Adult classic Bartter syndrome: a case report with 5-year follow-up and literature review.

Bartter syndrome (BS) is a rare, inherited salt-losing renal tubular disorder characterized by secondary hyperaldosteronism, hypokalemia, hypochloremia, metabolic alkalosis, and low-to-normal blood pressure. Classic BS, or BS Type 3, the most common subtype in the Asian population, is caused by a molecular defect in ClC-Kb, a voltage-gated chloride channel in renal tubules, due to CLCNKB gene mutation. Because the onset of BS is more common in children than in adults, the diagnosis, treatment outcomes, genotype/phenotype association, and follow-up of adult-onset BS Type 3 are limited. This case report describes the findings in a 20-year-old man who was admitted with hypokalemic paralysis, with clinical manifestations were similar to those of Gitelman syndrome (GS); however, the patient was later diagnosed to have BS Type 3 through genetic testing (NM_000085.4 (CLCNKB): c.1052G>T). A literature review showed that no homozygous mutations have been reported to date. After 5 years of treatment and follow-up, we found that this genotype requires high levels of potassium and is prone to urinary protein and metabolic syndrome. Distinguishing adult-onset BS from GS is challenging in clinical practice. However, genetic diagnosis can help solve this problem effectively, and genotypes play a guiding role in treatment planning.

A novel homozygous CLCNKB variant: An early presentation of classic Bartter syndrome in a neonate.

BACKGROUND: Bartter syndrome (BS) is a rare congenital salt-losing renal tubular transport disorder, characterized by salt wasting, polyuria, biochemical abnormalities, and acid-base homeostasis imbalance. The syndrome has five different genetic forms, and novel mutations of CLCNKB gene lead to type 3 BS also known as classic BS. In this case, we report clinical and molecular findings from a newborn baby with BS. CASE: A 10-day-old male infant born at 37 weeks of gestation by cesarean section following a pregnancy complicated with polyhydramnios, and fetal distress to a 30-year-old gravida 3, para 3 mother, with a 2500 g birth weight was brought to the pediatric emergency department due to weight loss and jaundice. The neonate was referred to the neonatal intensive care unit (NICU) with a preliminary diagnosis of hyponatremic dehydration (Na: 122 mmol/L, 10% dehydration) and hypokalemic hypochloremic metabolic alkalosis (K: 2.13 mmol/L, Cl: 63 mmol/L, pH: 7.62, pCO2 : 39 mmHg, HCO3 : 40.8 mmol/L, BE: 16.9 mmol/L), and hypocalcemia (ionized Ca: 0.72 mmol/L). On arrival to the NICU, symptomatic focal seizures, and polyuria complicated his course. Spot urine biochemistry revealed a renal salt wasting and hypercalciuria: Creatine 11.4 mg/dL Na: 51 mmol/L (54-150), K: 26 mmol/L (20-80), Cl: 164 mmol/L, fractional excretion of sodium (FENa): 3% (0.9-1.6), fractional excretion of chloride (FECl): 17% (<0.5%) and Ca/Cr: 0.33 (<0.2). Biochemical abnormalities disappeared through intravenous fluid and electrolyte therapy, but he could not achieve adequate weight gain, and polyuric urine output (6.5 cc/kg/h), and metabolic alkalosis continued as the enteral feedings advance. Patient's serum renin: 184 pg/mL (5-27 pg/mL) and aldosterone: 1670 pg/mL (1-180 ng/dL) were elevated. Polyuria, renal salt wasting, electrolyte and acid-base disturbances, and hyperreninemic hyperaldosteronism established the diagnosis as Bartter syndrome. An oral indomethacin (1 mg/kg/day) treatment, on the 8th day. ensured the weight gain, and normalized daily urine output. He achieved the goal of birth weight on the 30th day and he was 3520 g weight at discharge on day 42. The genetic tests of the patient as KCNJ1 SLC12A1 gene sequence analysis revealed a novel homozygous mutation in the 14th exon of the CLCNKB gene, the c.1334_1338del CTTTT (p. Ser445fs*4) variant was identified. DISCUSSION: The diagnosis of BS should be considered in the presence of a medical history of severe polyhydramnios of fetal origin. Postnatally, polyuria, signs of dehydration, renal salt wasting, and hypokalemic-metabolic alkalosis should prompt the clinician to request genetic testing for BS in the neonatal period. This case is presented to emphasize that early diagnosis of BS should be considered in newborns presenting with electrolyte abnormalities and metabolic alkalosis accompanying dehydration and favorable growth results can be achieved by starting indomethacin treatment in the early neonatal period. The clinical exome sequencing illustrated a novel missense variant in the CLCNKB gene leading to the molecular diagnosis of BS type 3.

