ATP6V1B1 regulates ovarian cancer progression and cisplatin sensitivity through the mTOR/autophagy pathway.

Early detection and effective chemotherapy for ovarian cancer, a serious gynecological malignancy, require further progress. This study aimed to investigate the molecular mechanism of ATPase H+-Transporting V1 Subunit B1 (ATP6V1B1) in ovarian cancer development and chemoresistance. Our data show that ATP6V1B1 is upregulated in ovarian cancer and correlated with decreased progression-free survival. Gain- and loss-of-function experiments demonstrated that ATP6V1B1 promotes the proliferation, migration, and invasion of ovarian cancer cells in vitro, while ATP6V1B1 knockout inhibits tumor growth in vivo. In addition, knocking down ATP6V1B1 increases the sensitivity of ovarian cancer cells to cisplatin. Mechanistic studies showed that ATP6V1B1 regulates the activation of the mTOR/autophagy pathway. Overall, our study confirmed the oncogenic role of ATP6V1B1 in ovarian cancer and revealed that ATP6V1B1 promotes ovarian cancer progression via the mTOR/autophagy axis.

Urinary Acidification Does Not Explain the Absence of Nephrocalcinosis in a Mouse Model of Familial Hypomagnesaemia with Hypercalciuria and Nephrocalcinosis (FHHNC).

Patients with mutations in Cldn16 suffer from familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) which can lead to renal insufficiency. Mice lacking claudin-16 show hypomagnesemia and hypercalciuria, but no nephrocalcinosis. Calcium oxalate and calcium phosphate are the most common insoluble calcium salts that accumulate in the kidney in the case of nephrocalcinosis, however, the formation of these salts is less favored in acidic conditions. Therefore, urine acidification has been suggested to limit the formation of calcium deposits in the kidney. Assuming that urine acidification is causative for the absence of nephrocalcinosis in the claudin-16-deficient mouse model, we aimed to alkalinize the urine of these mice by the ablation of the subunit B1 of the vesicular ATPase in addition to claudin-16. In spite of an increased urinary pH in mice lacking claudin-16 and the B1 subunit, nephrocalcinosis did not develop. Thus, urinary acidification is not the only factor preventing nephrocalcinosis in claudin-16 deficient mice.

A pH imbalance is linked to autophagic dysregulation of inner ear hair cells in Atp6v1ba-deficient zebrafish.

V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H+ secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H+ transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba-/-) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba-/- zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba-/- zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment.

High ATP6V1B1 expression is associated with poor prognosis and platinum­based chemotherapy resistance in epithelial ovarian cancer.

The vacuolar ATPase H+ transporting V1 subunit B1 (ATP6V1B1) belongs to the family of ATP6Vs, which functions to transport hydrogen ions. The expression of ATP6V1B1 and associated clinicopathological features have been linked to various cancers; however, its role in epithelial ovarian cancer (EOC) has remained to be explored. The present study aimed to unveil the function, molecular mechanisms and clinical significance of ATP6V1B1 in EOC. The mRNA levels of ATP6V1 subunits A, B1 and B2 in EOC tissues were determined using data from the Gene Expression Profiling Interactive Analysis database and RNA sequencing. Protein levels of ATP6V1B1 were evaluated through immunohistochemistry staining of EOC, borderline, benign and normal epithelial tissues. The association between ATP6V1B1 expression and clinicopathological features and prognosis of patients with EOC was analyzed. Furthermore, the biological role of ATP6V1B1 in ovarian cancer cell lines was also assessed. RNA sequencing and public dataset analyses revealed elevated ATP6V1B1 mRNA levels in EOCs. High ATP6V1B1 protein levels were also observed in EOC compared with those of borderline and benign tumors and nonadjacent normal epithelial tissues. High ATP6V1B1 expression was associated with the serous cell type, advanced International Federation of Gynecology and Obstetrics stage, high/advanced tumor grade, elevated serum cancer antigen 125 levels and platinum resistance (P<0.001, P<0.001, P=0.035, P=0.029 and P=0.011, respectively). High expression levels of ATP6V1B1 were also associated with poor overall and disease­free survival (P<0.001). Knockdown of ATP6V1B1 decreased cancer cell proliferation and colony­forming abilities (P<0.001) in vitro by inducing cell cycle arrest in G0/G1 phase. Significant upregulation of ATP6V1B1 was observed in EOC and the prognostic significance and association with chemotherapy resistance of ATP6V1B1 in EOC was demonstrated, rendering it an EOC­related biomarker for prognostic evaluation and chemotherapy resistance, as well as a potential therapeutic target for patients with EOC.

