A Novel CLCN5 Splice Site Mutation in a Boy with Incomplete Phenotype of Dent Disease

Abstract

Dent disease is a rare X-linked renal proximal tubulopathy presenting with low-molecular-weight proteinuria (LMWP), hypercalciuria, and nephrocalcinosis, other signs of incomplete renal Fanconi syndrome, and renal failure. Early identification of patients who harbor disease-associated mutations is important for effective medical care and avoidance of unnecessary interventions. We report the case of an asymptomatic 9-year-old boy who presented with proteinuria in routine examination. Further investigation revealed the presence of nephrotic range proteinuria, mostly LMWP and mild hypercalciuria without nephrocalcinosis, or other features of tubular dysfunction. Renal function, growth, and bone mineral density were within regular limits. The male gender and the presence of LMWP and hypercalciuria even in the absence of other findings prompted us to genetic investigation for Dent disease. A novel splice site mutation (c.416–2A > G) of the chloride voltage-gated channel 5 ( CLCN5 ) gene, responsible for Dent disease type 1 was identified. In silico analysis revealed that this mutation interferes with the mating of exons 4 and 5. Due to early molecular diagnosis, our patient did not undergo a renal biopsy, neither required aggressive pharmacological interventions. This case underscores the diversity and complexity of CLCN5 mutations and highlights the importance of early molecular testing in male patients with incomplete phenotype of Dent disease.

Introduction

Dent disease is a recessive X-linked inherited disorder of the proximal renal tubule. ^{1 2} Two genetic subtypes have been described, Dent disease type 1 caused by mutations in CLCN5 gene and accounting for 60% of the cases and Dent disease type 2 caused by mutations in OCRL gene and accounting for 15% of the cases. ³ In one-fourth of patients, neither CLCN5 nor OCRL mutations are detected. ³

In Dent disease type 1, the responsible gene CLCN5 encodes the chloride channel CIC-5, a transmembrane protein, that is mainly expressed in the epithelial cells of renal proximal tubule, the thick ascending limb of Henle's loop, and the intercalated cells of the collecting duct and promotes Cl ⁻ /H ⁺ exchange. ⁴ The absence of CIC-5 protein results in disruption of acidification of recycling endosomes in proximal tubule where reabsorption of proteins, minerals, and vitamins, including parathyroid hormone and vitamin D takes place ^{2 4 5 6} and in defective transport system in the thick ascending limb of Henle's loop where calcium reabsorption takes place. ^{7 8} Therefore, the cardinal clinical manifestations of Dent's disease include low-molecular-weight proteinuria (LMWP) and hypercalciuria that often lead to nephrolithiasis, nephrocalcinosis, and progressive renal failure. ² Patients may present with other manifestations of proximal tubule dysfunction, including aminoaciduria, glycosuria, hyperphosphaturia and potassium losses, resembling incomplete renal Fanconi syndrome, as well as metabolic bone disorders. ² The presence of all three criteria: LMWP, hypercalciuria, and at least one of the following: nephrocalcinosis, nephrolithiasis, chronic kidney disease, or other indices of renal Fanconi syndrome is diagnostic of the disease and warrant molecular testing. ² However, patients with Dent disease are often difficult to diagnose due to phenotype heterogeneity and usually undergo renal biopsy or are treated with immunosuppressive medications due to nephrotic range proteinuria before genetic diagnosis is made. ^{5 6}

In this case report, we report the case of a Greek boy presenting with asymptomatic nephrotic range proteinuria and mild hypercalciuria and absence of other diagnostic criteria of Dent disease. Clinical suspicion of Dent disease type 1 prompted us to early genetic testing before the performance of invasive diagnostic methods. We identified a novel CLCN5 splice site mutation (c.416–2A > G) that was further found to significantly disrupt the splicing site of intron 4 and exon 5 boundary with in silico analysis. This case adds to our understanding of Dent disease heterogeneity and suggests that early genetic testing in cases with high clinical suspicion, even in the absence of all diagnostic criteria, is beneficial for the care of the patients.

Case Report

Clinical Presentation

An asymptomatic 9-year-old boy was referred to our department for investigation of proteinuria that was randomly found in urine dipstick in routine laboratory examinations. He was the third child of nonconsanguineous Greek parents, delivered after a full-term uneventful pregnancy with a birth weight of 3.6 kg. At the age of 9 months, in a course of febrile viral infection, he had proteinuria (protein in urine dipstick: 3+), but it was attributed to the febrile course and was not repeated. Personal and family medical history was unremarkable.

