A novel heterozygous mutation in the ATP6V0A4 gene encoding the V-ATPase a4 subunit in an adult patient with incomplete distal renal tubular acidosis.
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ABSTRACT:
SUBMITTER: Imai E
PROVIDER: S-EPMC4886904 | biostudies-literature | 2016 Jun
REPOSITORIES: biostudies-literature
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A novel heterozygous mutation in the ATP6V0A4 gene encoding the V-ATPase a4 subunit in an adult patient with incomplete distal renal tubular acidosis.
Imai Eri E Kaneko Shuzo S Mori Takayasu T Okado Tomokazu T Uchida Shinichi S Tsukamoto Yusuke Y
Clinical kidney journal 20160324 3
A 40-year-old Japanese man who had a medical history of hypokalemic periodic paralysis 4 months prior was hospitalized to undergo a cholecystectomy. Hypokalemia, nephrocalcinosis and alkaluria suggesting distal renal tubular acidosis (dRTA) were detected, but metabolic acidosis was not evident. An ammonium chloride/furosemide-fludrocortisone/bicarbonate loading test demonstrated a remarkable disability in urinary H(+) excretion. A novel heterozygous mutation in the ATP6V0A4 gene encoding the vac ...[more]
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Project description:The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.
| S-EPMC5827428 | biostudies-literature
Project description:BackgroundAutosomal recessive distal renal tubular acidosis (dRTA) is a rare disease characterized by a hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. In some cases, neurosensorial deafness is associated. dRTA is developed during the first months of life and the main manifestations are failure to thrive, vomiting, dehydration, and anorexia.MethodsNine unrelated families were studied: seven children, a teenager, and an adult with dRTA. Hearing was preserved in four children. Coding regions of the genes responsible for recessive dRTA were analysed by Sanger sequencing.ResultsMolecular defects were found in the genes ATP6V1B1 and ATP6V0A4. We identified three homozygous variants in ATP6V1B: a frameshift mutation (p.Ile386Hisfs*56), a nucleotide substitution in exon 10 (p.Pro346Arg), and a new splicing mutation in intron 5. Three patients were homozygous for one novel (p.Arg743Trp) and one known (p.Asp411Tyr) missense mutations in the ATP6V0A4 gene. Three patients were compound heterozygous: one proband displayed two novel mutations, the frameshift mutation p.Val52Metfs*25, and a large deletion of exons 18-21; two probands showed the missense mutation p.Asp411Tyr and as a second mutation, p.Arg194Ter and c.1691+2dup, respectively.ConclusionATP6V0A4 and ATP6V1B1 genes were involved in recessive dRTA of Mexican families. All ATP6V1B1 mutations detected were homozygous and all patients developed sensorineural hearing loss (SNHL) early in infancy. ATP6V0A4 mutations were found in one infant and three children without SNHL, and in one teenager and one adult with SNHL confirming the phenotypic variability in this trait. The mutation p.Asp411Tyr detected in four Mexican families was due to a founder effect. Screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA in this population.
| S-EPMC4867564 | biostudies-literature
Project description:The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1-a4. This study investigated two naturally occurring point mutations in a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H+ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1's catalytic activity. In yeast expression studies, protein levels, H+ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H+ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H+ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H+ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).
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Project description:Autosomal recessive distal renal tubular acidosis (dRTA) is a severe disorder of acid-base homeostasis, often accompanied by sensorineural deafness. We and others have previously shown that mutations in the tissue-restricted a4 and B1 subunits of the H(+)-ATPase underlie this syndrome. Here, we describe an Atp6v0a4 knockout mouse, which lacks the a4 subunit. Using β-galactosidase as a reporter for the null gene, developmental a4 expression was detected in developing bone, nose, eye, and skin, in addition to that expected in kidney and inner ear. By the time of weaning, Atp6v0a4(-/-) mice demonstrated severe metabolic acidosis, hypokalemia, and early nephrocalcinosis. Null mice were hypocitraturic, but hypercalciuria was absent. They were severely hearing-impaired, as shown by elevated auditory brainstem response thresholds and absent endocochlear potential. They died rapidly unless alkalinized. If they survived weaning with alkali supplementation, treatment could later be withdrawn, but -/- animals remained acidotic with alkaline urine. They also had an impaired sense of smell. Heterozygous animals were biochemically normal until acid-challenged, when they became more acidotic than +/+ animals. This mouse model recapitulates the loss of H(+)-ATPase function seen in human disease and can provide additional insights into dRTA and the physiology of the a4 subunit.