Molecular Diagnosis of Primary Hyperoxaluria Type 1 and Distal Renal Tubular Acidosis in Moroccan Patients With Nephrolithiasis and/or Nephrocalcinosis.

Nephrolithiasis (NL) and urolithiasis (UL) are usual reasons for hospitalization and presentation in pediatric outpatient departments and their incidence continues to rise worldwide. In Morocco, a previous epidemiological study done in the Fez region between January 2003 and November 2013 reported a prevalence of 0.83% of childhood UL. In two studies, heritability accounted for almost half of all NL or nephrocalcinosis (NC) prevalence. Genetic factors must be considered in the etiological diagnosis of urinary lithiasis in Morocco since the frequency of consanguineous marriages is high. Hereditary tubular disorders, especially distal renal tubular acidosis (dRTA) and Dent disease, and metabolic disorders like idiopathic hypercalciuria and hyperoxaluria are the most common causes of medullary NC. Primary hyperoxaluria type 1 (PH1), which can generate an early onset of NC, and often chronic kidney disease (CKD) should always be considered and thoroughly diagnosed. The aim of this work was to establish a molecular diagnosis of PH1 and dRTA and, thus, to predict and explain the disease phenotype in a cohort of 44 Moroccan patients with NL and/or NC by analyzing the AGXT and ATP6V1B1 genes that cause NL and/or NC when mutated. Disease phenotype was molecularly explained and solved in six of 44 individuals with NL and/or NC (13.6%). In the pediatric subgroup of individuals, a causative mutation in 16.2% was identified, whereas in the adult cohort no pathogenic mutation was detected. In our patients, PH1 was objectified in 67% of cases followed by dRTA in 33% of cases. We suggest that prompt detection and prophylactic treatment of UL are necessary to limit the risk of everlasting renal damage and thus prevent or delay the progression to CKD.

Genotypic and phenotypic analysis in 51 Chinese patients with primary distal renal tubular acidosis.

The aim of this study was to analyze the genetic variants of 51 Chinese patients with distal renal tubular acidosis (dRTA) and explore the correlation between their genotype and phenotype. Eight variants of SLC4A1, 19 variants of ATP6V0A4, and 16 variants of ATP6V1B1 have been identified, and of which 14 were novel ones. Eleven patients with autosomal dominant dRTA, and four patients with autosomal recessive dRTA were caused by genetic defects in SLC4A1; 18 and nine patients with recessive dRTA were resulted by defects in ATP6V0A4 and ATP6V1B1 respectively; no causal gene was identified in seven patients. Mutation frequency of SLC4A1 in Chinese populations was more common than Europeans. The incidence of deafness in ATP6V0A4 and ATP6V1B1 groups was 16.7% and 54.5%, respectively. The frequency of CKD in adults, children and infants was 100%, 51%, and 3%, separately. Our study will further expand the mutation spectrum of primary dRTA and provide valuable references to genetic counseling of Chinese populations.

Identification of seven exonic variants in the SLC4A1, ATP6V1B1, and ATP6V0A4 genes that alter RNA splicing by minigene assay.

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1, or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the precursor messenger RNA (pre-mRNA) splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 7 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1 (c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108* and c.1572G>A, p.Pro524Pro) were identified to result in complete or incomplete exon skipping by either disruption of exonic splicing enhancers (ESEs) and generation of exonic splicing silencers, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro.

Molecular aspects and long-term outcome of patients with primary distal renal tubular acidosis.

