Acidosis and Deafness in Patients with Recessive Mutations in FOXI1

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Sven Enerbäck ,1 Daniel Nilsson,1 Noel Edwards,2 Mikael Heglind,1 Sumaya Alkanderi,2 Emma Ashton,3 Asma Deeb,4 Feras E.B. Kokash,5 Abdul R.A. Bakhsh,5 William van’t Hoff,6 Stephen B. Walsh,7 Felice D’Arco,5 Arezoo Daryadel,8,9 Soline Bourgeois,8,9 Carsten A. Wagner ,8,9 Robert Kleta ,6,7 Detlef Bockenhauer ,6,7 and John A. Sayer 2

1Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; 2Institute of Genetic Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom; 3North East Thames Regional Genetic Service Laboratories, London, United Kingdom; 4Pediatric Services, Mafraq Hospital, Abu Dhabi, United Arab Emirates; 5College of Medicine, Gulf Medical University, Ajman, United Arab Emirates; 6Great Ormond Street Hospital for Children, National Health Service Foundation Trust, London, United Kingdom; 7University College London Centre for Nephrology, London, United Kingdom; 8Institute of Physiology, University of Zürich, Zurich, Switzerland; and 9National Center for Competence in Research, National Center in Competence in Research Kidney.CH, Zurich, Switzerland

ABSTRACT Maintenance of the composition of inner ear fluid and regulation of electrolytes and acid-base homeostasis in the collecting duct system of the kidney require an overlapping set of membrane transport proteins regulated by the forkhead transcription factor FOXI1. In two unrelated consanguineous families, we identified three patients with novel homozygous missense mutations in FOXI1 (p.L146F and p.R213P) predicted to affect the highly conserved DNA binding domain. Patients presented with early-onset sensorineural deafness and distal renal tubular acidosis. In cultured cells, the mutations reduced the DNA binding affinity of FOXI1, which hence, failed to adequately activate genes crucial for normal inner ear function and acid- base regulation in the kidney. A substantial proportion of patients with a clinical diagnosis of inherited distal renal tubular acidosis has no identified causative mutations in currently known disease genes. Our data suggest that recessive mutations in FOXI1 can explain the disease in a subset of these patients.

J Am Soc Nephrol 29: 1041–1048, 2018. doi: https://doi.org/10.1681/ASN.2017080840

An important homeostatic function of the kidney is secretion. The intercalated cells of the collecting the regulation of acid-base balance. This occurs in the proximal tubule via bicarbonate reabsorption andinthecollectingductsystemvia proton Signiﬁcance Statement