| S-EPMC3427075 | biostudies-literature
Project description:The V-ATPase is a multisubunit complex that transports protons across membranes. Mutations of its B1 or a4 subunit are associated with distal renal tubular acidosis and deafness. In the kidney, the a4 subunit is expressed in intercalated cells of the distal nephron, where the V-ATPase controls acid/base secretion, and in proximal tubule cells, where its role is less clear. Here, we report that a4 KO mice suffer not only from severe acidosis but also from proximal tubule dysfunction with defective endocytic trafficking, proteinuria, phosphaturia and accumulation of lysosomal material and we provide evidence that these findings may be also relevant in patients. In the inner ear, the a4 subunit co-localized with pendrin at the apical side of epithelial cells lining the endolymphatic sac. As a4 KO mice were profoundly deaf and displayed enlarged endolymphatic fluid compartments mirroring the alterations in pendrin KO mice, we propose that pendrin and the proton pump co-operate in endolymph homeostasis. Thus, our mouse model gives new insights into the divergent functions of the V-ATPase and the pathophysiology of a4-related symptoms.
| S-EPMC3491836 | biostudies-other
Project description:We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.
| S-EPMC6087784 | biostudies-literature
Project description:BackgroundAutosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic variants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia and nephrocalcinosis. Although several intronic nucleotide variants in these genes have been detected, all of them fell in the apparent splice consensus sequence. In general, transcriptional analysis is necessary to determine the effect on function of the novel intronic variants located out of splicing consensus sequences. In recent years, functional splicing analysis using minigene construction was used to assess the pathogenicity of novel intoronic variant in various field.MethodsWe investigated a sporadic case of dRTA with a compound heterozygous mutation in the ATP6V0A4 gene, revealed by next generation sequencing. One variant was already reported as pathogenic; however, the other was a novel variant in intron 11 (c.1029 + 5G > A) falling outside of the apparent splicing consensus sequence. Expression of ATP6V0A4 was not detected in peripheral leukocytes by RT-PCR analysis. Therefore, an in vitro functional splicing study using minigene construction was conducted to analyze the splicing pattern of the novel variant.ResultsA minigene assay revealed that the novel intronic variant leads to a 104 bp insertion immediately following exon 11. In addition, this result was confirmed using RNA extracted from the patient's cultured leukocytes.ConclusionThese results proved the pathogenicity of a novel intronic variant in our patient. We concluded that the minigene assay is a useful, non-invasive method for functional splicing analysis of inherited kidney disease, even if standard transcriptional analysis could not detect abnormal mRNA.