BACKGROUND: Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder caused by impaired distal mechanisms of urinary acidification. Most cases are secondary to pathogenic variants in ATP6V0A4, ATP6V1B1, and SLC4A1 genes, which encode transporters regulating acid-base balance in the collecting duct. METHODS: Retrospective study of molecular and clinical data from diagnosis and long-term follow-up (10, 20, and 40±10 years) of 16 patients with primary dRTA diagnosed in childhood. RESULTS: Molecular analyses revealed nine patients had ATP6V0A4 pathogenic variants, five in ATP6V1B1, and two in SLC4A1. A novel intragenic deletion and a common ATP6V0A4 gene variant (c.1691 + 2dupT) in ATP6V0A4 occurred in two-thirds of these patients, suggesting a founder effect. Median age at diagnosis was 3.25 months (IQR 1, 13.5), which was higher in the SLC4A1 group. Median SDS height at diagnosis was -1.02 (IQR -1.79, 0.14). Delayed clinical diagnosis was significantly related to growth failure (P = 0.01). Median SDS height at 20 years follow-up was -1.23 (IQR -1.71, -0.48), and did not significantly improve from diagnosis (P = 0.76). Kidney function declined over time: at last follow-up, 43% had moderate to severe chronic kidney disease (CKD). Adequate metabolic control was not related to CKD development. Incidence of sensorineural hearing loss (SNHL) was high in ATP6V1B1 patients, though not universal. Patients harboring ATP6V0A4 variants also developed SNHL at a high rate (80%) over time. CONCLUSIONS: Patients with dRTA can develop moderate to severe CKD over time with a high frequency despite adequate metabolic control. Early diagnosis ameliorates long-term height prognosis.

Mutation analysis and phenotypic investigation in patients with primary distal renal tubular acidosis / 中华肾脏病杂志

Objective:To analyze the gene variants in patients with primary distal renal tubular acidosis (dRTA), and explore the correlation between the genotype and phenotype.Methods:The Sanger direct sequencing or whole-exome sequencing was used to identify causal variants and the variation pathogenicity was evaluated according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines in 44 dRTA patients (37 families) diagnosed in the Affiliated Qingdao Municipal Hospital of Qingdao University and the Affiliated Hospital of Qingdao University from April 2010 to September 2020. The clinical features of the patients were summarized, and the correlation between the genotype and phenotype was investigated.Results:Seven variants of SLC4A1 gene, 17 variants of ATP6V0A4 gene, and 15 variants of ATP6V1B1 gene were identified in 44 patients with dRTA, and of which 11 variants were new ones. According to ACMG guidelines, the pathogenic, likely pathogenic, benign variants among the 39 variants were 22, 16 and 1, respectively. Nine patients were autosomal dominant hereditary dRTA caused by SLC4A1 gene mutation, 4 patients with autosomal recessive hereditary dRTA complicated with Southeast Asian ovalocytosis and anemia were caused by SLC4A1 gene mutation, and 14 patients caused by ATP6V0A4 gene mutation and 8 patients caused by ATP6V1B1 gene mutation were autosomal recessive hereditary dRTA; Two children with dRTA were found to carry one monoallelic defect in ATP6V1B1, and no causal gene mutation was identified in 7 patients. One patient showed incomplete dRTA, and the other 43 patients showed complete dRTA. The prevalence of sensory neural hearing loss caused by ATP6V0A4 and ATP6V1B1 mutation were 2/14 and 6/10 respectively. The frequency of chronic kidney disease in adults, children and infants were 4/4, 2/4, and 1/36, separately. After the drug treatment based on potassium citrate and sodium citrate, the growth and development (28/40) and electrolyte disturbance (41/44) of most patients were significantly improved. Conclusions:The present study has identified 39 variants of SLC4A1, ATP6V0A4 and ATP6V1B1 genes in 44 patients with dRTA, including 11 novel ones. There is a close relationship between genotype and phenotype in dRTA patients and most patients' conditions were improved after proper treatment. This study enriches the human gene mutation database and provides valuable references for diagnosis, treatment and genetic counseling in patients with dRTA.

