Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract
Authors: Daw-Yang Hwang, Gabriel C. Dworschak, Stefan Kohl, Pawaree Saisawat, Asaf Vivante, Alina C. Hilger, Heiko M. Reutter, Neveen A. Soliman, Radovan Bogdanovic, Elijah O. Kehinde, Velibor Tasic, Friedhelm Hildebrandt
Journal: Kidney International (2014)

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease. CAKUT can be caused by monogenic mutations; however, data are lacking on their frequency. Genetic diagnosis has been hampered by genetic heterogeneity and lack of genotype–phenotype correlation. To determine the percentage of cases with CAKUT that can be explained by mutations in known CAKUT genes, we analyzed the coding exons of the 17 known dominant CAKUT-causing genes in a cohort of 749 individuals from 650 families with CAKUT. The most common phenotypes in this CAKUT cohort were vesicoureteral reflux in 288 patients, renal hypodysplasia in 120 patients, and unilateral renal agenesis in 90 patients. We identified 37 different heterozygous mutations (33 novel) in 12 of the 17 known genes in 47 patients from 41 of the 650 families (6.3%). These mutations include (number of families): BMP7 (1), CDC5L (1), CHD1L (5), EYA1 (3), GATA3 (2), HNF1B (6), PAX2 (5), RET (3), ROBO2 (4), SALL1 (9), SIX2 (1), and SIX5 (1). Furthermore, several mutations previously reported to be disease-causing are most likely benign variants. Thus, in a large cohort over 6% of families with isolated CAKUT are caused by a mutation in 12 of 17 dominant CAKUT genes. Our report represents one of the most in-depth diagnostic studies of monogenic causes of isolated CAKUT in children. Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease. CAKUT can be caused by monogenic mutations; however, data are lacking on their frequency. Genetic diagnosis has been hampered by genetic heterogeneity and lack of genotype–phenotype correlation. To determine the percentage of cases with CAKUT that can be explained by mutations in known CAKUT genes, we analyzed the coding exons of the 17 known dominant CAKUT-causing genes in a cohort of 749 individuals from 650 families with CAKUT. The most common phenotypes in this CAKUT cohort were vesicoureteral reflux in 288 patients, renal hypodysplasia in 120 patients, and unilateral renal agenesis in 90 patients. We identified 37 different heterozygous mutations (33 novel) in 12 of the 17 known genes in 47 patients from 41 of the 650 families (6.3%). These mutations include (number of families): BMP7 (1), CDC5L (1), CHD1L (5), EYA1 (3), GATA3 (2), HNF1B (6), PAX2 (5), RET (3), ROBO2 (4), SALL1 (9), SIX2 (1), and SIX5 (1). Furthermore, several mutations previously reported to be disease-causing are most likely benign variants. Thus, in a large cohort over 6% of families with isolated CAKUT are caused by a mutation in 12 of 17 dominant CAKUT genes. Our report represents one of the most in-depth diagnostic studies of monogenic causes of isolated CAKUT in children. Congenital anomalies of the kidney and urinary tract (CAKUT) are observed in three to six per 1000 live births and account for 40–50% of the etiology of chronic kidney disease (CKD) in children worldwide.1.Hildebrandt F. Genetic kidney diseases.Lancet. 2010; 375: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar,2.North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) The EMMES Corporation, Rockville, MD, USA2008Google Scholar CAKUT cover a wide range of structural malformations that result from a defect in the morphogenesis of the kidney and/or the urinary tract.3.Dressler G.R. The cellular basis of kidney development.Annu Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (478) Google Scholar, 4.Ichikawa I. Kuwayama F. Pope IV., J.C. et al.Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT.Kidney Int. 2002; 61: 889-898Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 5.Sanna-Cherchi S. Caridi G. Weng P.L. et al.Genetic approaches to human renal agenesis/hypoplasia and dysplasia.Pediatr Nephrol. 2007; 22: 1675-1684Crossref PubMed Scopus (88) Google Scholar The condition may appear as an isolated feature or as part of a syndrome in association with extra-renal manifestations.6.Eccles M.R. Schimmenti L.A. Renal-coloboma syndrome: a multi-system developmental disorder caused by PAX2 mutations.Clin Genet. 1999; 56: 1-9Crossref PubMed Scopus (129) Google Scholar,7.Bingham C. Bulman M.P. Ellard S. et al.Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease.Am J Hum Genet. 