Genetics of Type III Bartter Syndrome in Spain, Proposed Diagnostic Algorithm

9 p.The p.Ala204Thr mutation (exon 7) of the CLCNKB gene is a "founder" mutation that causes most of type III Bartter syndrome cases in Spain. We performed genetic analysis of the CLCNKB gene, which encodes for the chloride channel protein ClC-Kb, in a cohort of 26 affected patients from 23 families. The diagnostic algorithm was: first, detection of the p.Ala204Thr mutation; second, detecting large deletions or duplications by Multiplex Ligation-dependent Probe Amplification and Quantitative Multiplex PCR of Short Fluorescent Fragments; and third, sequencing of the coding and flanking regions of the whole CLCNKB gene. In our genetic diagnosis, 20 families presented with the p.Ala204Thr mutation. Of those, 15 patients (15 families) were homozygous (57.7% of overall patients). Another 8 patients (5 families) were compound heterozygous for the founder mutation together with a second one. Thus, 3 patients (2 siblings) presented with the c. -19-?_2053+? del deletion (comprising the entire gene); one patient carried the p.Val170Met mutation (exon 6); and 4 patients (3 siblings) presented with the novel p.Glu442Gly mutation (exon 14). On the other hand, another two patients carried two novel mutations in compound heterozygosis: one presented the p.Ile398_Thr401del mutation (exon 12) associated with the c. -19-?_2053+? del deletion, and the other one carried the c.1756+1G>A splice-site mutation (exon 16) as well as the already described p.Ala210Val change (exon 7). One case turned out to be negative in our genetic screening. In addition, 51 relatives were found to be heterozygous carriers of the described CLCNKB mutations. In conclusion, different mutations cause type III Bartter syndrome in Spain. The high prevalence of the p.Ala204Thr in Spanish families thus justifies an initial screen for this mutation. However, should it not be detected further investigation of the CLCNKB gene is warranted in clinically diagnosed families.This study was supported by two grants (PI09/90888 and PI11/01412) from the FIS of the Instituto de Salud Carlos III, Madrid, Spain, and the Eitb Maratoia-Bioef (BIO08/ER/020) the Basque Foundation for Health Innovation and Research (BIOEF, from the Basque Berrikuntza + Ikerketa + Osasuna Eusko Fundazioa). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Detection of <i>CLCNKB</i> deletions by the QMPSF and MLPA techniques.

<p>QMPSF and MLPA electropherograms for the <i>CLCNKB</i> gene from controls (upper panels) and patients (lower panels). (A) QMPSF half doses for exons 16, 17-18, 12 and 4. The arrow shows the peak for exon 12 with the small deletion c.1192_1203del12 (patient SOR0063). Exon 7 of the <i>HNF1B</i> gene is the internal control. (B) QMPSF half doses for exons 20, 6-7 and 9-10. (C) For MLPA, each peak represents the exons for the <i>CLCNKB</i> gene and 15 control probes. The arrows show half doses for all the <i>CLCNKB</i> exons (patient SOR0011, family SOR0024 and SOR0063).</p

Detection of novel <i>CLCNKB</i> mutations by direct sequencing.

<p>Figures represent the sequencing chromatograms from controls (upper panels) and patients (lower panels). (A) Heterozygous c.1192_1203del12 mutation in exon 12; arrows show the extension of the deletion. (B) Heterozygous c.1756+1G>A mutation at the splicing site in exon 16. (C) Heterozygous missense mutation c.1325A>G in exon 14.</p

Poor phenotype-genotype association in a large series of patients with Type III Bartter syndrome