| S-EPMC5716019 | biostudies-literature
Project description:Background and objectivesIncomplete distal renal tubular acidosis is a well known cause of calcareous nephrolithiasis but the prevalence is unknown, mostly due to lack of accepted diagnostic tests and criteria. The ammonium chloride test is considered as gold standard for the diagnosis of incomplete distal renal tubular acidosis, but the furosemide/fludrocortisone test was recently proposed as an alternative. Because of the lack of rigorous comparative studies, the validity of the furosemide/fludrocortisone test in stone formers remains unknown. In addition, the performance of conventional, nonprovocative parameters in predicting incomplete distal renal tubular acidosis has not been studied.Design, setting, participants, & measurementsWe conducted a prospective study in an unselected cohort of 170 stone formers that underwent sequential ammonium chloride and furosemide/fludrocortisone testing.ResultsUsing the ammonium chloride test as gold standard, the prevalence of incomplete distal renal tubular acidosis was 8%. Sensitivity and specificity of the furosemide/fludrocortisone test were 77% and 85%, respectively, yielding a positive predictive value of 30% and a negative predictive value of 98%. Testing of several nonprovocative clinical parameters in the prediction of incomplete distal renal tubular acidosis revealed fasting morning urinary pH and plasma potassium as the most discriminative parameters. The combination of a fasting morning urinary threshold pH <5.3 with a plasma potassium threshold >3.8 mEq/L yielded a negative predictive value of 98% with a sensitivity of 85% and a specificity of 77% for the diagnosis of incomplete distal renal tubular acidosis.ConclusionsThe furosemide/fludrocortisone test can be used for incomplete distal renal tubular acidosis screening in stone formers, but an abnormal furosemide/fludrocortisone test result needs confirmation by ammonium chloride testing. Our data furthermore indicate that incomplete distal renal tubular acidosis can reliably be excluded in stone formers by use of nonprovocative clinical parameters.
| S-EPMC5586565 | biostudies-literature
Project description:BACKGROUND:Primary distal renal tubular acidosis (dRTA) caused by mutations in the genes that codify for the H?+?-ATPase pump subunits is a heterogeneous disease with a poor phenotype-genotype correlation. Up to now, large cohorts of dRTA Tunisian patients have not been analyzed, and molecular defects may differ from those described in other ethnicities. We aim to identify molecular defects present in the ATP6V1B1, ATP6V0A4 and SLC4A1 genes in a Tunisian cohort, according to the following algorithm: first, ATP6V1B1 gene analysis in dRTA patients with sensorineural hearing loss (SNHL) or unknown hearing status. Afterwards, ATP6V0A4 gene study in dRTA patients with normal hearing, and in those without any structural mutation in the ATP6V1B1 gene despite presenting SNHL. Finally, analysis of the SLC4A1 gene in those patients with a negative result for the previous studies. METHODS:25 children (19 boys) with dRTA from 20 families of Tunisian origin were studied. DNAs were extracted by the standard phenol/chloroform method. Molecular analysis was performed by PCR amplification and direct sequencing. RESULTS:In the index cases, ATP6V1B1 gene screening resulted in a mutation detection rate of 81.25%, which increased up to 95% after ATP6V0A4 gene analysis. Three ATP6V1B1 mutations were observed: one frameshift mutation (c.1155dupC; p.Ile386fs), in exon 12; a G to C single nucleotide substitution, on the acceptor splicing site (c.175-1G?>?C; p.?) in intron 2, and one novel missense mutation (c.1102G?>?A; p.Glu368Lys), in exon 11. We also report four mutations in the ATP6V0A4 gene: one single nucleotide deletion in exon 13 (c.1221delG; p.Met408Cysfs*10); the nonsense c.16C?>?T; p.Arg6*, in exon 3; and the missense changes c.1739 T?>?C; p.Met580Thr, in exon 17 and c.2035G?>?T; p.Asp679Tyr, in exon 19. CONCLUSION:Molecular diagnosis of ATP6V1B1 and ATP6V0A4 genes was performed in a large Tunisian cohort with dRTA. We identified three different ATP6V1B1 and four different ATP6V0A4 mutations in 25 Tunisian children. One of them, c.1102G?>?A; p.Glu368Lys in the ATP6V1B1 gene, had not previously been described. Among deaf since childhood patients, 75% had the ATP6V1B1 gene c.1155dupC mutation in homozygosis. Based on the results, we propose a new diagnostic strategy to facilitate the genetic testing in North Africans with dRTA and SNHL.
| S-EPMC4225572 | biostudies-literature
Project description:Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.
| S-EPMC1735017 | biostudies-other