Distal renal tubular acidosis (dRTA) is defined by the inability of the distal nephron to acidify the urine and can Received August 5, 2017. Accepted November 15, 2017. be associated with sensorineural deafness. About one C.A.W., R.K., D.B., and J.A.S. have shared senior authorship. third of patients with inherited dRTA have no identified causative mutations in known disease genes. Here, we Published online ahead of print. Publication date available at report recessive mutations in the forkhead transcription www.jasn.org. factor FOXI1 as a novel cause of dRTA with deafness. fi Correspondence: Dr. Sven Enerbäck, Department of Medical Our ndings enable a precise diagnosis in affected Biochemistry and Cell Biology, Institute of Biomedicine, University of patients, inform genetic counseling as well as cascade Gothenburg, Medicinareg. 9A, SE405 30 Gothenburg, Sweden. screening, and provide insight into the transcriptional Email: [email protected] regulation of transport proteins in the distal nephron and inner ear by FOXI1. Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 1041–1048, 2018 ISSN : 1046-6673/2903-1041 1041 CLINICAL RESEARCH www.jasn.org duct use specifictransporterstoregulateacid-basebalance 1.2) (Figure 1A). In family 2, the proband (patient 2.1), a girl, that can secrete protons (type A intercalated cells) and bicar- was the only affected family member (Figure 1B). Key clinical bonate (type B intercalated cells).1 Impaired type A interca- features in all affected patients included early-onset sensori- lated cell function may lead to an inability to acidify the urine neural hearing loss requiring cochlear implants; a hypokale- and secrete acids, with consequent development of hyper- mic, hyperchloremic metabolic acidosis at presentation with chloremic non-anion gap metabolic acidosis. Mutations inappropriately high urine pH; failure to thrive; and bilateral known to cause inherited distal renal tubular acidosis nephrocalcinosis (Figure 1E), consistent with dRTA. The (dRTA) have been identified in transporters present in the deafness was profound and associated with dilation of the type A intercalated cells and include the B1 and a4 subunits vestibular aqueduct (Figure 1F, Supplemental Figures 1–4). of the vacuolar H+-ATPase (V-ATPase), anion exchanger 1 The dRTA was associated with hypercalciuria and nephrocal- (AE1), and the cytosolic carbonic anhydrase.2–4 Interestingly, cinosis noted at presentation but responded to treatment with mutations in the V-ATPase B1 subunit are typically associated both alkali and potassium supplements, with subsequent with sensorineural deafness as can be those in a4,2,5 emphasizing catchup in growth of the children. In addition, patients 1.1 functional similarities between the epithelium of the inner ear and 2.1 also developed medullary cystic changes within the and that of the collecting ducts of the kidney.6 This is due to kidney (Figure 1, E and F). Clinical data are summarized in the expression of a common set of membrane transport pro- Table 1. teins in distinct cell types of the epithelium of the inner ear and the renal collecting duct. Specifically, Forkhead-related Structural Analyses of Mutated FOXI1 Proteins cells of endolymphatic duct and sac epithelium of inner ear7 The novel FOXI1 homozygous missense mutations, p.L146F and intercalated cells express B1 and a4 subunits of the V- and p.R213P, were identified after whole-exome sequencing in ATPase proton pump as well as the anion exchange protein the affected patients with segregation of the alleles from the AE4 and pendrin.7,8 parents (Figure 1, C and D). The mutations were not found in A genetic impairment of urine acidification can be associated the Exome Aggregation Consortium database. Both missense with an altered pH/ionic composition in the inner ear fluid and changes were located within evolutionary highly conserved hence, prevent proper sound perception. Another common fea- residues of the FOXI1 protein (Figure 1G). Consistent with ture of these cell types is that they each express FOXI1—atran- FOX protein sequence alignments (Figure 1G, Supplemental scription factor that has been shown to be crucial for regulation of Figure 1), in silico modeling of FOXI1, using the crystal struc- AE1, AE4, and the V-ATPase subunits B1, a4, A, and E2.8 Con- ture of rat Foxa3,12 predicted that FOXI1 L146 occupies a ho- sistent with this, mice with a germline deletion of Foxi1 display mologous position to L142 in a-helix 2 of the Foxa3 DNA sensorineural deafness and dRTA9,10 together with reduced ex- binding domain (Figure 1, H and I). A stabilizing interaction pression of these target genes. Although a family has been between Foxa3 and its cognate DNA is formed between the identified in which Pendred syndrome (PS) segregates with a side-chain d1 carbon atom of L142 and the ribose DNA back- composite heterozygous genotype of single-allele mutations in bone.12 Identical results were obtained when modeling FOXI1 SLC26A4 and FOXI1,11 biallelic recessive loss-of-function mu- on the structures of human FOXM1,13 human FOXO4,14 hu- tations in FOXI1 have not been described in human patients. man FOXK2,15 and rat Foxd316 (Supplemental Figure 2), with In this report, we describe two novel missense mutations similar protein-DNA interactions identified for the homolo- that do not directly affect membrane transport proteins but gous leucine residues in these structures. The FOXI1 L146F rather, disrupt cell function at the transcriptional regulatory mutation was predicted to disrupt this stabilizing protein- level—in that they prevent the transcription factor FOXI1 DNA interaction, with the larger (approximately 30 Å3),17 from binding to regulatory DNA cis-elements of target gene rigid aromatic ring of phenylalanine predicted to impinge promoters. This leads to a severely reduced activation of those on the ability of the recognition helix (a-helix 3) to fully en- target genes, including membrane transport proteins, which gage the major groove of the target DNA (Figure 1J). The require FOXI1 interactions for proper expression. In this way, functional importance of a small hydrophobic residue at this these two novel loss-of-function mutations induce a severe position in the DNA binding domain is highlighted by the syndrome of deafness and acidosis with a phenotype very sim- almost complete conservation of a leucine residue in the ilar to that of mice that altogether lack Foxi1 expression. FOX protein family (Supplemental Figure 1). FOXI1 R213 was predicted to occupy a homologous position to L209 in the wing 2 domain of Foxa3 (Figure 1K). Similar to Foxa3 RESULTS L142, the side-chain b-carbon atom of Foxa3 L209 interacts with the ribose DNA backbone.12 Therefore, we hypothe- Patient Reports sized that FOXI1 R213 also served to stabilize the interaction Two consanguineous kindred with an autosomal recessive with its target DNA, similar to the role of R210, R211, and pattern of dRTA and sensorineural deafness were evaluated R214 in Foxa3 (Figure 1K). Substitution of the positively (Figure 1). In family 1, the proband (patient 1.1), a boy, was charged side chain of arginine with proline (p.R213P) will the eldest of three children, with one affected sister (patient destroy the predicted electrostatic interaction with the DNA,

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thereby disrupting DNA binding (Figure 1L). (Additional modeling information is detailed in Supplemental Appendix.)