2001; 68: 219-224Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar In addition, CAKUT may either be diagnosed sporadically or be described with familial aggregation in up to 15% of cases.8.Roodhooft A.M. Birnholz J.C. Holmes L.B. Familial nature of congenital absence and severe dysgenesis of both kidneys.N Engl J Med. 1984; 310: 1341-1345Crossref PubMed Scopus (150) Google Scholar,9.Bulum B. Ozcakar Z.B. Ustuner E. et al.High frequency of kidney and urinary tract anomalies in asymptomatic first-degree relatives of patients with CAKUT.Pediatr Nephrol. 2013; 28: 2143-2147Crossref PubMed Scopus (43) Google Scholar In familial cases, the mode of inheritance in most pedigrees is autosomal dominant with variable expressivity and reduced penetrance.10.McPherson E. Carey J. Kramer A. et al.Dominantly inherited renal adysplasia.Am J Med Genet. 1987; 26: 863-872Crossref PubMed Scopus (98) Google Scholar The pathogenesis of CAKUT is based on the disturbance of normal nephrogenesis and can be due to genetic abnormalities in the renal developmental genes that direct the process.1.Hildebrandt F. Genetic kidney diseases.Lancet. 2010; 375: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar,3.Dressler G.R. The cellular basis of kidney development.Annu Rev Cell Dev Biol. 2006; 22: 509-529Crossref PubMed Scopus (478) Google Scholar,4.Ichikawa I. Kuwayama F. Pope IV., J.C. et al.Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT.Kidney Int. 2002; 61: 889-898Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar,5.Sanna-Cherchi S. Caridi G. Weng P.L. et al.Genetic approaches to human renal agenesis/hypoplasia and dysplasia.Pediatr Nephrol. 2007; 22: 1675-1684Crossref PubMed Scopus (88) Google Scholar,11.Chen F. Genetic and developmental basis for urinary tract obstruction.Pediatr Nephrol. 2009; 24: 1621-1632Crossref PubMed Scopus (47) Google Scholar, 12.Schedl A. Renal abnormalities and their developmental origin.Nat Rev Genet. 2007; 8: 791-802Crossref PubMed Scopus (292) Google Scholar, 13.Song R. Yosypiv I.V. Genetics of congenital anomalies of the kidney and urinary tract.Pediatr Nephrol. 2011; 26: 353-364Crossref PubMed Scopus (126) Google Scholar To date, 23 monogenic CAKUT-causing genes have been identified that result in isolated CAKUT or syndromic CAKUT with mild extra-renal manifestations.14.Chen T. Li Q. Xu J. et al.Mutation screening of BMP4, BMP7, HOXA4 and HOXB6 genes in Chinese patients with hypospadias.Eur J Hum Genet. 2007; 15: 23-28Crossref PubMed Scopus (41) Google Scholar, 15.Weber S. Taylor J.C. Winyard P. et al.SIX2 and BMP4 mutations associate with anomalous kidney development.J Am Soc Nephrol. 2008; 19: 891-903Crossref PubMed Scopus (158) Google Scholar, 16.Groenen P.M. Vanderlinden G. Devriendt K. et al.Rearrangement of the human CDC5L gene by a t(6;19)(p21;q13.1) in a patient with multicystic renal dysplasia.Genomics. 1998; 49: 218-229Crossref PubMed Scopus (38) Google Scholar, 17.Brockschmidt A. Chung B. Weber S. et al.CHD1L: a new candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT).Nephrol Dial Transplant. 2012; 27: 2355-2364Crossref PubMed Scopus (28) Google Scholar, 18.Abdelhak S. Kalatzis V. Heilig R. et al.A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family.Nat Genet. 1997; 15: 157-164Crossref PubMed Scopus (520) Google Scholar, 19.Horikawa Y. Iwasaki N. Hara M. et al.Mutation in hepatocyte nuclear factor-1 beta gene (TCF2) associated with MODY.Nat Genet. 1997; 17: 384-385Crossref PubMed Scopus (744) Google Scholar, 20.Lindner T.H. Njolstad P.R. Horikawa Y. et al.A novel syndrome of diabetes mellitus, renal dysfunction and genital malformation associated with a partial deletion of the pseudo-POU domain of hepatocyte nuclear factor-1beta.Hum Mol Genet. 1999; 8: 2001-2008Crossref PubMed Scopus (307) Google Scholar, 21.Sanyanusin P. Schimmenti L.A. McNoe L.A. et al.Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.Nat Genet. 1995; 9: 358-364Crossref PubMed Scopus (575) Google Scholar, 22.Skinner M.A. Safford S.D. Reeves J.G. et al.Renal aplasia in humans is associated with RET mutations.Am J Hum Genet. 2008; 82: 344-351Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23.Lu W. van Eerde A.M. Fan X. et al.Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux.Am J Hum Genet. 2007; 80: 616-632Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 24.Kohlhase J. Wischermann A. Reichenbach H. et al.Mutations in the SALL1 putative transcription factor gene cause Townes-Brocks syndrome.Nat Genet. 1998; 18: 81-83Crossref PubMed Scopus (339) Google Scholar, 25.Ruf R.G. Berkman J. Wolf M.T. et al.A gene locus for branchio-otic syndrome maps to chromosome 14q21.3-q24.3.J Med Genet. 