ATP6V1B1 recurrent mutations in Algerian deaf patients associated with renal tubular acidosis.

Hereditary distal renal tubular acidosis (dRTA) is a rare disorder characterized by metabolic acidosis due to impaired renal acid excretion. To date, three genes (ATP6V1B1, ATP6V0A4 and SLC4A1) have been reported to be responsible for this genetic disorder. Notably, mutations of ATP6V1B1 gene, which encode B1-subunit of H + -ATPase pump cause distal renal tubular acidosis often, associated with sensorineural hearing loss (SNHL). Furthermore, enlarged vestibular aqueduct (EVA) was also described in some patients with ATP6V1B1 mutations. Four Algerian unrelated patients presented with dRTA and SNHL were recruited. The ATP6V1B1 gene was preferentially analyzed in all these patients by Sanger sequencing. We identified two previously reported variants in ATP6V1B1 gene: a frameshift mutation (c.1155dupC: p.(Ile386Hisfs*56) in exon 12 and a splicing mutation in intron 2 (c.175-1G > C: p?). Both mutations were homozygous in affected members. Interestingly, one patient with p.(Ile386Hisfs*56) mutation presented profound SNHL and bilateral enlarged vestibular aqueduct (EVA). Our study indicates the importance contribution of ATP6V1B1 gene mutations to the pathogenesis of the dRTA in the Algerian population and will contribute to introducing principles to predict the characteristics of the dRTA in patients. Thus, screening for this gene could allow rapid patient management and provide adequate genetic counseling.

Novel compound heterozygous ATP6V1B1 mutations in a Chinese child patient with primary distal renal tubular acidosis: a case report.

BACKGROUND: Distal renal tubular acidosis (dRTA) is a heterogeneous disorder characterized by normal anion gap metabolic acidosis. Autosomal recessive dRTA is usually caused by mutations occurring in ATP6V1B1 and ATP6V0A4 genes,encoding subunits B1 and a4 of apical H+-ATPase, respectively. The heterogeneous clinical manifestations of dRTA have been described in different ethnic groups harboring distinct mutations. Most of the reported cases are from Europe and Africa. At present, the prevalence of primary dRTA is still poorly elucidated in Chinese population. CASE PRESENTATION: A 2-year and six-month-old female patient was hospitalized because of recurrent hypokalemia, hyperchloremic metabolic acidosis and growth retardation. Laboratory investigations presented a normal anion gap hyperchloremic metabolic acidosis, hypokalemia, and inappropriate alkaline urine. Renal ultrasound indicated bilateral nephrocalcinosis. Bilateral sensorineural hearing loss (SNHL) was confirmed with moderately severe (45 dB) on the left ear and severe (80 dB) on the right ear, which was accompanied with enlarged vestibular aqueduct (EVA) on both sides. According to these findings, a diagnosis of dRTA was made. To identify the pathogenic gene mutation, all coding regions of ATP6V1B1 and ATP6V0A4 gene, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. The splicing variants were verified in peripheral blood leucocytes of the patient by RT-PCR. As a result, two novel heterozygous mutations in ATP6V1B1 were identified in the child. One mutation was a successive 2-nucleotide deletion in exon 2(c.133-134delTG), which caused a marked nonsense mediated mRNA decay. The other was a guanine to adenine substitution of the first nucleotide of intron 8(c.785 + 1 G > A), which led to the exclusion of exon 8. After treatment with sodium citrate, potassium citrateand citric acid, metabolic acidosis and hypokalemia were corrected, but her hearing decreased gradually during the 2 years and had to accept the use of bilateral hearing aids. CONCLUSIONS: We described two novel dRTA associated mutations in ATP6V1B1 identified in a Chinese child patient accompanying with SNHL and EVA. Our study will help to expand the understanding of this rare disease in Chinese population.

Five Novel Mutations in Chinese Children with Primary Distal Renal Tubular Acidosis.

