**Background:** Dent disease (DD) is a rare X-linked renal tubulopathy characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, and progressive renal failure. Approximately 60% of cases are due to inactivating variants in the CLCN5 gene (Dent disease 1, DD1), and about 15% are due to variants in the OCRL gene (Dent disease 2, DD2). While many variants are presumed to affect protein function, some may also disrupt pre-mRNA splicing, a process that can lead to exon skipping, intron retention, or other aberrant transcripts. This study aimed to identify novel CLCN5 and OCRL variants in a cohort of DD patients and to experimentally test the splicing effects of selected variants using minigene assays.
**Methods:** Thirteen unrelated male patients with a clinical diagnosis of DD (10 from Spain, 2 from Cuba, 1 from Uruguay) and 27 relatives were investigated. Diagnostic criteria included LMWP (defined by excessive urinary β2-microglobulin or total proteinuria) plus at least one of hypercalciuria (>4 mg/kg/d) or nephrocalcinosis/nephrolithiasis. Age at diagnosis ranged from 0.8 to 19 years. Genomic DNA was extracted from peripheral blood, and coding exons and flanking intronic sequences of CLCN5 and OCRL were analyzed by Sanger sequencing. Variants were compared to reference sequences (NG_007159.3 for CLCN5, NG_008638.1 for OCRL) and queried against gnomAD, 1000 Genomes, dbSNP, and HGMD. Bioinformatics tools (MutPredSplice, SPANR, HSF, NNSplice, CADD-splice, SpliceAI) were used to predict splicing effects. Fifteen variants (12 CLCN5 missense from literature/databases, plus 3 from the patient cohort) were selected for functional testing based on proximity to splice sites (<70 nucleotides) and prediction by at least two tools. Minigenes were constructed by cloning CLCN5 fragments (exons 3, 7, 9, 10-11) and OCRL fragments (exons 11-12, 15) into the pET01 vector. Site-directed mutagenesis introduced mutations. COS7 and HEK293T cells were transfected with wild-type (WT) or mutant minigenes, and RNA was extracted after 24 h. RT-PCR was performed using vector-specific primers, and products were analyzed by agarose gel electrophoresis and sequencing.
**Key Results:** All 13 patients had LMWP, 10 had hypercalciuria, and 5 had nephrolithiasis/nephrocalcinosis. Sequence analysis revealed nine CLCN5 variants (four missense, four frameshift, one nonsense) and four OCRL variants (two missense, two intronic). Six CLCN5 variants (c.1641G>T; p.W547C, c.976G>C; p.G326R, c.1600T>A; p.Y534N, c.2026delA; p.T676Lfs*2, c.1560_1561delTC; p.L521Cfs*6, c.966delC; p.F322Lfs*37) and two OCRL variants (c.1056+1G>A, c.1467-1G>A) were novel. In ten families, mothers were carriers. Bioinformatics predicted pathogenicity for all novel variants. Minigene assays showed that three CLCN5 variants altered splicing: c.1535G>A (p.G512D) and c.1537G>A (p.G513R) caused skipping of exon 10, while c.1641G>T (p.W547C) caused skipping of exons 10 and 11. These variants generated overlapping exonic splicing silencer (ESS) motifs. The other nine CLCN5 variants tested (in exons 3, 7, 9) did not affect splicing. For OCRL, c.1056+1G>A inactivated the donor splice site of intron 11, resulting in skipping of exon 11. c.1467-1G>A inactivated the acceptor splice site of intron 14, leading to incorporation of a truncated exon 15 missing 20 nucleotides from the 5' end, due to use of a cryptic acceptor site 18 nucleotides downstream. Both OCRL variants produced no WT transcript.
**Clinical Implications:** This study expands the genotypic spectrum of Dent disease by identifying six novel CLCN5 and two novel OCRL variants. Functional analysis demonstrated that three presumed missense CLCN5 variants and two canonical splice-site OCRL variants disrupt pre-mRNA splicing, emphasizing that exonic variants can have dual effects (amino acid change and splicing alteration). The findings underscore the necessity of functional splicing assays for accurate variant classification, as bioinformatics predictions alone may be insufficient. Understanding splicing defects may aid in genetic diagnosis and counseling, and could inform future therapeutic strategies such as antisense oligonucleotide therapy to correct splicing. The study also highlights that variants in the ASH-RhoGap domain of OCRL1 (e.g., p.P693L) may be associated with extra-renal symptoms like cataracts and developmental delay, while those in the phosphatase domain (e.g., p.R318C) typically present with renal-only phenotype.