Functional Analyses of Mutated FOXI1 Proteins Wefurther investigated the functional effect of the mutations in vitro. First, we assessed protein expression levels of the transfected wild type and the mutations p.L146F and p.R213P of FOXI1 in the human kidney cell line HEK 293T, which did not differ (Figure 2A). Second, we compared trans-activation of wild-type and mutant FOXI1 using previously validated promoter constructs8,10 for the human a4 subunit of the H+-ATPase (ATP6V0A4), the human anion-exchange protein pendrin (SLC26A4), and the human anion-exchange protein 1 (AE1/SLC4A1). Consistent with our previous data,8,10 wild- type FOXI1 increased reporter gene activity several fold above empty vector–transfected cells, whereas the p.L146F or p.R213P mutant constructs show signiﬁcantly reduced capability to trans-activate these promoters (Figure 2B). In an electrophoretic mobility shift assay, we assessed binding of wild-type and mutant FOXI1 proteins to DNA in the form of a canonical FOXI1 binding cis-element. Wild-type FOXI1 bound to the oligonucleotide (Figure 2C, lane 1), whereas no such interaction was detected for the p.L146F or p.R213P mutants (Figure 2C, lanes 2 and 3, respectively). Third, we used GFP fusion constructs

the red arrow, and FOXI1 R213 in W2 is indicated by the black arrow. H shows superposition of the human FOXI1 homology model (green) with the crystal structure of Foxa3 (orange) bound to DNA. (I) The recognition helix (a3) of Foxa3 is Figure 1. Clinical, molecular and modeling data of FOXI1 families and their mutations. A positioned in the major groove of the cognate and B show the family pedigrees and status with respect to the FOXI1 mutation genotype. DNA and stabilized in part by a 3.4-Å interaction Squares denote family members who are men/boys, and circles denote family members between Foxa3 L142 and the sugar-phosphate who are women/girls. Shaded symbols denote affected members with homozygous mu- backbone. (J) Mutation of the homologous leu- tation, and semishaded symbols denote heterozygous carriers. Arrows denote the probands cine residue in FOXI1 to phenylalanine (L146F; (patient 1.1 in family 1 and patient 2.1 in family 2). C and D show sequence chromatograms colored magenta) is predicted to disrupt DNA of the affected and parental genotype. Mutations are marked with arrowheads. E and F binding. K shows a close-up view of the W2 do- show renal ultrasound scans showing medullary nephrocalcinosis in all affected and cystic main of Foxa3 and FOXI1. L209 in Foxa3 forms a change within the kidneys in patients 1.1 and 2.1. Computed tomography (CT) volumetric 4.3-Å interaction with the DNA backbone. (L) The rendering of the right labyrinth in patient 2.1 revealed (F, white arrow) dilation of the en- structurally homologous FOXI1 R213 is predicted dolymphatic sac compared with (F, lower right CT scan) healthy control. Note the normal to facilitate DNA binding, similar to the role of appearance of the cochlea (C) and lateral semicircular canals (SCs) in patient 2.1. G depicts R210, R211, and R214 in W2 of Foxa3. Mutation the secondary structural elements of the Foxa3 DNA binding domain (a-helices, cylinders; of FOXI1 R213 to proline (R213P; colored ma- b-sheets, arrows; and wing domains wing 1 [W1] and W2) above sequence alignments with genta) will destroy the electrostatic interaction FOXI1 proteins from various species. FOXI1 L146 in helix 2 (a-helix 2 [a2]) is indicated by with DNA.