AIM: To analyze the variants of the potential causative genes in five Chinese patients with primary distal renal tubular acidosis (dRTA) from five unrelated families, and to explore their possible genotype-phenotype correlations, so as to raise the awareness of the disease. METHODS: Variants were identified by next generation sequencing. Clinical features and biochemical findings at the first presentation, as well as at follow-up visits were also investigated. One hundred unrelated healthy subjects were selected to evaluate each of the novel mutations found in this study. RESULTS: A total of seven different mutations in the ATP6V0A4, ATP6V1B1, and SLC4A1 genes, the three main causative genes of dRTA, were detected in 4/5 patients. In patient I a novel heterozygous intronic mutation (c.639 + 1G>A) in the ATP6V0A4 gene was identified along with a heterozygous nonsense variant (c.580C>T, p.Arg194*). Two novel heterozygous missense mutations of the ATP6V1B1 gene (c.409C>T, p.Pro137Ser; c.904C>T, p.Arg302Trp) were identified in patient II. In patient III 2 novel heterozygous duplications (c.1504dupT, p.Tyr502Leufs*22; c.2351dupT, p.Phe785Ilefs*28) were found. Thus, these three patients all were compound heterozygotes leading to dRTA. These findings are consistent with the known autosomal recessive inheritance pattern of this disease. Furthermore, a de novo heterozygous missense mutation previously reported (c.1765C>A, p.Arg589Ser) in the SLC4A1 gene was observed in patient IV. No mutations in any of the known dRTA-related causative genes were found in the patient V. CONCLUSIONS: In the present study we identified 7 mutations, including 5 novel variants, in the three genes previously correlated with dRTA, enriching the human gene mutation database (HGMD). In addition, our lack of findings in these three genes for patient V suggests that other genes may contribute to dRTA in some cases.

Clinical and genetic analysis of distal renal tubular acidosis in three Chinese children.

OBJECTIVE: Primary distal renal tubular acidosis (dRTA) is a rare genetic disease characterized by distal tubular dysfunction leading to metabolic acidosis and alkaline urine. Growth retardation is a major concern in these children. The disease is caused by defects in at least three genes (SLC4A1, ATP6V0A4, and ATP6V1B1) involved in urinary distal acidification. Several series of dRTA patients from different ethnic backgrounds have been genetically studied, but genetic studies regarding Chinese population is rare. Our aim was to investigate the clinical features and genetic basis of primary dRTA in Chinese children. METHODS: Three unrelated patients with dRTA participated in our study. Next-generation sequencing was performed, and the findings were validated using the Sanger sequencing method. RESULTS: All patients exhibited hyperchloraemic metabolic acidosis, abnormally high urine pH, hypokalemia, and nephrocalcinosis. Growth retardation was observed in all patients. During the follow-up (range 1-4 years), alkali replacement therapy corrected the systemic metabolic acidosis, and two patients demonstrated normal growth. rhGH therapy was administered to patient-3 at the age of 6 years, and his growth rate was significantly improved (growth velocity 9.6 cm/yr). In total, 5 mutations were identified in our cohort of three patients, and four mutations were novel. CONCLUSIONS: We report the clinical and molecular characteristics of dRTA patients from China. The four novel mutations detected in our study extend the spectrum of gene mutations associated with primary dRTA. Furthermore, our study confirms the effect of early treatment in improving growth for dRTA patient and provides insight into the effects of rhGH on dRTA patients who were diagnosed late and exhibiting a persistent growth delay despite appropriate therapy.

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Distal renal tubular acidosis. Clinical manifestations in patients with different underlying gene mutations.

