated endocytosis and it uses the proton gradient generated by the proton pump vacuolar ATPase (V-ATPase) to transport chloride into the vesicular lumen , . More than 190 different mutations have been reported so far . Phenotypic heterogeneity characterizes the disease and although proximal renal tubular dysfunction is the predominant clinical phenotype, differences in disease expression between families and individuals of the same family remain poorly understood  (full text).
Locus heterogeneity of DD has been confirmed by our and other groups  , and in 15% of cases it is associated with mutations in the OCRL gene (DD2) that is also mutated in Lowe Syndrome (LS)  (full text). To date, around 55 pts with DD2 have been reported.
Whereas DD1 only affects the kidney, the spectrum of symptoms in DD2 can range from apparent exclusive kidney manifestations to the involvement of other organs, notably brain and muscle in overlap with LS.
Further genetic heterogeneity is assumed to exist, considering the presence of pts with the distinctive phenotype of DD without mutation in either of these genes (about 25% of all DD pts). However, no causative variants in the presumable third gene have been discovered so far.
To evaluate the rate of success of currently avaiable genetic tests for the diagnosis of Dent disease.
Material and methods
From 2008 to 2014 DNA samples from 130 unrelated pediatric (71%) and adult (29%) patients with a clinical suspicion of DD were analyzed. We defined as a probable DD phenotype when patients presented with LMWP and hypercalciuria in association with at least one of the following: nephrocalcinosis, nephrolithiasis, phosphaturic tubulopathy, bone disorders, chronic kidney disease, and family history of nephropathy, and “possible” when patients presented with LMWP with at least one of the other signs also in the absence of hypercalciuria.
Genomic DNA was analysed after obtaining patients’ written informed consent.Patients’ DNA samples were firstly subjected to High Resolution Melting analysis and Sanger sequencing for detecting mutations in the CLCN5 gene, secondarly if no mutation was detected, OCRL gene was analysed by Sanger sequencing.
Among 130 patients, we detected 23 CLCN5 mutations, of which 14 were novel (figure 1), giving a detection rate 18%. Of the 107 patients with no CLCN5 mutation, we selected 34 for mutational screening of OCRL gene. The selection was based on the presence of a probable DD phenotype independently from the presence of extra-renal symptoms. We detected 15 OCRL mutations, of which 2 were novel (figure 1), giving a detection rate of 44%. Twelve patients who were six pairs of brothers in pediatric age were all positive. The remaining CLCN5 and OCRL negative patients may represent a selected sample where conduct Whole Exome Sequencing for discovering further DD gene/s.
Our results suggest that a well assessed clinical suspicion is determinant for the success of molecular diagnosis of DD. We think that the CLCN5 negative patients not investigated for the presence of OCRL mutations due to the lack of a clear DD phenotype may represent a heterogeneous pool of patients with phenotypic variants of already known hereditary renal disease. Targeted sequencing using a panel of disease genes for renal tubulopathies and nephrolithiasis should be more appropriated as molecular diagnostic procedure.
Last minute findings: Whole Exome Sequencing data
Targetted Whole Exome Sequencing pilot study was performed on DNA samples of seven DD male patients with no detectable mutations in the CLCN5 or OCRL genes.
Data analysis was performed using a prioritization strategy that includes:
1) eight prediction algorithms: SIFT, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, MetaLR, MetaSVR
2) three conservation scores: PhyloP, GERP++ and SiPhy
3) allele frequencies observed in the 1000 Genomes Project phase 3 data
The results are reported in figure 2
*Dent Disease Italian Network
Gian Marco Ghiggeri and Giancarlo Barbano (Division of Nephrology, Dialysis and Kidney Transplantation, Pediatric Institute G. Gaslini, Genova), Francesco Emma and Gianluca Vergine (Division of Nephrology and Dialysis, Pediatric Hospital Bambin Gesù, Rome), Giuseppe Vezzoli (Division of Nephrology, Dialysis and Hypertension, IRCCS San Raffaele Hospital, Milan), Marilena Cara (Division of Nephrology, Camposampiero General Hospital, Camposampiero) Gabriele Ripanti (Division of Pediatrics and Neonatology, San Salvatore Hospital, Pesaro), Anita Ammenti (Pediatric Institute, University of Parma), Licia Peruzzi (Division of Nephrology, Dialysis and Transplantation, Regina Margherita Hospital,Turin), Giacomo Colussi (Unit of Nephrology, Varese Hospital, Varese), Mario Giordano (Nephrology and Pediatric Dialysis, Pediatric Hospital, Bari) Maria Rosa Caruso (Unit of Nephrology, Bergamo Hospital, Bergamo), Ilse Maria Ratsch (Pediatric Institute, University of Ancona), Giuseppina Marra and Fabio Paglialonga (Nephrology Unit, IRCCS foundation, Ca’ Granda Ospedale Maggiore Policlinico, University of Milano), Angela La Manna (Department of Pediatrics, 2° University of Napoli)