<div><p>Introduction</p><p>Type III Bartter syndrome (BS) is an autosomal recessive renal tubule disorder caused by loss-of-function mutations in the <i>CLCNKB</i> gene, which encodes the chloride channel protein ClC-Kb. In this study, we carried out a complete clinical and genetic characterization in a cohort of 30 patients, one of the largest series described. By comparing with other published populations, and considering that 80% of our patients presented the p.Ala204Thr Spanish founder mutation presumably associated with a common phenotype, we aimed to test the hypothesis that allelic differences could explain the wide phenotypic variability observed in patients with type III BS.</p><p>Methods</p><p>Clinical data were retrieved from the referral centers. The exon regions and flanking intronic sequences of the <i>CLCNKB</i> gene were screened for mutations by polymerase chain reaction (PCR) followed by direct Sanger sequencing. Presence of gross deletions or duplications in the region was checked for by MLPA and QMPSF analyses.</p><p>Results</p><p>Polyuria, polydipsia and dehydration were the main common symptoms. Metabolic alkalosis and hypokalemia of renal origin were detected in all patients at diagnosis. Calciuria levels were variable: hypercalciuria was detected in 31% of patients, while 23% had hypocalciuria. Nephrocalcinosis was diagnosed in 20% of the cohort. Two novel <i>CLCNKB</i> mutations were identified: a small homozygous deletion (c.753delG) in one patient and a small deletion (c.1026delC) in another. The latter was present in compound heterozygosis with the already previously described p.Glu442Gly mutation. No phenotypic association was obtained regarding the genotype.</p><p>Conclusion</p><p>A poor correlation was found between a specific type of mutation in the <i>CLCNKB</i> gene and type III BS phenotype. Importantly, two <i>CLCNKB</i> mutations not previously described were found in our cohort.</p></div

Poor phenotype-genotype association in a large series of patients with Type III Bartter syndrome

Altres ajuts: Departamento de Salud del Gobierno Vasco (2014111064) i Departamento de Educación del Gobierno Vasco (IT795-13)Type III Bartter syndrome (BS) is an autosomal recessive renal tubule disorder caused by loss-of-function mutations in the CLCNKB gene, which encodes the chloride channel protein ClC-Kb. In this study, we carried out a complete clinical and genetic characterization in a cohort of 30 patients, one of the largest series described. By comparing with other published populations, and considering that 80% of our patients presented the p.Ala204Thr Spanish founder mutation presumably associated with a common phenotype, we aimed to test the hypothesis that allelic differences could explain the wide phenotypic variability observed in patients with type III BS. Methods. Clinical data were retrieved from the referral centers. The exon regions and flanking intronic sequences of the CLCNKB gene were screened for mutations by polymerase chain reaction (PCR) followed by direct Sanger sequencing. Presence of gross deletions or duplications in the region was checked for by MLPA and QMPSF analyses. Results. Polyuria, polydipsia and dehydration were the main common symptoms. Metabolic alkalosis and hypokalemia of renal origin were detected in all patients at diagnosis. Calciuria levels were variable: hypercalciuria was detected in 31% of patients, while 23% had hypocalciuria. Nephrocalcinosis was diagnosed in 20% of the cohort. Two novel CLCNKB mutations were identified: a small homozygous deletion (c.753delG) in one patient and a small deletion (c.1026delC) in another. The latter was present in compound heterozygosis with the already previously described p.Glu442Gly mutation. No phenotypic association was obtained regarding the genotype. Conclusion. A poor correlation was found between a specific type of mutation in the CLCNKB gene and type III BS phenotype. Importantly, two CLCNKB mutations not previously described were found in our cohort

Long-term prognosis and clinical characteristics of 15 patients with CLCNKB mutations after 17 [14–22]^* years of follow-up (expressed as number or mean ± SD).

<p>Long-term prognosis and clinical characteristics of 15 patients with CLCNKB mutations after 17 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173581#pone.0173581.ref014" target="_blank">14</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173581#pone.0173581.ref002" target="_blank">2</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173581#pone.0173581.ref002" target="_blank">2</a>]^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173581#t003fn003" target="_blank">*</a>} years of follow-up (expressed as number or mean ± SD).</p

Description of the <i>CLCNKB</i> mutations observed in our cohort and their <i>in silico</i> pathogenicity prediction.

<p>Description of the <i>CLCNKB</i> mutations observed in our cohort and their <i>in silico</i> pathogenicity prediction.</p