2003; 40: 515-519Crossref PubMed Scopus (37) Google Scholar, 26.Hoskins B.E. Cramer C.H. Silvius D. et al.Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome.Am J Hum Genet. 2007; 80: 800-804Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 27.Gimelli S. Caridi G. Beri S. et al.Mutations in SOX17 are associated with congenital anomalies of the kidney and the urinary tract.Hum Mutat. 2010; 31: 1352-1359Crossref PubMed Scopus (52) Google Scholar, 28.Hart T.C. Gorry M.C. Hart P.S. et al.Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy.J Med Genet. 2002; 39: 882-892Crossref PubMed Scopus (378) Google Scholar, 29.Jenkins D. Bitner-Glindzicz M. Malcolm S. et al.De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure.J Am Soc Nephrol. 2005; 16: 2141-2149Crossref PubMed Scopus (104) Google Scholar, 30.Van Esch H. Groenen P. Nesbit M.A. et al.GATA3 haplo-insufficiency causes human HDR syndrome.Nature. 2000; 406: 419-422Crossref PubMed Scopus (433) Google Scholar, 31.Gbadegesin R.A. Brophy P.D. Adeyemo A. et al.TNXB mutations can cause vesicoureteral reflux.J Am Soc Nephrol. 2013; 24: 1313-1322Crossref PubMed Scopus (50) Google Scholar, 32.Vivante A. Mark-Danieli M. Davidovits M. et al.Renal hypodysplasia associates with a WNT4 variant that causes aberrant canonical WNT signaling.J Am Soc Nephrol. 2013; 24: 550-558Crossref PubMed Scopus (40) Google Scholar, 33.Sanna-Cherchi S. Sampogna R.V. Papeta N. et al.Mutations in DSTYK and dominant urinary tract malformations.N Engl J Med. 2013; 369: 621-629Crossref PubMed Scopus (96) Google Scholar, 34.Barak H. Huh S.H. Chen S. et al.FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man.Dev Cell. 2012; 22: 1191-1207Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 35.Saisawat P. Kohl S. Hilger A.C. et al.Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association.Kidney Int. e-pub ahead of print; advance online publication 23 October 2013. 2013Google Scholar Only a few studies have screened large cohorts of CAKUT patients for disease-causing mutations.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar, 37.Thomas R. Sanna-Cherchi S. Warady B.A. et al.HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort.Pediatr Nephrol. 2011; 26: 897-903Crossref PubMed Scopus (95) Google Scholar, 38.Madariaga L. Moriniere V. Jeanpierre C. et al.Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes.Clin J Am Soc Nephrol. 2013; 8: 1179-1187Crossref PubMed Scopus (71) Google Scholar, 39.Heidet L. Decramer S. Pawtowski A. et al.Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases.Clin J Am Soc Nephrol. 2010; 5: 1079-1090Crossref PubMed Scopus (204) Google Scholar, 40.Ulinski T. Lescure S. Beaufils S. et al.Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort.J Am Soc Nephrol. 2006; 17: 497-503Crossref PubMed Scopus (203) Google Scholar, 41.Edghill E.L. Bingham C. Ellard S. et al.Mutations in hepatocyte nuclear factor-1beta and their related phenotypes.J Med Genet. 2006; 43: 84-90Crossref PubMed Scopus (264) Google Scholar These studies screened for one to five disease-causing genes and some were preselected for chronic renal insufficiency or severe disease phenotypes.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar, 37.Thomas R. Sanna-Cherchi S. Warady B.A. et al.HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort.Pediatr Nephrol. 2011; 26: 897-903Crossref PubMed Scopus (95) Google Scholar, 38.Madariaga L. Moriniere V. Jeanpierre C. et al.Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes.Clin J Am Soc Nephrol. 2013; 8: 1179-1187Crossref PubMed Scopus (71) Google Scholar Hence, data are lacking on the frequency of monogenic forms of CAKUT in large cohorts. To address these issues we investigated the frequency of mutations in 17 known dominant CAKUT-causing genes in a phenotypically nonselective international cohort of 749 CAKUT individuals out of 650 different families. We show that mutations in known CAKUT-causing genes are present in more than 6% of these families, and we outline possible pitfalls in analyzing autosomal dominant single-gene disorders. Our cohort of 749 individuals from 650 different families with CAKUT originated from Eastern Europe (63.6%), Western Europe (12.7%), Arab countries (10%), India (7.9%), Romany populations (1.5%), and Asia (0.7%) (Supplementary Table S1 online). There were 414 male (55%) and 331 female (44.2%) individuals. The most common CAKUT phenotype was vesicoureteral reflux (n=288), followed by renal hypodysplasia (n=120) and unilateral renal agenesis (n=90). A total of 161 individuals from 100 families were considered as having familial CAKUT according to