A case report and literature analysis of an infant with Batter syndrome caused by CLCNKA and CLCNKB gene mutation / 中国热带医学

@#Abstract: Bartter syndrome (BS) is a kind of inherited metabolic disease characterized by electrolyte and endocrine disorder, resulting from genetic gene mutation or deletion. Clinically, it manifests as vomiting, constipation, feeding difficulties, weight loss, growth retardation. The laboratory examination shows hypokalemia, metabolic alkalosis, hyperreninemia, aldosteronism, etc. As an autosomal recessive genetic disease, BS has an extremely low incidence rate, making diagnosis and treatment quite challenging. In recent years, with the progress of gene detection and other technologies, China has made great progress in the study of BS, more and more BS has been diagnosed accurately. According to known gene mutation types, it can be divided into type 1, type 2, type 3, type 4, type 5 and Gitelman syndrome, a total of 6 types, with type 4 further divided into type 4a and type 4b. At present, the most common type of children with BS in clinical practice is type 3, which is the classic type of BS. This paper reports a case of Bartter syndrome type 4b (BS4b). After two rounds of three-generation family gene sequencing, it was discovered that the infant had a combined mutation of both the CLCNKA and CLCNKB alleles, in addition to Alport syndrome, and both parents were carriers of this type of gene defect. The child was finally diagnosed as BS4b and Alport syndrome. This report, combined with the patient's clinical features, diagnosis and treatment process, as well as related literature analysis, aims to provide experience for understanding and diagnosis and treatment of this disease.

Twelve exonic variants in the SLC12A1 and CLCNKB genes alter RNA splicing in a minigene assay.

Background: Bartter syndrome (BS) is a rare renal tubular disease caused by gene variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND or MAGED2 genes. There is growing evidence that many exonic mutations can affect the pre-mRNA normal splicing and induce exon skipping by altering various splicing regulatory signals. Therefore, the aim of this study was to gain new insights into the consequences of exonic mutations associated with BS on pre-mRNA splicing. Methods: We analyzed all the missense, nonsense and synonymous variants described in six pathogenic genes by bioinformatics programs and identified candidate mutations that may promote exon skipping through a minigene system. Results: Results of the study showed that 12 of 14 candidate variants distributed in SLC12A1 (c.728G>A, C.735C>G, c.904C>T, c.905G>A, c.1304C>T, c.1493C>T, c.2221A>T) and CLCNKB (c.226C>T, c.228A>C, c.229G>A, c.229G>C, c.1979C>A) were identified to induce splicing alterations. These variants may not only disrupt exonic splicing enhancers (ESEs) but also generate new exonic splicing silencers (ESSs), or disturb the classic splicing sites. Conclusion: To our knowledge, this is a comprehensive study regarding alterations in pre-mRNA of exonic variants in BS pathogenic genes. Our results reinforce the necessity of assessing the consequences of exonic variants at the mRNA level.

Thirteen novel CLCNKB variants and genotype/phenotype association study in 42 Chinese patients with Bartter syndrome type 3.