BACKGROUND: To evaluate whether there are differences in the phenotype of primary distal renal tubular acidosis (dRTA) patients according to the causal defective gene. METHODS: Twenty-seven non-oriental patients with genetically confirmed dRTA were grouped according to the identified underlying mutations in either ATP6V1B1 (n = 10), ATP6V0A4 (n = 12), or SLC4A1 (n = 5) gene. Demographic features, growth impairment, biochemical variables and presence of deafness, nephrocalcinosis, and urolithiasis at diagnosis were compared among the three groups. RESULTS: Patients with SLC4A1 mutations presented later than those with ATP6V1B1 or ATP6V0A4 defects (120 vs. 7 and 3 months, respectively). Hearing loss at diagnosis was present in the majority of patients with ATP6V1B1 mutations, in two patients with ATP6V0A4 mutations, and in none of cases harboring SLC4A1 mutations. Serum potassium concentration (X ± SD) was higher in SLC4A1 group (3.66 ± 0.44 mEq/L) than in ATP6V0A4 group (2.96 ± 0.63 mEq/L) (p = 0.046). There were no differences in the other clinical or biochemical variables analyzed in the three groups. CONCLUSIONS: This study indicates that non-oriental patients with dRTA caused by mutations in the SLC4A1 gene present later and have normokalemia or milder hypokalemia. Hypoacusia at diagnosis is characteristically associated with ATP6V1B1 gene mutations although it may also be present in infants with ATP6V0A4 defects. Other phenotypical manifestations do not allow predicting the involved gene.

Mutation analysis of 5 children with primary distal renal tubular acidosis / 中华肾脏病杂志

Objective To analyze the mutations of causal genes in 5 children with primary distal renal tubular acidosis (dRTA),and explore their association of genotype and phenotype,so as to raise the awareness of the disease.Methods The whole exome sequencing was used to identify mutations in these 5 children from 5 families.Results A total of 4 different mutations of ATP6V0A4 gene were found in 2 dRTA children,including a novel heterozygous intron mutation (c.639 + 1G＞ A),a reported heterozygous nonsense variant (c.580C ＞T,p.Arg194*) and 2 novel heterozygous duplications (c.1504dupT,p.Tyr502Leufs*22;c.2351dupT,p.Phe785Ilefs*28).Two novel heterozygous missense mutations of ATP6V 1B 1 gene (c.409C ＞ T,p.Pro 137Ser;c.904C ＞ T,p.Arg302Trp) were identified in the third child,and a heterozygous missense mutation of SLC4A1 gene (c.1765C ＞ A,p.Arg589Ser) previously reported was found in the fourth child.No mutation of the dRTA-related causal genes was found in the fifth child.Furthermore,the mutations of causal genes in each of the first three children were compound heterozygous,which were consistent with the autosomal recessive inheritance pattern,and the variant from the fourth child was de novo.Conclusions The present study has found 7 mutations,including 5 novel variants,which enriches the human gene mutation database (HGMD) and contributes to a better understanding of the disease mechanisms.

Clinical and molecular findings in three Moroccan families with distal renal tubular acidosis and deafness: Report of a novel mutation of ATP6V1B1 gene.

BACKGROUND: Primary distal renal tubular acidosis (dRTA) is a rare genetic condition characterized by an impaired acid excretion by the intercalated cells in the renal collecting duct. Recessive forms of this disease are caused by mutations in tow major genes: ATP6V1B1 and ATP6V0A4. Causal mutations in ATP6V1B1 gene are classically associated with early sensorineural hearing loss, however cases of tubular acidosis with early deafness have also been described in patients with mutations in the ATP6V0A4 gene. METHODS: The phenotype and genotype of three Moroccan consanguineous families with dRTA and deafness were assessed. Molecular analysis was performed by PCR amplification and direct sequencing of exon 12 of ATP6V1B1 gene. RESULTS: A novel c.1169dupC frameshift mutation of ATP6V1B1 gene was identified in one family and the c.1155dupC North African mutation in the tow other families. DISCUSSION AND CONCLUSION: In this report, we propose first line genetic testing based on screening of these two mutations both located in exon 12 of ATP6V1B1 gene in Moroccan patients with recessive form of dRTA associated to precocious hearing loss. Molecular diagnosis of dRTA leads to appropriate treatment and prevention of renal failure in affected individuals and to provide genetic counseling for families at risk.