clinical questionnaires in our cohort. These families had two to six affected individuals. The most common familial CAKUT phenotypes include vesicoureteral reflux (n=68) and duplex system (n=29), followed by renal hypodysplasia (n=19) and others. For detailed cohort characteristics see Supplementary Table S1 online. Download .doc (.37 MB) Help with doc files Supplementary Information By targeted resequencing of 170 coding exons of 17 genes known to cause autosomal dominant CAKUT we identified 144,382 single-nucleotide variants and 39,081 insertion–deletion variants in the 650 families. After variant filtering, as described in the Materials and Methods, we retained 341 variants as potentially deleterious alleles. Of these, 152 were confirmed by Sanger sequencing, whereas the others represented low-representation artifacts of multiplex polymerase chain reaction (PCR). In order to distinguish benign variants from likely disease-causing mutations we carefully evaluated each variant individually on the basis of the criteria described in the Materials and Methods section. Overall, 105 variants did not meet our criteria for being probably disease-causing. Among these, 43 variants were previously reported as mutations in individuals with CAKUT (Supplementary Table S2 online), and 62 variants were not previously reported (Supplementary Table S3 online) in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). In 749 patients with CAKUT from 650 families, disease-causing heterogeneous dominant mutations were identified in 41 unrelated families (6.3%) (Table 1). Mutations were detected in the following genes: BMP7 (one family), CDC5L (one family), CHD1L (five families), EYA1 (three families), GATA3 (two families), HNF1B (five families), PAX2 (five families), RET (three families), ROBO2 (four families), SALL1 (nine families), SIX2 (one family), and SIX5 (one family) (Table 1). No causative mutations were identified in the genes SOX17, UMOD, BMP4, SIX1, and UPK3A. In total, 33 of the 37 mutations were most likely novel pathogenic mutations.Table 1Genotypes and phenotypes of 41 families with mutations in 17 known autosomal dominant CAKUT-causing genesGeneFamily-individualSexEthnicityRenal phenotypeNucleotide changeaAll mutations are heterozygousAmino-acid changeConservationEVS allelesbExome variant server database (http://evs.gs.washington.edu/EVS/)SIFTcSorting Intolerant From Tolerant (http://sift.bii.a-star.edu.sg)Mutation-tasterdMutationTaster (http://www.mutationtaster.org)PP-2ePolyPhen-2 prediction score ranges from 0 (= benign) to 1 (= probably damaging) (http://genetics.bwh.harvard.edu/pph2/)ReferencesMmGgXtDrBMP7A3068-21MEER UVJOc.661G>Ap.E221KEEEE0/13,006TDC0.192A3068-22ML HDCDC5LA4171-11MEEL RAc.2014C>Tp.P672SPPP/0/13,006TDC0.393A4171-21ML RACHD1LA5061-21MWER MCDK, L UVJOc.998C>Gp.P333RPP/P0/13,006DDC0.953CHD1LA549-21FAsiB kidney malrotationc.1199A>Gp.E400GEE/E0/13,006DDC0.997CHD1LA3902-21fIndividual A3902-21 had clinical findings compatible with Down syndromeMIndPUVc.1551A>Gp.I517MII/I0/13,006DDC0.505CHD1LA3925-21FIndR RDc.1551A>Gp.I517MII/I0/13,006DDC0.505CHD1LA3219-21MIndHorseshoe kidneys, R DSc.1551A>Gp.I517MII/I0/13,006DDC0.505EYA1A1522-21gIndividuals A1077-21 and A1522-21 were published previously by our group and were used as positive controlsMAraR UPJOc.647C>Tp.P216LPPPP0/13,006DDC0.07945*EYA1F1438-21hIndividual F1438-21 had hearing impairment and facial dysmorphismFWEB VUR, B RHDc.966+1G>ANA0/13,006EYA1A1542-21iIndividual A1542-21 had hypospadiasMAraL UPJOc.1733C>Tp.S578LSSSS0/13,006DDC0.984GATA3A4733-21FEEB VURc.766C>Gp.R256GRRRR0/12,988DDC0.404GATA3A1319-21FEEB VURc.889C>Ap.Q297KQQQQ0/13,006DDC0.439HNF1BA3967-21MIndB VUR, NBc.234G>Cp.E78DEEEE0/13,004DDC0.992HNF1BA2921-21MEEL RHD, R MCDKc.477delTp.M160*0/13,00646A2921-12FUnspecified CAKUTHNF1BA3069-21FEEL VURc.499G>Ap.A167TAAAA0/13,006DDC0.999HNF1BA3840-21MIndVUR, PUVc.542G>Ap.R181QRRRR0/13,006DDC0.888HNF1BA2326-21MWEL UPJO, subcapsular cystsc.823C>Tp.Q275*0/13,006A2326-11Msubcapsular cystsHNF1BA4672-21jIndividual A4672-21 had compound heterozygous mutations in SLC3A1 (p.T216M, p.M467T)FEER RHD, cystinuriac.1024T>Cp.S342PSSSS0/13,006DDC0.767PAX2A3148-21MWEB RHD, RCTc.76dupp.V26Gfs*280/12,98047PAX2A2334-21kIndividual A2334-21 was diagnosed with a ganglioneuroblastomaFWEB RHDc.211A>Gp.R71GRRRR0/12,958DDC0.888PAX2A1087-21MEEB UVJOc.320C>Tp.P107LPPPP0/13,006DDC0.999PAX2A3872-21MIndB RHDc.343C>Tp.R115X0/13,006PAX2A1743-12FWERCTc.408delp.N136Kfs*230/13,006A1743-21FRCTRETA3836-21lIndividual A3836-21 had growth retardation and skeletal anomaliesFIndB RHDc.667G>Ap.V223MVVVV0/12,958DDC0.642RETA1077-21gIndividuals