PURPOSE: Analyze the genotype of 42 Chinese patients with Bartter syndrome type 3 (BS3) and investigate their correlation between genotype and phenotype. METHODS: Identify CLCNKB gene variants by the next-generation sequencing and the multiplex ligation-dependent probe amplification (MLPA), and then evaluate their mutation effects according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines. RESULTS: Thirty-six different variants in CLCNKB gene, including 13 novel ones, were found. The whole gene deletion of CLCNKB gene was the most frequent mutation (40%), and the rate of large deletions is up to 55%. Among 36 variants, six (c.1244T>A, c.1468G>A, c.849_851delCTT, c.359G>T, c.1052G>T, and c.1309G>A) and three (c.228A>C, c.1294_1295TA>CT, and c.1333T>G) variants were classified as "likely pathogenic variants" and "variants with uncertain significance (VUS)," respectively. The other 27 variants were classified as "pathogenic variants". The most common symptoms included: growth retardation (38/42), polydipsia and polyuria (35/42), constipation (31/42), and vomiting (27/42). All patients presented with hypokalemia, hypochloremia, and metabolic alkalosis. The genotype and phenotype association study revealed that who had mutations probably resulting in complete loss of function of both alleles might have severer phenotype. After the treatment that based on indomethacin and potassium chloride, most patients could achieve obvious recovery of growth rate and restoration of hypokalemia. CONCLUSIONS: The present study have found 36 variants of CLCNKB gene, including 13 novel ones, which enrich the human gene mutation database and provide valuable references to diagnosis, treatment, and the genetic counseling of Chinese population.

Variant analysis and genotype/phenotype association study in 42 Chinese patients with Bartter syndrome type 3 / 中华肾脏病杂志

Objective To analyze the variants of 42 Chinese patients with Bartter syndrome type 3 (BS3) and explore the characteristics of genotype and phenotype. Methods Forty-two genetically diagnosed patients from 40 Han and one Hui families were collected in the Affiliated Hospital of Qingdao University and the Affiliated Qingdao Municipal Hospital of Qingdao University during the period of June 2012 to October 2018. The second-generation sequencing and multiplex ligase probe-dependent amplification (MLPA) technique were used to analyze the CLCNKB gene variation and its characteristics in children with BS3. The clinical data were collected, and the therapeutic effect and growth improvement were observed and followed up. Thirty eight patients were divided into severe (n=26) and light (n=12) groups according to the severity of genetic variation. The clinical phenotypic characteristics of the two groups were compared. Results Thirty-six variants including 16 novel ones of CLCNKB gene were found. The whole gene deletion of CLCNKB gene was the most frequent mutation (40％), and the rate of large deletions was up to 55％. The most common symptoms included development retardation (38/42), polydipsia and polyuria (35/42), constipation (31/42) and vomiting (27/42). All patients presented with hypokalemia, hypochloremia and metabolic alkalosis. After the medicine treatment that based on indomethacin and potassium chloride, most patients could achieve obvious recovery of growth rate and restoration of hypokalemia. The severe group showed more severe metabolic alkalosis than the light group. Conclusions Thirty-six variants of CLCNKB gene have been found in this study, including 16 novel ones, which enrich the human gene mutation database (HGMD) and provide valuable references to diagnosis, treatment and the genetic counseling of Chinese population.

Mutation spectrum of Chinese patients with Bartter syndrome.

OBJECTIVE: Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. METHODS: Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. RESULTS: 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. CONCLUSION: The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population.

Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome.

KEY POINTS: The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype-phenotype association has not been established. Low-level expression of the human ClC-Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC-Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C-terminal in-frame green fluorescent protein fusion may slow down the proteasome-mediated proteolysis. Barttin co-expression necessarily improves the stability, membrane trafficking and gating of ClC-Kb. CLCNKB mutations in barttin-binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC-Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. ABSTRACT: Mutations in the CLCNKB gene encoding the human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype-phenotype association has not been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

Gene analysis, treatment, and follow-up of sixteen Chinese patients with Bartter syndrome / 中华肾脏病杂志

Objective To analyze the mutations of causal genes in sixteen Chinese patients with suspicious Bartter syndrome,and follow up their treatment results.Methods Mutations were identified by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA).Clinical and biochemical features at the first presentation as well as follow-up results were reviewed.Results 15 different CLCNKB gene mutations were identified in sixteen patients with BS,including 11 novel ones.A novel missense mutation and a novel small deletion were found from SLC12A1 gene.A novel gross deletion was found in CLCNKA gene.A recurrent missense mutation was identified from BSND gene.The whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32％),and the rate of gross deletion was up to 50 percent in this group of Chinese patients.The most common clinical manifestations were development retardation (15/16),polydipsia and polyuria (15/16).All of the patients were detected with hypokalemia,hypochloremia and metabolic alkalosis.Indomethacin treatment had significant improvement to the stature and weight restoration.Conclusion The present study has found 19 mutations,including 14 novel ones,which enriches the human gene mutation database (HGMD) and provides valuable references to the genetic counseling and diagnosis of Chinese population.