On admission, weight and height were in 25th and 50th percentiles, respectively, blood pressure and vital signs and physical examination were normal. The presence of nephrotic range proteinuria, that is, the loss of more than 40 mg/m ² /h of protein in a 24-hour urine collection or the presence of 2 g of protein/g of urine creatinine on a single-spot urine collection ⁹ was confirmed; the patient's protein-to-creatinine ratio was 2.2 g/g in random urine sample and 24-hour protein excretion ranged from 34 to 52 mg/m ² /h. Calcium-to-creatinine ratio ranged from 0.28 to 0.34 in random samples (normal < 0.25) and urine calcium excretion of 8.5 mg/kg/24 hours (normal < 4). There was no hypoalbuminemia or hyperlipidemia, and renal and liver function tests, arterial blood gases, C3 and C4 serum levels, and thyroid function tests were normal. Calcium and phosphate serum levels, 25-OH-vitamin D and parathyroid hormone were within normal limits. Antinuclear and anti-DNA antibodies were negative. Bone mineral density was normal by densitometry. Further investigation of proteinuria revealed a high component of LMWP and constantly elevated β-2 microglobulin values up to 43,000 to 71,700 mg/L (normal < 0.2) suggesting a tubular defect. Other findings of tubular dysfunction were not identified. There was no aminoaciduria, glycosuria, uricosuria, or phosphaturia (tubular reabsorption of phosphate > 91% and renal threshold phosphate concentration to glomerular filtration rate 4.44). The boy had a normal kidney ultrasound without nephrocalcinosis.

There was no family history of renal diseases. However, urinalysis in both parents and siblings revealed isolated mild hypercalciuria and mild LMWP in the mother. The patient's father and siblings did not have LMWP or hypercalciuria. Due to persistent LMWP and hypercalciuria and despite the absence of other diagnostic criteria for Dent's disease, we decided to proceed to genetic testing for potential CLCN5 and OCRL mutations.

Genetic Testing and Mutation Analysis

Informed consent for genetic testing from patient's family members was obtained. Genomic DNA was extracted from the patient's whole blood, and the molecular analyses of the patient's mutation were performed with direct Sanger sequencing of the coding sequence of the CLCN5 gene including approximately 20 nucleotides of the 5′ and 3′ flanking introns. The OCRL gene was also assessed at the same time in a similar way. An alteration in the DNA sequence of CLCN5 gene was identified, namely the hemizygous mutation NM_001127899.3: c.416–2A > G at the boundary of exon 5 and intron 4 (splice site). The mutation, located within an intronic sequence (intron 4) ( Fig. 1A ) of CLCN5 gene removed the normal AG motif in the 3′ acceptor splice site ( Fig. 1B ). No other pathogenic mutation was detected in either CLCN5 or OCRL gene. This CLCN5 mutation was not listed in Human Gene Mutation Database (HGMD; http://www.hgmd.org ) and in ClinVar ( http://www.ncbi.nlm.nih.gov/clinvar ) nor reported in the literature before. ³ Analysis of parental DNA was not feasible; however, hypercalciuria and mild LMWP in the mother may be due to a carrier state, implicating it was not a de novo mutation.

Fig. 1.

Patient's mutation location and splice site defect. ( A ) Distribution of exons and introns of the CLCN5 gene in human genome. The nomenclature of exons was adapted from the study of Ludwig et al. ⁴ The arrows indicate the various transcriptional start sites and the letters (a–e) indicate the isoforms that are produced by alternative splicing. ( B ) The genomic bases of exon 5 of CLCN5 gene (NM_001127899.3) and flanking intron sequences are presented and the hemizygous X chromosome mutation c.416–2A > G is depicted with arrowheads. Predictions of the scores of splice acceptor and donor sites of CLCN5 exon 5 of the wild-type and mutated genomic bases calculated using Human Splicing Finder, version 3 and MaxEntScan are displayed below each splice site.

To further interpret the pathogenicity of this mutation and its role in mRNA splicing, we performed two in silico splice site analyses via the Human Splicing Finder software (version 3.1) and MaxEntScan. ^{10 11} Based on these algorithms the c.416–2A > G mutation was confirmed to disrupt the obligator acceptor (3′) splice site in intron 4–exon 5 boundary. In each algorithm, the splice score given by the wild-type sequence was compared with the splice site score given by the mutated sequence. The scores of a splice acceptor site of exon 5 were markedly decreased by the c.416–2a > G mutation ( Fig. 1B ). Physiological alternative CLCN5 splicing produces five different splice variants that produce two different CIC-5 protein isoforms, a long (816 amino acids) and a shorter one (714 amino acids). ^{4 12} The location of this mutation suggests that all transcript variants are affected ( Fig. 1A ).

