nature publishing group Articles Clinical Investigation Primary distal renal tubular acidosis: novel findings in patients studied by next-generation sequencing Juan Gómez1, Helena Gil-Peña2, Fernando Santos2,3, Eliecer Coto1,4, Ana Arango1, Olaya Hernandez3, Julián Rodríguez2, Inmaculada Nadal5, Virginia Cantos6, Sara Chocrón7, Inés Vergara8, Álvaro Madrid7, Carlos Vazquez9, Luz E González10, Fiona Blanco11 and the RenalTube Group BACKGROUND: Primary distal renal tubular acidosis (DRTA) is expressed by alpha intercalated cells of distal tubule as well as a rare disease caused by loss-of-function mutations in at least by inner ear and endolymphatic sac cells, and this explains why three genes (ATP6V0A4, ATP6V1B1, and SLC4A1) involved in uri- mutations in this gene are related to DRTA with early-onset nary distal acidification. The next-generation sequencing (NGS) sensorineural hearing loss (SNHL) (2). The A4 subunit, coded technique facilitates the search for mutations in DRTA patients by the ATP6V0A4 gene, is part of the V0 transmembrane and helps to characterize the genetic and clinical spectrum of domain involved in proton translocation through the cellular the disease. membrane. Mutations in this gene were first related to DRTA METHODS: Ten DRTA patients were studied. They had normal with no deafness or late-onset SNHL (3), although it is cur- serum anion gap (AG), metabolic acidosis with simultaneous rently established that deafness cannot discriminate between positive urinary AG, and inability to maximally acidify the urine. A4 and B1 subunits’ defects (4). Few cases have been related The exons of the three genes were sequenced in two pools by with defects in the human AE1 (HCO3-/Clex changer) chan- ultrasequencing. Putative mutations were confirmed by corre- nel, expressed not only by alpha intercalated cells but also by sponding Sanger sequencing of each exon. erythrocytes. The AE1 kidney isoform (kAE1) has the same RESULTS: We found 13 mutations in nine patients. ATP6V0A4: amino acid sequence as the red cell isoform (eAE1), but the Intron16+2insA; p.R807Q; p.Q276fs; p.P395fs; Intron7-2T>C. N-terminal end is truncated. Although both kAE1 and eAE1 ATP6V1B1: p.I386fs; p.R394Q. SLC4A1: p.V245M; p.R589C; isoforms are coded by the SLC4A1 gene, they have different p.R589H; p.G609A. One case was a compound heterozygous promoter regions and alternative splicing, which regulate with a known mutation in ATP6V1B1 (p.G609R) and a patho- the expression and sequence characteristics of the kidney genic variation at SLC4A1 (p.E508K). One patient was negative and erythrocyte isoforms. Thus, mutations in the SLC4A1 for mutations. gene cause either DRTA and/or hemolytic anemia with red CONCLUSION: This study evidences that NGS is labor and cell morphology anomalies (5). The inheritance of this type cost effective for the analysis of DRTA genes. Our results show of DRTA is complex with both autosomal-dominant (6) and for the first time SLC4A1 gene mutations in Spanish patients autosomal-recessive (5) transmissions. Approximately 94 and disclose that compound heterozygosity at two different mutations (Human Gene Mutation Database) in ATP6V0A4, genes can be responsible for DRTA. ATP6V1B1, or SLC4A1 genes have been found to cause DRTA. The identification of these mutations has facilitated a better understanding of the molecular defects underlying DRTA with rimary distal renal tubular acidosis (DRTA) (1) is caused regard to the alterations in the structure (2,3,5–7) and intra- Pby a genetic defect that impairs the ability of kidneys to cellular traffic (4,8) of the involved transporters and channels. maximally acidify the urine in the presence of normal serum To date, the mutational screening of these genes relied in the anion gap hyperchloremic metabolic acidosis. Autosomal- amplification and Sanger sequencing of the 58 coding exons, recessive defects in the H+ ATPase activity cause most of the what represents a huge effort in technical labor and cost. Next- primary DRTA cases. H+ ATPase is a highly conserved proton generation sequencing (NGS) would facilitate the rapid and pump formed by two domains, V1 and V0. The B1 isoform, cost-saving search for mutations in these complex diseases coded by the ATP6V1B1 gene, forms part of the V1 domain (9). The Ion Torrent Personal Genome Machine (PGM) is a that captures protons from cell cytoplasm. This isoform is semiconductor NGS technology that has been proved useful The first two authors contributed equally to this work. 1Department of Molecular Genetics, Hospital Universitario Central de Asturias, Oviedo, Asturias, Spain; 2Department of Pediatrics, Hospital Universitario Central de ­Asturias, Oviedo, Asturias, Spain; 3Department of Pediatrics, Universidad de Oviedo, Oviedo, Asturias, Spain; 