Sequenciamento total do exoma como ferramenta de diagnóstico de acidose tubular renal distal / Whole-exome sequencing as a diagnostic tool for distal renal tubular acidosis

Resumo Objetivo A acidose tubular renal distal (ATRd) é caracterizada por acidose metabólica devido à excreção renal de ácido prejudicada. O objetivo deste artigo é apresentar o diagnóstico genético de quatro crianças com ATRd com uso do sequenciamento total do exoma. Métodos Selecionamos duas famílias não relacionadas, quatro crianças com ATRd e seus pais, para fazer o sequenciamento total do exoma. A audição foi preservada em ambas as crianças da família um, porém em nenhuma criança da família dois, na qual um par de gêmeas teve perda auditiva severa. Fizemos o sequenciamento total do exoma em dois conjuntos de amostras e confirmamos os achados com o método de sequenciamento de Sanger. Resultados Duas mutações foram identificadas nos genes ATP6V0A4 e ATP6V1B1. Na família um, detectamos uma nova mutação no éxon 13 do gene ATP6V0A4 com uma alteração em um nucleotídeo único GAC → TAC (c.1232G>T) que causou substituição de ácido aspártico por tirosina na posição 411. Na família dois, detectamos uma mutação recorrente do homozigoto com inserção de um par de bases (c.1149_1155insC) no éxon 12 do gene ATP6V1B1. Conclusão Nossos resultados confirmam o valor do sequenciamento total do exoma para o estudo de nefropatias genéticas complexas e permitem a identificação de mutações novas e recorrentes. Adicionalmente, demonstramos claramente pela primeira vez a aplicação desse método molecular em doenças tubulares renais.

Abstract Objective Distal renal tubular acidosis (dRTA) is characterized by metabolic acidosis due to impaired renal acid excretion. The aim of this study was to demonstrate the genetic diagnosis of four children with dRTA through use of whole-exome sequencing. Methods Two unrelated families were selected; a total of four children with dRTA and their parents, in order to perform whole-exome sequencing. Hearing was preserved in both children from the first family, but not in the second, wherein a twin pair had severe deafness. Whole-exome sequencing was performed in two pooled samples and findings were confirmed with Sanger sequencing method. Results Two mutations were identified in the ATP6V0A4 and ATP6V1B1 genes. In the first family, a novel mutation in the exon 13 of the ATP6V0A4 gene with a single nucleotide change GAC → TAC (c.1232G>T) was found, which caused a substitution of aspartic acid to tyrosine in position 411. In the second family, a homozygous recurrent mutation with one base-pair insertion (c.1149_1155insC) in exon 12 of the ATP6V1B1 gene was detected. Conclusion These results confirm the value of whole-exome sequencing for the study of rare and complex genetic nephropathies, allowing the identification of novel and recurrent mutations. Furthermore, for the first time the application of this molecular method in renal tubular diseases has been clearly demonstrated.

Whole-exome sequencing as a diagnostic tool for distal renal tubular acidosis.

OBJECTIVE: Distal renal tubular acidosis (dRTA) is characterized by metabolic acidosis due to impaired renal acid excretion. The aim of this study was to demonstrate the genetic diagnosis of four children with dRTA through use of whole-exome sequencing. METHODS: Two unrelated families were selected; a total of four children with dRTA and their parents, in order to perform whole-exome sequencing. Hearing was preserved in both children from the first family, but not in the second, wherein a twin pair had severe deafness. Whole-exome sequencing was performed in two pooled samples and findings were confirmed with Sanger sequencing method. RESULTS: Two mutations were identified in the ATP6V0A4 and ATP6V1B1 genes. In the first family, a novel mutation in the exon 13 of the ATP6V0A4 gene with a single nucleotide change GAC → TAC (c.1232G>T) was found, which caused a substitution of aspartic acid to tyrosine in position 411. In the second family, a homozygous recurrent mutation with one base-pair insertion (c.1149_1155insC) in exon 12 of the ATP6V1B1 gene was detected. CONCLUSION: These results confirm the value of whole-exome sequencing for the study of rare and complex genetic nephropathies, allowing the identification of novel and recurrent mutations. Furthermore, for the first time the application of this molecular method in renal tubular diseases has been clearly demonstrated.

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness.

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H(+) secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding ß1 subunit of H(+)-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H(+) ions contributing to dRTA with sensorineural deafness.