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Table 1. Clinical and genetic characteristics of affected patients Patient Identiﬁcation 1.1a 1.2a 2.1b Sex Boy Girl Girl Ethnicity UAE UAE Iraq Mutationc Nucleotided c.436C.T c.436C.Tc.638G.C Amino acid substitution p.Leu146Phe p.Leu146Phe p.Arg213Pro Allele frequency Not present in EXaC Not present in EXaC Not present in EXaC dRTA presentation Age at diagnosis, yr 8 6 ,1 Symptoms Rickets Rickets Failure to thrive, rickets Weight, kg (SDS) 18 (22.9) 16 (21.9) 9 (23.5) Height, cm (SDS) 119 (21.6) 105 (22.0) 83.5 (22.5) Blood biochemistries pH 7.30 7.30 7.30

tCO2, mmol/L 16 17 14 Potassium, mmol/L 3.6 3.4 3.5 eGFR, ml/min per 1.73 m2 87 99 110 Urine biochemistries pH 8 8 8 Calcium-to-creatinine ratio, mmol/mmol 0.20 (,0.6)d 0.26 (,1.1)e 4.25 (,1.4) (upper limit of normal) Radiology Nephrocalcinosis (age detected)f Yes (10 yr) Yes (9 yr) Yes (2 yr) Medullary cysts (age detected) Yes (10 yr) Yes (9 yr) Yes (8 yr) Deafness Age at diagnosis, yr 1 1 2 Cochlear implant (age inserted) Yes (4 yr) Yes (4 yr) Yes (13 yr)

UAE, United Arab Emirates; EXaC, Exome Aggregation Consortium; SDS, SD score; tCO2, total carbon dioxide in blood. aData at presentation for patients 1.1 and 1.2 are from the first presentation in the tertiary center in the UAE at ages 8 and 6 years old, respectively. bData at presentation for patient 2.1 are from the first presentation in the United Kingdom at age 2 years old when she was partially treated. Data from the initial presentations are not available. cMutations were homozygous in all three patients. dNumbers refer to NCBI reference sequence: NM_012188.4. eUrine calcium-to-creatinine ratio values pretreatment are unavailable; normocalciuria was seen in the context of correction of metabolic acidosis. fThe first renal imaging of patients 1.1 and 1.2 was at ages 10 and 9 years old, respectively. Nephrocalcinosis and cysts may have been present earlier. to determine the intracellular distribution of the wild-type and V-ATPase, and thus, FOXI1 mutations are important in the mutant FOXI1 proteins. Distinct nuclear localization was ob- differential diagnosis of dRTA. The phenotypic mimicry is served for wild-type and mutant FOXI1 proteins (Figure 2D), not surprising, considering the previously reported impor- precluding defective nuclear import as a cause for the reduced tant role of Foxi1 in the regulation of genes associated with trans-activational capacity of the p.L146F and p.R213P mu- dRTA.3,5,10,18 The discrepancy with respect to the absence or tants. A morphometric analysis of data from Figure 2D is later onset of sensorineural deafness in patients with a4 or shown in Supplemental Figure 5, and an immunochemis- more typically, B1 mutations is likely due to FOXI1 mutations try-based approach to intracellular localization of FOXI1 affecting the regulation of several important membrane and the p.L146F and p.R213P mutants are shown in Supple- transporters, including those critical for inner ear function, mental Figure 6. Taken together, these experiments show that whereas isolated impairment of the a4 subunit and B1 accord- these mutants have a similar intracellular distribution as wild- ingly may generate a less severe phenotype that, to some extent, type FOXI1. Our patients with loss-of-function mutations in can be tolerated in the ear. This emphasizes the difference in FOXI1 recapitulate the phenotype described in a mouse phenotypic scope between a mutation in a distinct membrane model that lacks Foxi1 expression with early-onset sensori- transport protein and that of a transcription factor that regulates neural deafness and dRTA.7,10 several target genes. In murine studies, in the intercalated cells of the renal tu- bules and the Forkhead-related cells of the endolymphatic duct DISCUSSION and sac of the inner ear, Foxi1 regulates expression of B1 and a4 subunits of the V-ATPase proton pump and also, the anion- The human phenotype that we report mimics the renal find- exchange proteins AE1, AE4, and pendrin. Without expression ings of patients with mutations in the B1 or a4 subunits of the of a functional Foxi1 protein, there is a failure to express these