4Red de Investigación Renal – REDINREN, Madrid, Madrid, Spain; 5­Department of Pediatrics, Complejo Hospitalario de Navarra, Pamplona, Navarra, Spain; 6Department of Pediatrics, Hospital Infanta Elena, Huelva, Andalucía, Spain; 7­Department of Pediatrics, Hospital Vall d`Hebron, Barcelona, Barcelona, Spain; 8Department of Pediatrics, Hospital Materno-Infantil Teresa Herrera, A Coruña, Galicia, Spain; 9Department of Molecular Genetics, Complejo Hospitalario Universitario Insular Materno Infantil, Las Palmas de Gran Canaria, Canarias, Spain; 10Department of Pediatrics, Fundación Cardio Infantil, Bogotá, Colombia; 11Department of Pediatrics, IIS-Fundación Jiménez Díaz, Madrid, Madrid, Spain. Correspondence: Helena Gil-Peña ([email protected]) Received 8 May 2015; accepted 22 August 2015; advance online publication 27 January 2016. doi:10.1038/pr.2015.243 496 Pediatric Research Volume 79 | Number 3 | March 2016 Copyright © 2016 International Pediatric Research Foundation, Inc. Copyright © 2016 International Pediatric Research Foundation, Inc. Foundation, Research ©2016InternationalCopyright Pediatric semiconductor chip-based NGS of the of NGS chip-based semiconductor a validated and designated we this study, (10,11).In disorders spectrum several of Mendelian the mutational characterize to ( 600 311and inpatients reported was Consanguinity SNHL. to associated were cases Three nephrocalcinosis. mEq/l) and ( sequencing Sanger by confirmed them were all of and tions, muta putative of fulfilled our criteria that fied variants nine inboth pools ( variants control detected all the known We screening. mutation partas of in sequenced all patients be Sanger should these exons Thus, and 16of 11,10,and exons to responding inboth pools, reads cor null poor or gave the 67amplicons of run in summarized is PGM Ion of the data main The Caucasian. cases were of rest The African, respectively. and Hispanic, Hindu, 600)were and 311,414, (patients sequencing Sanger by conventional tified findings were hypokalemia (serum2.9 potassium: were findings (blood pH:7.19 in shown are patients the 10DRTA of features Clinical biochemical and (12). primary tubulopathies of study to aimed the effort international acollaborative in RenalTube, and maximally acidifymaximally the urine (urine pH7.03 to inability and urinary gap positive anion simultaneous with SIFT software (value = 0). To find out the way of segregation of segregation way out the find To (value = 0).software SIFT (value = 1) by software safety not of and the PolyPhen to ing the the at R394Q carrying 306was the mutation wild-type base. Patient the 2.391vs. 7.730with of score strength site asplice with zone acceptor inaconstitutive revealed the base involved which software, Predictor Site Splice the Alternative by nicity pathoge described, corroborated we been previously not has 7−2T>C Intron the variation 7−2 T>C(nondescribed). As the of splicing affecting mutations two confirmed 786,we patient ATP6V0A4 in aGnucleotide of deletion 158 disclosed ahomozygosis Case mutation. of the same is carrier who mother affected his database), (Renaltube DRTA of familial background have He G609Rinthe describeda single mutation for heterozygous was 493 Patient polymorphism. a was it that concluded we DRTA, of manifestations biochemical clinicalor have didnot the father As the father. from inherited variation to alsocarry 122was confirmed the Patient a carriers,suggesting mutation are their parents of Neither R589H,respectively). (R589C and in missense change cases innine ( tions revealed muta sequencing Sanger by corroboration terior RESULTS Table 1 Table 2 ATP6V0A4 SLC4A1 SLC4A1 SLC4A1 ATP6V1B1 ). Control cases harboring a known mutation iden mutation aknown cases harboring ). Control ; Supplementary Figure S2 Figure Supplementary gene, causing the frameshift change p.P395fs. In In p.P395fs. change the frameshift causing gene, gene with a predictive deleterious effect accord deleterious apredictive with gene genes on 10 patients with primary DRTA enrolled enrolled primary with DRTA 10patients on genes , respectively ( , respectively Table 1 gene mRNA, Intron 16+2 insA (2) and Intron Intron and (2) insA 16+2 Intron mRNA, gene ± gene (16) and also the variation E508K at E508Kat also the variation (16) and gene 0.09; serum bicarbonate: 14.1 serum0.09; bicarbonate: Table 2 SLC4A1 Supplementary Table S2 Table Supplementary . Patients presented metabolic acidosis acidosis metabolic presented . Patients Supplementary Figure S1 Figure Supplementary ). Patients 122 and 323 harbor a a 323harbor 122and ). Patients hotspot p.589 position (13,14) (13,14) position p.589 hotspot denovo Table 2 online). NGS and pos and NGS online). ATP6V0A4 ATP6V0A4 origin inboth origin cases. ). Besides, identi we SLC4A1