A1077-21 and A1522-21 were published previously by our group and were used as positive controlsFAraL RA, R UPJOc.2110G>Tp.V704FVVVV0/13,006TDC0.90148*RETA1318-21FEEL DS, VUR, ureterocelec.3079C>Gp.L1027VLLLL0/13,006DDC0.996ROBO2A1220-21FIndR UPJO, stonec.340G>Tp.G114WGGGG0/12,438DDC1ROBO2A3839-21MIndPUVc.724A>Gp.T242ATTTT0/11,902DDC0.224ROBO2A3372-21MEER MCDKc.808C>Gp.P270APPPP0/11,930DDC0.988ROBO2A521-11MEEB VURc.3712G>Ap.D1238NDDDD0/12,130DDC0.251SALL1A3935-21MIndPUVc.220G>Ap.V74IVVVV0/12,996DDC0.007SALL1A2333-21MWEB VUR, MCDKc.548C>Gp.T183RTTTT0/12,996DDC0.296SALL1A2898-21FEEL UPJOc.602A>Gp.Q201RQQQQ0/12,996DDC0.968SALL1A617-21FEEB VUR gr III, Rt duplexc.703G>Ap.A235TAAAA0/12,996DDC0.782SALL1A3070-21MEEL UPJOSALL1A4448-21FEEB VURc.1738A>Gp.I580VIIII0/12,996DDC0.035SALL1A5083-21FEEL VURc.1738A>Gp.I580VIIII0/12,996DDC0.035SALL1A3687-12mIndividual A3687-12 had polytheliaFEEL DSc.2582C>Ap.S861*0/12,996A3687-21MR RHDSALL1F1434-21nIndividual F1434-21 had juvenile rheumatoid arthritis, facial dysmorphism and ectopic testes.MWER RA, L VURc.3006_3009delp.C1003Tfs*410/12,996SIX2A3904-21MIndPUVc.859G>Ap.V287MVVVV0/13,006DDC0.987SIX5A959-21MEER DS, VU, L UVJOc.1817C>Tp.P606LP/-P0/12,946DDC0.994Abbreviations: AA, accessory auricle; Alb, Albania; B, bilateral; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; D, deleterious; DC, disease-causing; Dr, Danio rerio; Dm, Drosophila melanogaster; DS, duplex collecting system; F, female; Ger, Germany; Gg, Gallus gallus; Ind, India; Kuw, Kuwait; L, left; M, male; Mac, Macedonia; MCDK, Multicystic dysplastic kidney; Mm, Mus musculus; NA, not applicable; NB, neurogenic bladder; PUV, posterior urethral valve; R, right; RA, renal agenesis; RC, renal coloboma; RCT, renal cysts; RD, renal dysplasia; RHD, renal hypodysplasia; Ser, Serbia; T, tolerated; UK, United Kingdom; UPJO, ureteropelvic junction obstruction; UVJO, ureterovesical junction obstruction; VUR, vesicoureteral reflux; Xt, Xenopus tropicalis; '–', no alignment at this position; '/', no sequence data in this species.Nucleotide change numbering refers to the cDNA position of the following transcripts: BMP4 (NM_001202.3), BMP7 (NM_001719.2), CDC5L (NM_001253.2), CHD1L (NM_004284.3), EYA1 (NM_000503.4), GATA3 (NM_001002295.1), HNF1B (NM_000458.2), PAX2 (NM_003990), RET (NM_020975.4), ROBO2 (NM_001128929.2), SALL1 (NM_002968.2), SIX1 (NM_005982.3), SIX2 (NM_016932.4), SIX5 (NM_175875.4), UMOD (NM_003361.2) and UPK3A (NM_006953.3) where +1 corresponds to the A of ATG start translation codon.a All mutations are heterozygousb Exome variant server database (http://evs.gs.washington.edu/EVS/)c Sorting Intolerant From Tolerant (http://sift.bii.a-star.edu.sg)d MutationTaster (http://www.mutationtaster.org)e PolyPhen-2 prediction score ranges from 0 (= benign) to 1 (= probably damaging) (http://genetics.bwh.harvard.edu/pph2/)f Individual A3902-21 had clinical findings compatible with Down syndromeg Individuals A1077-21 and A1522-21 were published previously by our group and were used as positive controlsh Individual F1438-21 had hearing impairment and facial dysmorphismi Individual A1542-21 had hypospadiasj Individual A4672-21 had compound heterozygous mutations in SLC3A1 (p.T216M, p.M467T)k Individual A2334-21 was diagnosed with a ganglioneuroblastomal Individual A3836-21 had growth retardation and skeletal anomaliesm Individual A3687-12 had polythelian Individual F1434-21 had juvenile rheumatoid arthritis, facial dysmorphism and ectopic testes. Open table in a new tab Abbreviations: AA, accessory auricle; Alb, Albania; B, bilateral; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; D, deleterious; DC, disease-causing; Dr, Danio rerio; Dm, Drosophila melanogaster; DS, duplex collecting system; F, female; Ger, Germany; Gg, Gallus gallus; Ind, India; Kuw, Kuwait; L, left; M, male; Mac, Macedonia; MCDK, Multicystic dysplastic kidney; Mm, Mus musculus; NA, not applicable; NB, neurogenic bladder; PUV, posterior urethral valve; R, right; RA, renal agenesis; RC, renal coloboma; RCT, renal cysts; RD, renal dysplasia; RHD, renal hypodysplasia; Ser, Serbia; T, tolerated; UK, United Kingdom; UPJO, ureteropelvic junction obstruction; UVJO, ureterovesical junction obstruction; VUR, vesicoureteral reflux; Xt, Xenopus tropicalis; '–', no alignment at this position; '/', no sequence data in this species. Nucleotide change numbering refers to the cDNA position of the following transcripts: BMP4 (NM_001202.3), BMP7 (NM_001719.2), CDC5L (NM_001253.2), CHD1L (NM_004284.3), EYA1 (NM_000503.4), GATA3 (NM_001002295.1), HNF1B (NM_000458.2), PAX2 (NM_003990), RET (NM_020975.4), ROBO2 (NM_001128929.2), SALL1 (NM_002968.2), SIX1 (NM_005982.3), SIX2 (NM_016932.4), SIX5 (NM_175875.4), UMOD (NM_003361.2) and UPK3A (NM_006953.3) where +1 corresponds to the A of ATG start translation codon. We examined a large international cohort of 650 unrelated families with CAKUT for the presence of mutations in 17 autosomal dominant known CAKUT-causing genes. We identified 37 different heterozygous mutations in 12 different genes in 41 of the 650 families (6.3%). Thirty-three of the 37 mutations detected were novel. Our findings also revealed that some variants previously reported as disease-causing cannot be accepted as such on the basis of a finding that shows lack of segregation of these genetic variants in families with multiple affected individuals. For example, the BMP4 variant p.S91C and the SIX2 variant p.P241L have been reported to lead to CAKUT among five unrelated patients.15.Weber S. Taylor J.C. Winyard P. et al.SIX2 and BMP4 mutations associate with anomalous kidney development.J Am Soc Nephrol. 2008; 19: 891-903Crossref PubMed Scopus (158) Google Scholar We detected these two variants among 13 unrelated families in our cohort and five of them did not segregate with the disease—that is, not all affected family members have the variant. These findings reveal that these two variants cannot be considered as disease-causing variants. These findings encourage us to adhere to our strict definition of disease-causing variants as outlined in 'Materials and Methods' and are consistent with the findings that many alleles published as disease-causing may not reliably have such a role.42.Bell C.J. Dinwiddie D.L. Miller N.A. et al.Carrier testing for severe childhood recessive diseases by next-generation sequencing.Sci Transl Med. 2011; 3: 65ra64Crossref Scopus (540) Google Scholar,43.Xue Y. Chen Y. Ayub Q. et al.Deleterious- and disease-allele prevalence in healthy individuals: insights from current predictions, mutation databases, and population-scale resequencing.Am J Hum Genet. 2012; 91: 1022-1032Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar We found that nine variants (43 individuals) in previously CAKUT-related publications and 50 human gene mutation database-unreported variants (62 individuals) did not fulfill our criteria (Supplementary Tables S2 and S3 online, respectively). This work, to the best of our knowledge, is the most extensive genetic screening of known CAKUT-causing genes. SALL1, HNF1B, and PAX2 were the most prevalent disease-causing genes in our cohort. This is in line with the predominance of HNF1B and PAX2 mutations that has been described in patients with renal hypodysplasia.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar,37.Thomas R. Sanna-Cherchi S. Warady B.A. et al.HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort.Pediatr Nephrol. 2011; 26: 897-903Crossref PubMed Scopus (95) Google Scholar,39.Heidet L. Decramer S. Pawtowski A. et al.Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases.Clin J Am Soc Nephrol. 2010; 5: 1079-1090Crossref PubMed Scopus (204) Google Scholar HNF1B and PAX2 were previously reported to be disease-causing in 5–20% of CAKUT cases.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar, 37.Thomas R. Sanna-Cherchi S. Warady B.A. et al.HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort.Pediatr Nephrol. 2011; 26: 897-903Crossref PubMed Scopus (95) Google Scholar, 38.Madariaga L. Moriniere V. Jeanpierre C. et al.Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes.Clin J Am Soc Nephrol. 2013; 8: 1179-1187Crossref PubMed Scopus (71) Google Scholar, 39.Heidet L. Decramer S. Pawtowski A. et al.Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases.Clin J Am Soc Nephrol. 2010; 5: 1079-1090Crossref PubMed Scopus (204) Google Scholar, 40.Ulinski T. Lescure S. Beaufils S. et al.Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort.J Am Soc Nephrol. 2006; 17: 497-503Crossref PubMed Scopus (203) Google Scholar, 41.Edghill E.L. Bingham C. Ellard S. et al.Mutations in hepatocyte nuclear factor-1beta and their related phenotypes.J Med Genet. 2006; 43: 84-90Crossref PubMed Scopus (264) Google Scholar The finding that PAX2 and HNF1B mutations were seen at a higher frequency in previous studies on CAKUT is most likely explained by the fact that these studies were carried out in CAKUT cohorts preselected for CKD and in prenatal findings with severe renal anomalies.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar, 37.Thomas R. Sanna-Cherchi S. Warady B.A. et al.HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort.Pediatr Nephrol. 