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target genes, and as a consequence, this results in severely impaired cellular function, with altered ionic/pH composition of inner ear fluid, and a significantly reduced capacity of the kidneys to secrete an acid load.9,10 The inner ear phenotype seen in mice lacking Foxi1 is remarkably similar to what we show here for one of the probands (Figure 1F), with dRTA and an enlargement of the endolymphatic sac and duct resulting in an enlarged vestibular aqueduct (EVA) syndrome (Supplemental Figures 3 and 4).7,9 The biochemical analysis of mutated FOXI1 protein (Figure 2) showed a severe reduction in the ability to interact with bona fide FOXI1 cis-elements with consequent reduced expression of target genes. This is consistent with the in silico analysis showing impaired interactions of mutant FOXI1 with the major groove of target DNA (p.L146F) and disturbance of electrostatic interactions with DNA (p.R213P). Together, these data show that, in patients homozygous for these missense FOXI1 mutations, there is very limited FOXI1 DNA binding activity, likely resulting in a failure to trans-activate a set of membrane transport proteins needed for normal inner ear and kidney function. The patients in this study did not show any signs of goiter, and available data on thyroid function were normal. Further- more, we show that patients with recessive loss-of-function mutation in FOXI1 display dRTA and deafness with EVA, suggesting that complete loss of FOXI1 activity Figure 2. Effect of FOXI1 mutations on protein stability, promoter transactivation, induces a unique and distinct phenotype. DNA binding, and cellular localization. (A) Western blot (WB) analysis of FOXI1 In a previous report, PS associated with a protein expression in whole-cell lysates from HEK 293T cells transfected with digenic pattern of inheritance, in which a FOXI1 wild type (WT), L146F mutant, R213P mutant, or empty vector (EV). To composite heterozygous genotype with determine equal protein loading, the membrane was stripped and reprobed for mutations in FOXI1 and SLC26A4 segre- b-actin. *Incomplete stripping of signal from the FOXI1 labeling. B shows reporter gates with deafness and an EVA.11 In this gene activity, expressed in relative luciferase units (RLUs), on cotransfection of patient, goiter was not noted, and the con- increasing amounts (15, 30, and 75 ng) of FOXI1 expression plasmid and 25 ng of tributing missense mutation in FOXI1 was ATP6V0A4, pendrin, or AE1 promoter reporter constructs in HEK 293T cells. Level located approximately 50 amino acids C of significance, calculated by two-way ANOVA using GraphPad Prism 5, is indicated. **P,0.01; ***P,0.001; ****P,0.001. C shows electrophoretic mobility terminally of the DNA binding domain. fi shift assay using in vitro–transcribed/translated FOXI1 WT, L146F, or R213P with In addition, the authors identify ve patients EV as control and radiolabeled oligonucleotides harboring an FOXI1 binding site. with PS or nonsyndromic EVAwith a single Bound and unbound oligonucleotides (oligos) are marked. Lower panel shows a mutation in one of the FOXI1 alleles, similar WB of the transcribed/translated products. D illustrates the intracellular localiza- to another study.19 Although the finding tion of FOXI1-GFP fusion proteins after transfection of HEK 293T cells with FOXI1 of rare FOXI1 variants in this patient WT, L146F, or R213P mutants or EV. Green channel, GFP signal (insets show group is intriguing, the lack of a family zoomed in views of a single cell); red channel, nuclear staining (To-PRO-3); merge history in these patients argues against m channel, all channels together. Scale bar, 10 m. the pathogenicity of single heterozygous mutations, consistent with the apparent