Clinical Progress

At present, 5 years after initial presentation, the child is 14 years old and has a normal renal function (eGFR: 115 mL/min/1.73 m ² ), despite his proteinuria (urine protein-to-creatinine ratio: 1.5) which remains mainly LMWP (β-2 microglobulin 108,000 mg/L, normal < 0.2), while there are no signs of nephrolithiasis or nephrocalcinosis. He maintains normal growth and his bone metabolism and density are within normal limits. Due to early molecular diagnosis, our patient did not undergo a renal biopsy and did not receive any aggressive treatments for his proteinuria. He is receiving citrate supplementation for urine acidification and prevention of nephrolithiasis.

Discussion

We report a case of Dent disease type 1 with a novel splice site CLCN5 mutation in an asymptomatic boy with mild presentation consisting of nephrotic range proteinuria, mainly LMWP and hypercalciuria in the absence of any of the other criteria, such as nephrocalcinosis, Fanconi syndrome, or impaired calcium and bone metabolism. Due to early molecular testing, we could diagnose Dent disease before the implementation of invasive diagnostic methods, such as renal biopsy or unnecessary therapeutic trials. This child preserves adequate growth, normal bone density, and renal function today at the age of 14 years.

The cardinal feature of our patient was nephrotic range tubular LMWP, that is, the most constant feature of patients with Dent disease. ¹³ Usually, tubular proteinuria is expected to be lower than the nephrotic range ⁶ ; however, several cases with Dent disease have been reported to have nephrotic range tubular or mixed proteinuria, ^{6 14 15} and recent reports suggest even frank glomerular proteinuria with podocyte involvement in certain CLCN5 mutations. ¹⁶ Such clinical presentation in male patients, especially in the absence of hypoalbuminemia and edema, warrants investigation for tubular proteinuria and exclusion of Dent's disease before initiation of immunosuppressive treatments. ^{6 13}

Hypercalciuria is the second most common abnormality in patients with Dent disease. ¹³ Our patient had only mild hypercalciuria without further consequences during the follow-up period. Cases of Dent disease with atypical phenotype and absence of hypercalciuria have been reported. ^{8 15 17} Thus, Dent disease should be taken into consideration even in the absence of hypercalciuria in a patient with LMWP. ⁸

Apart from LMWP and hypercalciuria, the other clinical manifestations of Dent disease show wide variability. In studies of cohorts of patients, nephrolithiasis is reported in 17 to 32%, nephrocalcinosis in 11 to 56%, proximal tubular defects and signs of incomplete Fanconi syndrome in 48 to 73%, failure to thrive in 23 to 30%, and rickets and/or delayed bone age in 9 to 19% of cases. ^{3 13 18} Indeed, densitometric studies often show a reduction in bone mineral density ^{19 20 21} and several mild skeletal changes of rickets have been observed in patients. ^{18 22} Our patient has normal growth, without any signs of renal Fanconi's syndrome and normal bone mineral density. He is only on supportive care with adequate water intake and citrates to prevent nephrolithiasis.

In this case, we documented a novel CLCN5 mutation that affects an mRNA splice site. To date, more than 260 distinct CLCN5 mutations have been reported in patients with Dent's disease type 1 in the literature ³ and in HGMD (last accessed January 2019). The mutation c.416–2A > G has not been reported previously. Splicing mutations represent ∼10% of the reported mutations so far. ³ Splicing modifies pre-mRNA after transcription, by removing introns and bringing exons together to form the mature mRNA that is suitable for translation. ²³ In the boundaries of introns and exons, there are specific sequence motifs that are required for proper splicing. The most important motifs are located in the acceptor and donor splice sites at positions −1, −2, +1, and +2 of the intron, respectively. ^{23 24} The mutation in our patient is located in the acceptor splice site at position −2 and disrupts the normal AG motif. Mutations in these sequences surrounding the splice site junction can lead to alternate splicing and adversely affect the translated protein. ²⁵

In our patient, we performed in silico analysis using widely available algorithms to determine the pathogenicity of the c.416–2A > G mutation. We found that the splice acceptor site of intron 4 in the boundary of exon 5 is significantly disrupted, suggesting that this mutation leads to interruption of translation and in a truncated protein. Also, the location of this mutation is important since it affects all the physiological CLCN5 transcripts splice variants. However, we cannot estimate the severity of this mutation, since a splice site mutation does not always completely abrogate the splicing process. ^{26 27} Also, the fact that our patient has a mild disease phenotype does not necessarily predict that his mutation is also mild, since in patients with Dent disease, substantial intrafamiliar phenotype variability of the same mutation and no genotype–phenotype correlations with the clinical outcome have been observed. ^{2 8 20}

In conclusion, genetic testing in males with isolated proteinuria even in the absence of the remaining diagnostic criteria could spare these patients with Dent disease from invasive diagnostic investigation or aggressive therapies. ^{6 8 17 28} Dent disease–associated mutations may be complex, there is often no genotype to phenotype correlation and often require additional in silico analyses to interpret their significance and increase our comprehension of the mechanisms causing this disease.