J Pediatr (Rio J). 2015;91(6):583---589
Whole-exome sequencing as a diagnostic tool for distalrenal tubular acidosis
Paula Cristina Barros Pereiraa, Flvia Medeiros Meloa,Luiz Armando Cunha De Marcoa,b, Eduardo Arajo Oliveiraa,c,Dbora Marques Mirandaa,c, Ana Cristina Simes e Silvaa,c,
a Instituto Nacional de Cincia e Tecnologia --- Medicina Molecular (INCT-MM), Universidade Federal de Minas Gerais (UFMG), BeloHorizonte, MG, Brazilb Department of Surgery, Faculty of Medicine, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazilc Department of Pediatrics, Unit of Pediatric Nephrology, Interdisciplinary Laboratory of Medical Investigation, Faculty ofMedicine, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil
Received 11 November 2014; accepted 25 February 2015Available online 22 July 2015
KEYWORDSATP6V0A4;ATP6V1B1;Children;Distal renal tubularacidosis;Genetics;Whole-exomesequencing
AbstractObjective: Distal renal tubular acidosis (dRTA) is characterized by metabolic acidosis due toimpaired renal acid excretion. The aim of this study was to demonstrate the genetic diagnosisof four children with dRTA through use of whole-exome sequencing.Methods: Two unrelated families were selected; a total of four children with dRTA and theirparents, in order to perform whole-exome sequencing. Hearing was preserved in both childrenfrom 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 withSanger sequencing method.Results: Two mutations were identified in the ATP6V0A4 and ATP6V1B1 genes. In the first fam-ily, a novel mutation in the exon 13 of the ATP6V0A4 gene with a single nucleotide changeGAC TAC (c.1232G>T) was found, which caused a substitution of aspartic acid to tyrosinein position 411. In the second family, a homozygous recurrent mutation with one base-pairinsertion (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 rareand complex genetic nephropathies, allowing the identification of novel and recurrent muta-
tions. Furthermore, for the first time the application of this molecular method in renal tubulardiseases has been clearly demonstrated. 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.
Please cite this article as: Pereira PC, Melo FM, De Marco LA, Oliveira EA, Miranda DM, Simes e Silva AC. Whole-exome sequencing as adiagnostic tool for distal renal tubular acidosis. J Pediatr (Rio J). 2015;91:583---9.
Corresponding author.E-mail: [email protected] (A.C. Simes e Silva).
http://dx.doi.org/10.1016/j.jped.2015.02.0020021-7557/ 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.
584 Pereira PC et al.
PALAVRAS-CHAVEATP6V0A4;ATP6V1B1;Criancas;Acidose tubular renaldistal;Gentica;Sequenciamento totaldo exoma
Sequenciamento total do exoma como ferramenta de diagnstico de acidose tubularrenal distal
ResumoObjetivo: A acidose tubular renal distal (ATRd) caracterizada por acidose metablica devidoa excreco renal de cido prejudicada. O objetivo deste artigo apresentar o diagnsticogentico de quatro criancas com ATRd utilizando o sequenciamento total do exoma.Mtodos: Selecionamos duas famlias no relacionadas, totalizando quatro criancas com ATRde seus pais, para realizar o sequenciamento total do exoma. A audico foi preservada em ambasas criancas da famlia um, porm em nenhuma crianca da famlia dois, na qual um par de gmeasteve perda auditiva severa. Realizamos o sequenciamento total do exoma em dois conjuntos deamostras e confirmamos os achados com o mtodo de Sequenciamento de Sanger.Resultados: Duas mutaces foram identificadas nos genes ATP6V0A4 e ATP6V1B1. Na famliaum, detectamos uma nova mutaco no xon 13 do gene ATP6V0A4 com uma alteraco em umnucleotdeo nico GAC TAC (c.1232G>T) que causou substituico de cido asprtico portirosina na posico 411. Na famlia dois, detectamos uma mutaco recorrente do homozigotocom inserco de um par de bases (c.1149 1155insC) no xon 12 do gene ATP6V1B1.Concluso: Nossos resultados confirmam o valor do sequenciamento total do exoma para oestudo de nefropatias genticas complexas, permitindo a identificaco de mutaces novas erecorrentes. Adicionalmente, demonstramos claramente pela primeira vez a aplicaco dessemtodo molecular em doencas tubulares renais. 2015 Sociedade Brasileira de Pediatria. Publicado por Elsevier Editora Ltda. Todos os direitosreservados.
istal renal tubular acidosis (dRTA) is a rare and com-lex renal disease due to a defect in the excretion ofcid load (H+ and ammonium ions) in alpha-intercalatedells of the collecting duct. The acid load accumulationn the distal nephron results in consumption and reduc-ion of the bicarbonate/CO2 buffer in blood.1 The mainlinical features of dRTA are vomiting, diarrhea, and/oronstipation, loss of appetite, polydipsia, and polyuria.hronic acidosis and secondary alterations such as vomiting,olyuria, and dehydration affect growth, leading to fail-re to thrive. Ultrasound studies can show nephrocalcinosisnd/or nephrolithiasis.2 In general, dRTA has good prognosisf it is diagnosed at an early age and alkaline treatment isontinued. Untreated, dRTA causes growth retardation andickets in children and osteomalacia in adults. Deteriorationf renal function can occur over the years.3
Distal RTA can be transmitted as either an autosomalominant or an autosomal recessive trait.4 The autosomalominant phenotype typically courses mildly in adolescencer adulthood;4 one parent suffers from and is the carrier ofhe disease, or it is due to de novo mutation. Mutations in theLC4A1 gene in families with autosomal dominant dRTA haveeen identified.2,5,6 The symptoms in the autosomal reces-ive phenotype predominantly appear at infancy or earlyhildhood, in which growth retardation is very common. Thisariant can occur with or without deafness, and parents areot affected.2 Autosomal recessive dRTA is associated with
utations in any of the following genes: SLC4A1,7 ATP6V0A4,
nd ATP6V1B1.2,8 Individuals without hearing defects usu-lly carry mutations in the ATP6V0A4 gene, while those witheafness have ATP6V1B1 gene mutations. In approximately
0% of the patients with dRTA, no mutations were found inny of these related genes.3 Indeed, there are dRTA patientsith deafness without ATP6V1B1 gene mutations, and oth-rs with normal hearing who do not have ATP6V0A4 geneutations.3 These findings suggest that other transporters
r channels might cause dRTA. In terms of complexity, it isnown that some patients with mutations in the ATP6V0A4ene develop deafness only in the second decade of life.hus, there remains much to be elucidated in terms ofhenotype---genotype correlations.8---10 So far, more than 20utations in ATP6V0A4 are already known.Whole-exome sequencing provides coverage of more
han 95% of the exons, which contain 85% of disease-causingutations in Mendelian disorders and many disease-redisposing single nucleotide polymorphisms (SNPs)hroughout the genome.11,12 Whole-exome sequencing isorthwhile to evaluate the disease pathogenesis and to
ecognize new pathogenic genes or mutations associatedo disorder, especially in Mendelian disorders.11,12 In thisegard, the present study aimed to evaluate the usefulnessf whole-exome sequencing for genetic diagnosis of dRTA.
atients and methods
ubjects and clinical assessment
our children with confirmed dRTA from two unrelated fam-lies were selected for this study. All patients were followedp at the Pediatric Nephrology Unit of the Federal Univer-
ity of Minas Gerais (UFMG), Brazil. The first family (Family) consisted of two affected siblings, a girl and a boy, withRTA but without deafness, and their unaffected parents.he second family (Family 2) had a monozygotic twin pair