Clinical features and genetic analysis of three patients with Gitelman syndrome / 中华内分泌代谢杂志

[Summary] The clinical data of 3 inpatients clinically diagnosed as Gitelman syndrome ( GS ) were collected. The genomic DNA was isolated from the peripheral blood and the primers were designed to amplify all the exons and flanking introns in the SLC12A3 and CLCNKB genes by PCR. Direct sequencing of PCR products in the two genes was performed in all patients. Three patients manifested with recurrent hypokalemia, hypomagnesemia, hypocalciuria, hypochloremic metabolic alkalosis, but normal blood pressure. Gene sequencing results showed that one novel mutation p. L891V was identified in SLC12A3 gene in case 2. Seven and 12 types of polymorphic loci in the CLCNKB gene were found in case 1 and case 3, respectively. However, mutations were not found in the SLC12A3 and CLCNKB gene.

Mixed Bartter-Gitelman syndrome: an inbred family with a heterogeneous phenotype expression of a novel variant in the CLCNKB gene.

Patients with renal diseases associated with salt-losing tubulopathies categorized as Gitelman and classic form of Bartter syndrome have undergone genetic screening for possible mutation capture in two different genes: SLC12A3 and CLCNKB. Clinical symptoms of these two diseases may overlap. Bartter syndrome and Gitelman syndrome are autosomal recessive salt-losing tubulopathies with hypokalemia, metabolic alkalosis, hyperreninemia, hyperplasia of the juxtaglomerular apparatus, hyperaldosteronism, and, in some patients, hypomagnesemia. Here we describe four patients from an inbred family with a novel missense variant in the CLCNKB gene. All of patients are asymptomatic; yet they have the typical metabolic abnormality of salt losing tubulopathies. One of those patients had hypomagnesaemia while others not. Clinical and laboratory data of all patients was described. All 4 patients have a homozygous c.490G > T missense variant in exon 5 of the CLCNKB gene. This variant alters a glycine into a cysteine on amino acid position 164 of the resulting protein (p.Gly164Cys). The c.490G > T variant is a novel variant not previously described in other patients nor controls. Polyphen analysis predicts the variation to be possibly damaging. Analysis of SLC12A3 was normal. Here in we are describing a novel homozygous c.490G > T missense variation was identified in exon 5 of the CLCNKB gene was identified in an Emirati patients with a mild manifestation of Bartter - Gitelman syndrome.

Translational read-through of a nonsense mutation causing Bartter syndrome.

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

Translational Read-Through of a Nonsense Mutation Causing Bartter Syndrome

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

Analysis of CLCNKB gene mutation in a family with classic Bartter syndrome / 中华肾脏病杂志

Objective To investigate the mutations of CLCNKB gene in a family with classic Bartter syndrome. Methods Genetic DNA was extracted from peripheral blood leucocytes of family members.The coding exons and intron exon junctions of CLCNKB gene were amplyfied by PCR and sequenced directly.Fifty unrelated healthy subjects were selected to exclude the possibility of polymorphism. Results A heterozygous(missense)mutation(482T>G,L161R)was detected in the exon 4 of patients.The hetemzygous mutation(L161R)was found in the mother,while no mutation was found in the father of this family.L161R had not been reported and was a novel mutation when referring to literatures and human genomic database home and abroad.Conclusion A new CLCNKB gene mutation(L161R)is identified for the first time.

Study on CLCNKB gene mutation in a late-onset Chinese patient with classic Bartter syndrome / 中华肾脏病杂志

Objective To investigate the underlying mutation in a late-onset Chinese patient with classic Bartter syndrome. Methods The mutation analysis of CLCNKB gene was performed by the PCR direct sequencing. The patient's parents and siblings were studied as well. Fifty normal volunteers were analyzed as control group. Results The heterozygous deletion mutation cDNA 753delG and heterozygous missense mutation G433E were detected in the patient. Her father was found to carry heterozygous G433E and her mother to carry cDNA 753delG mutation respectively. Her brother carried heterozygous G433E and her sister was normal. Conclusions Two mutations of the CLCNKB gene in this Chinese patient with late-onset classic Bartter syndrome are identified. The cDNA 753delG mutation has not been reported previously.