2011; 26: 897-903Crossref PubMed Scopus (95) Google Scholar, 38.Madariaga L. Moriniere V. Jeanpierre C. et al.Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes.Clin J Am Soc Nephrol. 2013; 8: 1179-1187Crossref PubMed Scopus (71) Google Scholar Our data are consistent with previous publications describing that oligosyndromic CAKUT-causing genes can lead to an isolated CAKUT phenotype.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar The fact that we did not identify mutations in SOX17, UMOD, BMP4, SIX1, and UPK3A suggests that mutations in these genes are rarer. The identification of SALL1 mutations in >1% of our cohort suggests that this gene may be a more common cause of CAKUT than previously believed.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar It should be emphasized that in the current study we did not screen our cohort for copy number variations. It was previously shown that some of the known CAKUT-causing genes may be disrupted by deletions or duplications, such as heterozygous HNF1B deletion.36.Weber S. Moriniere V. Knuppel T. et al.Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study.J Am Soc Nephrol. 2006; 17: 2864-2870Crossref PubMed Scopus (284) Google Scholar Moreover, in a recent study involving 522 patients with CAKUT, 72 distinct known or novel copy number variations in 87 (16.6%) patients were identified, suggesting that kidney malformations can, in part, result from pathogenic genomic imbalances.44.Sanna-Cherchi S. Kiryluk K. Burgess K.E. et al.Copy-number disorders are a common cause of congenital kidney malformations.Am J Hum Genet. 2012; 91: 987-997Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Our study supports the observation that CAKUT are a genetically very heterogeneous group of diseases with diverse clinical phenotypes. We provide further evidence that the role of specific oligosyndromic CAKUT genes (i.e., SALL1) has a higher contribution in CAKUT than previously thought. The numbers of the known CAKUT genes are expanding with the recent discovery of several novel genes, including FGF20, TNXB, WNT4, DSTYK and TRAP1,31.Gbadegesin R.A. Brophy P.D. Adeyemo A. et al.TNXB mutations can cause vesicoureteral reflux.J Am Soc Nephrol. 2013; 24: 1313-1322Crossref PubMed Scopus (50) Google Scholar, 32.Vivante A. Mark-Danieli M. Davidovits M. et al.Renal hypodysplasia associates with a WNT4 variant that causes aberrant canonical WNT signaling.J Am Soc Nephrol. 2013; 24: 550-558Crossref PubMed Scopus (40) Google Scholar, 33.Sanna-Cherchi S. Sampogna R.V. Papeta N. et al.Mutations in DSTYK and dominant urinary tract malformations.N Engl J Med. 2013; 369: 621-629Crossref PubMed Scopus (96) Google Scholar, 34.Barak H. Huh S.H. Chen S. et al.FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man.Dev Cell. 2012; 22: 1191-1207Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 35.Saisawat P. Kohl S. Hilger A.C. et al.Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association.Kidney Int. e-pub ahead of print; advance online publication 23 October 2013. 2013Google Scholar which were not included in our study because they were described after the completion of our study. We expect the list of CAKUT-causing genes to keep growing with the increasing application of next-generation sequencing techniques. Identification of the monogenetic causes of CAKUT will have important implications in assessing the risk toward progression into end-stage renal disease (ESRD) for this group of diseases that causes ∼50% of all ESRD in the first two decades of life. We obtained blood samples and pedigrees from individuals with CAKUT after their informed consent. The study was approved by the institutional review board of the University of Michigan Medical School and the Boston Children's Hospital. Patients were included in the study if a diagnosis compatible with CAKUT was established by a pediatric nephrologist. The study comprised 749 individuals from 650 families with CAKUT from 25 different pediatric nephrology units worldwide (see Supplementary Table 1 online). Excluded from the study were patients with CAKUT associated with prominent involvement of other organs (syndromic CAKUT). DNA was extracted according to the standard method from peripheral blood obtained from all study participants. As previously described by our group, multiplexed PCR-based amplified products using Fluidigm Access-Array technology were followed by barcoding and next-generation resequencing on an Illumina MiSeq platform.49.Halbritter J. Diaz K. Chaki M. et al.High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.J Med Genet. 