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Table 2. Filtering criteria used for single-nucleotide variants than those reported here display pathologic phenotypes in and insertion and deletion selection response to loss-of-function mutations in FOXI1. Family 1 All three patients in this study were noted to have medullary Whole-exome data filtering steps Family 2 (2.1) (1.1 and 1.2) renal cysts, raising the question of whether FOXI1 could also be No. of variants 176,834 80,397 involved in cystogenesis. However, an association of dRTAwith Variants of high confidence 158,256 79,590 cysts is well recognized: in our own cohort of children with SNVs/indel #1% in HapMap18 14,363 6190 dRTA, over one third developed cysts during childhood.21 and 1000 Genome database Typically, this is ascribed to hypokalemia,22 although other Predicted deleterious 1076 2347 factors, such as hypercalciuria/nephrocalcinosis, have also Biologic context (kidney disease, 217 8 been implicated.23 Observations in more patients with dRTA renal tubular acidosis) are needed to better assess the frequency of cysts in FOXI1- Present on both alleles in all 11based dRTA compared with other genetic forms. affected individualsa Approximately one third of patients with dRTA have no iden- SNV, single-nucleotide variant; indel, insertion and deletion selection. fi 21,24,25 aAffected members after screening of kindred on the basis of clinical and ti ed causative mutation in recognized disease genes and biochemical features. thus, should be tested for FOXI1 mutations to help establish a precise diagnosis with consequent genetic counseling—especially in patients with cases involving hearing and possibly, fertility lack of a clinical phenotype in the parents of our patients. problems in men.20 Additional evidence against the pathogenicity of isolated heterozygous loss of function of FOXI1 is provided by observations in mice with a heterozygous germline deletion of Foxi1 CONCISE METHODS that also have no phenotype.7,9 On the basis of this, we find it FOXI1 very unlikely that a single mutation in one allele will This study was approved by the following institutional review boards: cause disease. However, in combination with another single- Institute of Child Health/Great Ormond Street Hospital Research allele mutation (for instance, in a gene that regulates Ethics Committee 05/Q0508/6 and Newcastle and North Tyneside FOXI1 SLC26A4), a single-allele mutation in could be part Research Ethics Committee 2003/163. of the pathogenetic mechanism underlying PS or nonsyn- Parents provided written informed consent. Three subjects with dromic EVA. dRTA and sensorineural deafness without identified mutations in Foxi1 Of interest, in mice lacking , males have been shown to known disease genes were investigated. We performed whole-exome 20 be infertile. This is due to a failure of clear cells in the epi- sequencing using peripheral blood DNA from affected family mem- fl didymis to acidify the extracellular luminal uid. This leads bers. Sanger sequencing was used to validate the variants identified by to a pathologic post-testicular sperm maturation, and as a whole-exome sequencing in both affected children. In silico modeling Foxi1 consequence, spermatozoa from null males fail to reach and biochemical analysis were performed with human FOXI1 protein fi the female genital tract in suf cient number to allow fertiliza- as a control compared with the two mutated FOXI1 proteins. tion.20 Whether mutations in FOXI1 will be associated with reduced fertility in men also remains an open question, be- Exome Capture and Next Generation Sequencing cause for the one patient in our study who was a boy, analysis Whole-exome sequencing of affected patients 1.1 and 1.2 was per- of spermatozoa function was not available. FOXI1 is highly formed by GATC Biotech using the Illumina HiSeq-2000 (Illumina expressed in human renal kidney cortex (RNA-Seq expression Inc., San Diego, CA). The genomic DNA was captured on the Sure- data from GTEx; https://www.gtexportal.org). Other human tis- SelectXT Human All Exon V5 Kit (Agilent, CA) to capture the target sues that express FOXI1 at more modest levels include breast sequence of exonic regions and noncoding RNAs in the human ge- tissue, salivary gland, prostate, and skin (https://www.gtexportal. nome. The paired end Illumina libraries were captured in solution org/home/gene/ENSG00000168269.7). The human protein at- according to the Agilent SureSelect protocol with 125-bp read length. las reveals high levels of FOXI1 protein expression in kidney and Perkin Elmer provided commercially available exome sequencing for low expression levels in nasopharynx, bronchus, salivary gland, patient 2.1 (exome capture: Agilent SureSelect V4; read length/library and breast tissue (https://www.proteinatlas.org/ENSG00000168269- construction: 75–100 bp per paired end library and reads; sequence FOXI1/tissue). Thus, it is possible that other tissues/cell types coverage: approximately 35 times mean coverage; detection of sequencing fragments via the Illumina Hiseq2000). Table 3. PCR primers used for Sanger sequencing FOXI1 FWD CAACCCCTACCTCTGGTTCA Exon 1 Single-Nucleotide Polymorphism Detection The base quality of each sequence read was inspected for low-quality calls FOXI1 REV GGGGAGGAAGGAAGTGAGTC Exon 1 FOXI1 FWD CATCTGTCACCTTGGCTTT Exon 2 and subsequently removed before proceeding with further processing. FOXI1 REV GGGGAGAAGGAGTTGAAGGT Exon 2 Using a sliding window approach, bases with low quality were removed 9 9 Experimental details regarding in silico modeling and cell-based experiments from the 3 and 5 ends. Bases were removed if the average quality was are in Supplemental Appendix. FWD, forward; REV, reverse. below the threshold of 15. Finally, only reads with both forward and

1046 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1041–1048, 2018 www.jasn.org CLINICAL RESEARCH reversereadwereusedforthenextanalysisstep.Mappingtothereference 4. Sly WS, Whyte MP, Sundaram V, Tashian RE, Hewett-Emmett D, database (hg19, NCBI37) was performed using the Burrows–Wheeler Guibaud P, Vainsel M, Baluarte HJ, Gruskin A, Al-Mosawi M, Sakati N, fi Alignment method with default parameters. The single-nucleotide poly- Ohlsson A: Carbonic anhydrase II de ciency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular aci- morphism and insertion and deletion selection calling was performed dosis and cerebral calcification. N Engl J Med 313: 139–145, 1985 fi using GATK Uni edGenotyper using a Bayesian genotype likelihood 5. Stover EH, Borthwick KJ, Bavalia C, Eady N, Fritz DM, Rungroj N, model to estimate simultaneously the most likely genotypes and allele Giersch AB, Morton CC, Axon PR, Akil I, Al-Sabban EA, Baguley DM, frequency in a population of N samples. Variants detected were annotated Bianca S, Bakkaloglu A, Bircan Z, Chauveau D, Clermont MJ, Guala A, on the basis of their gene context using snpEff. The total reads were Hulton SA, Kroes H, Li Volti G, Mir S, Mocan H, Nayir A, Ozen S, approximately 145 million, with the percentage of mapped reads of Rodriguez Soriano J, Sanjad SA, Tasic V, Taylor CM, Topaloglu R, Smith AN, Karet FE: Novel ATP6V1B1 and ATP6V0A4 mutations in autoso- 99%. Data were pipelined to allow insertion and deletion selection and mal recessive distal renal tubular acidosis with new evidence for single-nucleotide polymorphism vcf files to be generated, which were hearing loss. J Med Genet 39: 796–803, 2002 viewed using Ingenuity Variant Analysis software (Qiagen) (Tables 2 6. Stankovic KM, Brown D, Alper SL, Adams JC: Localization of pH regu- and 3). Additional information is in Supplemental Appendix. lating proteins H+ATPase and Cl-/HCO3- exchanger in the guinea pig inner ear. Hear Res 114: 21–34, 1997 7. Hulander M, Kiernan AE, Blomqvist SR, Carlsson P, Samuelsson EJ, ACKNOWLEDGMENTS Johansson BR, Steel KP, Enerbäck S: Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice. Development 130: 2013–2025, 2003 We thank the patients and their family members for their help 8. Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, in this study. J.A.S. is funded by the Medical Research Council Enerbäck S: The forkhead transcription factor Foxi1 is a master regulator (MR/M012212/1), Kidney Research UK, Kids Kidney Re- of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and search, Northern Counties Kidney Research Fund and the epididymis. PLoS One 4: e4471, 2009 9. Hulander M, Wurst W, Carlsson P, Enerbäck S: The winged helix tran- Newcastle upon Tyne Hospitals NHS Charity. Funding for scription factor Fkh10 is required for normal development of the inner this study to R.K. and D.B. was kindly provided by the Euro- ear. Nat Genet 20: 374–376, 1998 pean Union, FP7 (grant agreement 2012-305608 “European 10. Blomqvist SR, Vidarsson H, Fitzgerald S, Johansson BR, Ollerstam A, Consortium for High-Throughput Research in Rare Kidney Brown R, Persson AE, Bergström G, Enerbäck S: Distal renal tubular Diseases (EURenOmics)“) and Kids Kidney research, Kidney acidosis in mice that lack the forkhead transcription factor Foxi1. J Clin Invest 113: 1560–1570, 2004 Research UK and The John Moorhead Trust. S.E. is supported 11. Yang T, Vidarsson H, Rodrigo-Blomqvist S, Rosengren SS, Enerback S, by the Swedish Research Council (2014–2516), The Knut and Smith RJ: Transcriptional control of SLC26A4 is involved in Pendred Alice Wallenberg Foundation, Sahlgrenska’s University Hos- syndrome and nonsyndromic enlargement of vestibular aqueduct pital (LUA-ALF), The Inga Britt and Arne Lundgren Founda- (DFNB4). Am J Hum Genet 80: 1055–1063, 2007 tion, The Torsten Söderberg Foundation, Novo Nordisk 12. Clark KL, Halay ED, Lai E, Burley SK: Co-crystal structure of the HNF-3/ fork head DNA-recognition motif resembles histone H5. Nature 364: Foundation, and The King Gustaf Vand Queen Victoria Free- 412–420, 1993 mason Foundation. CAW is supported by the Swiss National 13. 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