2012; 49: 756-767Crossref PubMed Scopus (98) Google Scholar Sanger DNA sequencing was further conducted for single mutation conformation. All coding exons and adjacent splice sites of the following 17 autosomal dominant genes that are known to cause non-syndromic or oligosyndromic CAKUT were screened: BMP4, BMP7, CDC5L, CHD1L, EYA1, GATA3, HNF1B, PAX2, RET, ROBO2, SALL1, SIX1, SIX2, SIX5, SOX17, UMOD, and UPK3A. We designed 252 target-specific primer pairs to cover all 170 coding exons and intron/exon boundaries of the 17 known dominant CAKUT-causing genes (PCR primers are available on request). The maximum amplicon size was chosen as 150–300bp. Universal primer sequences 5′-ACACTGACGACATGGTTCTACA-(target-specific forward)-3′ and 5′-TACGGTAGCAGAGACTTGGTCT-(target-specific reverse)-3′ were added at the 5′ end to all target-specific forward and reverse primers, respectively. Primers were pooled to generate six-plex primer pools per PCR with a final concentration of 1μM per primer. Every sample master mix contained 50ng genomic DNA, 1 × FastStart High Fidelity Reaction Buffer with MgCl2, 5% dimethyl sulfoxide, dNTPs (200μM each), 'FastStart High Fidelity Enzyme Blend', and 1 × 'Access Array' loading reagent (Roche, Indianapolis, IN). A total of 48 different DNA samples were mixed with 48 different six-plex primer pools on one 48.48 Access Array followed by thermal cycling. Subsequently harvested amplicon pools were submitted to another PCR-step to tag PCR products with 48 different barcodes and Illumina sequence-specific adaptors as previously described.49.Halbritter J. Diaz K. Chaki M. et al.High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.J Med Genet. 2012; 49: 756-767Crossref PubMed Scopus (98) Google Scholar Barcoded PCR products were pooled from 125 individuals and submitted to next-generation resequencing on an Illumina MiSeq platform. A total of six 2 × 250bp paired-end runs of Illumina MiSeq were performed according to the manufacturer's protocol. Detected variants were confirmed by Sanger sequencing. Segregation analysis was performed if DNA from family members was available. Read alignment and variant detection were carried out using CLC Genomics Workbench software (CLC-bio, Aarhus, Denmark) as described previously by our group.49.Halbritter J. Diaz K. Chaki M. et al.High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing.J Med Genet. 2012; 49: 756-767Crossref PubMed Scopus (98) Google Scholar After applying filtering criteria, the numbers of remaining variants (in parentheses) were as follows: (1) exclude minor variant frequency <10% (56,410); (2) exclude dbSNP135 with minor allele frequency >1% (23,491); (3) non-synonymous changes and splice variants (7252); (4) keep variant with minor variant frequency >30% (2511); (5) keep if same variant present in <5% of the study cohort (341). We considered variants as probably disease-causing according to the following inclusion and exclusion criteria: Inclusion criteria were as follows: (1) truncating mutation (stop-gained, abrogation of obligatory splice site, frameshift); or (2) missense mutation if one of the following applied: (a) continuous evolutionary conservation to D. rerio; or (b) the given disease-causing allele is supported by functional data. Exclusion criteria (superseding inclusion criteria) are as follows: (1) lack of segregation of a 'mutant' allele to all affected family members; (2) no continuous evolutionary conservation to D. rerio; (3) allele is present in at least one of 6500 individuals of the Exome Variant Server database. We thank the physicians Drs L Braun (Erfurt), D Bockenhauer (London), H Fehrenbach (Memmingen), A Fekete (Budapest), J Gellermann (Berlin), J Goodship (Newcastle), J Hoefele (Munich), B Hoppe (Köln), P Hübner (Frankfurt), AS Kumar (Chennai), A Lemmer (Erfurt), R Mallmann (Essen), J Misselwitz (Jena), D Müller (Berlin), A Ribmann (Magdeburg), G Rönnefarth (Jena), P Senguttuvan (Chennai), A Schulte-Everding (Münster), and the participating families. FH is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and the Warren E. Grupe Professor of Pediatrics. This research was supported by grants from the National Institutes of Health (to FH; R01-DK088767) and by the March of Dimes Foundation (6FY11-241). Table S1. Demographic and phenotypic composition of the 650 families with CAKUT (749 individuals). Table S2. Variants previously reported in HGMD that were excluded by our criteria. Table S3. Previous unreported variants excluded by our criteria. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki