Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association
2013; Elsevier BV; Volume: 85; Issue: 6 Linguagem: Inglês
10.1038/ki.2013.417
ISSN1523-1755
AutoresPawaree Saisawat, Stefan Kohl, Alina C. Hilger, Daw‐Yang Hwang, Heon Yung Gee, Gabriel C. Dworschak, Velibor Tasić, Tracie Pennimpede, S. Natarajan, Ethan D. Sperry, Danilo Swann Matassa, Nataša Stajić, Radovan Bogdanović, Ivo de Blaauw, Carlo Marcelis, Charlotte H. W. Wijers, Enrika Bartels, Eberhard Schmiedeke, Dominik Schmidt, Stefanie Märzheuser, Sabine Grasshoff‐Derr, Stefan Holland‐Cunz, Michael Ludwig, Markus M. Nöthen, Markus Draaken, Erwin Brosens, Hugo A. Heij, Dick Tibboel, Bernhard G. Herrmann, Benjamin D. Solomon, Annelies de Klein, Iris A.L.M. van Rooij, Franca Esposito, Heiko Reutter, Friedhelm Hildebrandt,
Tópico(s)Renal cell carcinoma treatment
ResumoCongenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole-exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor–associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat-shock protein 90–related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle's loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or VACTERL association with CAKUT. Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole-exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor–associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat-shock protein 90–related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle's loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or VACTERL association with CAKUT. Congenital abnormalities of the kidney and urinary tract (CAKUT) occur in 3–6 per 1000 live births. CAKUT are the most frequent cause for chronic kidney disease in children (∼50%)1.NAPRTCS: 2011 Annual Dialysis Report.https://web.emmes.com/study/ped/annlrept/annualrept2011.pdfDate: 2011Google Scholar,2.Smith J.M. Stablein D.M. Munoz R. et al.Contributions of the transplant registry: the 2006 Annual Report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS).Pediatr Transplant. 2007; 11: 366-373Crossref PubMed Scopus (208) Google Scholar in the United States. The acronym 'CAKUT' comprises heterogeneous malformations involving the kidney (e.g., renal agenesis, hypodysplasia) and the urinary tract (e.g., vesicoureteral reflux (VUR), ureteropelvic junction obstruction).3.Pope J.Ct. Brock 3rd, J.W. Adams M.C. et al.How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT.J Am Soc Nephrol. 1999; 10: 2018-2028PubMed Google Scholar These congenital anomalies are related because a part of their pathogenesis is an impaired co-development of nephrogenic tissues derived from the metanephric mesenchyme and the ureteric bud.4.Ichikawa I. Kuwayama F. JCt Pope 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 Twenty monogenic causes of isolated CAKUT in humans have been published to date, as reviewed recently by Yosypiv.5.Yosypiv I.V. Congenital anomalies of the kidney and urinary tract: a genetic disorder?.Int J Nephrol. 2012; 2012: 909083Crossref PubMed Scopus (78) Google Scholar However, they only account for ∼10–20% of all cases indicating a broad genetic heterogeneity of CAKUT. A recent study on copy number variations in a large cohort of individuals with CAKUT and two publications identifying novel monogenic causes of CAKUT bring further evidence that CAKUT is a condition of extensive genetic heterogeneity.6.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, 7.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, 8.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 CAKUT most frequently occur isolated, but they might be associated with extrarenal phenotypes, for instance, with VACTERL association (MIM #192350). The acronym 'VACTERL' describes the combination of at least three of the following congenital anomalies: vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). VACTERL association is a rare disease that occurs mostly sporadic in 1/10,000–40,000 live births.9.Solomon B.D. VACTERL/VATER association.Orphanet J Rare Dis. 2011; 6: 56Crossref PubMed Scopus (284) Google Scholar Its etiology is enigmatic, although animal models suggest an involvement of Sonic hedgehog signaling.10.Vaze D. Mahalik S. Rao K.L. Novel association of VACTERL, neural tube defect and crossed renal ectopia: sonic hedgehog signaling: a point of coherence?.Congenit Anom (Kyoto). 2012; 52: 211-215Crossref PubMed Scopus (6) Google Scholar In humans, ZIC3 mutations are the cause of a closely related nonclassic VACTERL condition (VACTERL-X, MIM #314390).11.Wessels M.W. Kuchinka B. Heydanus R. et al.Polyalanine expansion in the ZIC3 gene leading to X-linked heterotaxy with VACTERL association: a new polyalanine disorder?.J Med Genet. 2010; 47: 351-355Crossref PubMed Scopus (65) Google Scholar,12.Chung B. Shaffer L.G. Keating S. et al.From VACTERL-H to heterotaxy: variable expressivity of ZIC3-related disorders.Am J Med Genet Pt A. 2011; 155A: 1123-1128Crossref PubMed Scopus (42) Google Scholar In addition, there are six case reports published of individuals with VACTERL association in conjunction with mitochondrial dysfunction, as summarized recently by Siebel and Solomon.13.Siebel S. Solomon B.D. Mitochondrial factors and VACTERL association-related congenital malformations.Mol Syndromol. 2013; 4: 63-73Crossref PubMed Scopus (20) Google Scholar To identify new recessive genes that cause isolated CAKUT or CAKUT in VACTERL association, we performed homozygosity mapping and whole-exome resequencing (WER) in 24 affected individuals with CAKUT from 16 families, and in 4 individuals with CAKUT in VACTERL. By homozygosity mapping in a family of two siblings (A3403) with unilateral and bilateral VUR III°, respectively (Figure 1a and b and Table 1), we identified a short 5.2-Mb segment of homozygosity on chromosome 5 (Figure 1c), indicating distant consanguinity of the parents. This finding suggested that in this family CAKUT are most likely caused by a homozygous recessive mutation in an unknown CAKUT gene. We performed WER in individual A3403-21, as described previously by the authors.14.Chaki M. Airik R. Ghosh A.K. et al.Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.Cell. 2012; 150: 533-548Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar,15.Zhou W. Otto E.A. Cluckey A. et al.FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair.Nat Genet. 2012; 44: 910-915Crossref PubMed Scopus (169) Google Scholar In order not to miss either a homozygous mutation in a short run of homozygosity or a compound heterozygous mutation (which, as in this case, cannot be excluded a priori in families with remote consanguinity),16.Ten Kate L.P. Scheffer H. Cornel M.C. et al.Consanguinity sans reproche.Human genetics. 1991; 86: 295-296Crossref PubMed Scopus (14) Google Scholar we considered variants not only in the homozygosity peak but within regions of genetic linkage for both siblings (coverage≥4; minor variant frequency≥0.2). After variant filtering, we retained 38 variants in 13 genes for Sanger confirmation and segregation analysis (Supplementary Table S1 online). Only a single homozygous missense mutation (R469H) in the gene TRAP1 on chromosome 16p13.3 survived the variant filtering process and segregation analysis (Figure 1d). This homozygous variant in TRAP1 in A3403-21 and A3403-22 was positioned in an ∼1.5-Mb run of apparent homozygosity that was not detected by homozygosity mapping (Figure 1c), because the threshold for detection of 'homozygosity peaks' is 2.1Mb.17.Hildebrandt F. Heeringa S.F. Ruschendorf F. et al.A systematic approach to mapping recessive disease genes in individuals from outbred populations.PLoS Genet. 2009; 5: e1000353Crossref PubMed Scopus (133) Google ScholarTable 1Mutations of TRAP1 in five families with isolated CAKUT or CAKUT in VACTERL associationFamily -Individ. (sex)Ethnic originNucleotide alterationaTRAP1 cDNA mutations are numbered according to human cDNA reference sequence NM_016292.2, where +1 corresponds to the A of ATG start translation codon.Deduced protein changeContinuous amino-acid sequence conservationMutTbMutationTaster score. Range: 0–1.0, 1.0 being most deleterious.Poly-Phen2cPolyPhen2 (HumVar) score. Range: 0–1.0, 1.0 being most deleterious.SIFTdSIFT score. Range: 0–1.0, 0 being most deleterious.MAF in EVSeMinor allele frequency in 8600 alleles of Americans of European descent.Exon (state; segregation)Urinary tract phenotypesOther phenotypesA3403-21 (F)-22 (F)Serbianc.1406G>Ap.R469HE. coli (C. elegans has L)0.990.9970.000.77%13 (Hom; Fa, Mo)-21: VUR-III° R -22: VUR-III° R and LNoneA4252 -21 (F)Central Europeanc.1406G>Ap.R469HE. coli (C. elegans has L)0.990.9970.000.77%13 (Hom; Mo; partial maternal isodisomy)Double kidney RVUR LVACTERL association including esophageal atresia IIIb, anal atresia, vestibular fistulaA3051-21 (M)Macedonianc.127_137dupc.1324G>Ap.R46Sfs*75p.E442KNAD. rerioNA0.99NA0.003NA0.3Absent0.08%2 (het; Mo) 12 (het; Fa)MCDK LNoneA4884 -21 (F)Dutchc.757A>Gc.1573C>Tp.I253Vp.L525FE. coli (X. tropicalis has V, S. cerevisiae has L) E. coli0.990.990.4330.9420.000.000.91%Absent7 (het; Mo)14 (het; Fa)Renal agenesis RVACTERL association including cervical/thoracic hemivertebrae, 5 dysplastic short ribs R, anal atresia with rectoperineal fistula, ASD type II, esophageal atresia, abnormal position of thumbsEA1717-21 (F)Dutchc.1330T>Ac.1663G>Ap.Y444Np.V555IC. elegansC. intestinalis0.990.990.9850.1150.030.390.91%fOne individual is homozygous for this allele.Absent12 (het; Fa)14 (het; Mo)Pyelectasis and VUR LVACTERL association including anal atresia, esophageal atresia, ASD, VSD, hypoplastic/absent humerus, persistent L vena cava superior, cloacaAbbreviations: ASD, atrial septum defect; CAKUT, congenital abnormalities of the kidney and urinary tract; cDNA, complementary DNA; E. coli, Escherichia coli; EVS, Exome Variant Server; F, female; Fa, mutation segregating from the father; L, left; NA, not applicable; M, male; MAF, minor allele frequency; MCDK, multicystic dysplastic kidney; Mo, mutation segregating from the mother; MutT, MutationTaster; R, right; SIFT, sorting intolerant from tolerant; TRAP1, tumor necrosis factor (TNF) receptor–associated protein 1; VSD, ventricular septum defect; VUR-III°, vesicoureteral reflux third degree.a TRAP1 cDNA mutations are numbered according to human cDNA reference sequence NM_016292.2, where +1 corresponds to the A of ATG start translation codon.b MutationTaster score. Range: 0–1.0, 1.0 being most deleterious.c PolyPhen2 (HumVar) score. Range: 0–1.0, 1.0 being most deleterious.d SIFT score. Range: 0–1.0, 0 being most deleterious.e Minor allele frequency in 8600 alleles of Americans of European descent.f One individual is homozygous for this allele. Open table in a new tab Abbreviations: ASD, atrial septum defect; CAKUT, congenital abnormalities of the kidney and urinary tract; cDNA, complementary DNA; E. coli, Escherichia coli; EVS, Exome Variant Server; F, female; Fa, mutation segregating from the father; L, left; NA, not applicable; M, male; MAF, minor allele frequency; MCDK, multicystic dysplastic kidney; Mo, mutation segregating from the mother; MutT, MutationTaster; R, right; SIFT, sorting intolerant from tolerant; TRAP1, tumor necrosis factor (TNF) receptor–associated protein 1; VSD, ventricular septum defect; VUR-III°, vesicoureteral reflux third degree. In family A4252 with CAKUT in VACTERL, we performed WER in an affected individual (A4252-21). This girl was born with a right double kidney and duplex ureter, left VUR, esophageal atresia type IIIb, and anal atresia with a vestibular fistula (Figure 1e and f and Table 1). Although there was no consanguinity reported in this family, homozygosity mapping showed unusually broad homozygosity peaks on chromosome 16 on the p-terminus and q-terminus (5.5 and 9.6Mb, respectively; Figure 1g). In this case, we hypothesized that CAKUT in VACTERL is caused by a homozygous mutation within these homozygous regions. When evaluating WER data in this individual, the 512,733 variants initially detected (minor variant frequency ≥0.55; coverage≥2) were reduced to only 11 variants within the 'homozygosity peaks' on chromosome 16 and 18 (Supplementary Table S2 online). The only variant that was confirmed by Sanger sequencing and that altered a conserved amino-acid residue was TRAP1 R469H, the same allele as in family A3403. By comparison of SNPs in the affected girl and her parents, we demonstrated that partial maternal isodisomy of chromosome 16 with two recombinants (one located on the p-arm and one located on the q-arm) was the underlying cause of homozygosity for TRAP1 R469H (Figure 1g–j). The TRAP1 allele c.1406G>A, p.R469H alters an evolutionary highly conserved amino-acid residue, and it is predicted to be deleterious for protein function by publically available software programs (Table 1 and Supplementary Figure S1 online). In the Exome Variant Server database, R469H has a minor allele frequency (MAF) of 0.9% in Americans of European descent. In our cohort of 675 individuals with CAKUT, most of them European, the MAF is 1.9%. The three affected individuals from two unrelated families with homozygous TRAP1 R469H, as well as six additional heterozygous carriers, share haplotypes at the TRAP1 locus (Supplementary Figure S2 online), which speaks for TRAP1 R469H being a European founder mutation. Download .jpg (.18 MB) Help with files Supplementary Figure 1 Download .jpg (.28 MB) Help with files Supplementary Figure 2 We subsequently analyzed the coding sequence of TRAP1 in a cohort of 675 individuals with isolated CAKUT (Supplementary Table S3 online) and 300 individuals with classic VACTERL association (i.e., VACTERL-X and other related disorders have been excluded) using a bar-coded multiplex PCR approach and consecutive next-generation sequencing, as described previously by the authors.18.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 As a control group, we included 800 individuals with the distinct renal phenotype of nephronophthisis. We detected six additional recessive mutations in TRAP1 in a compound heterozygous state in three additional unrelated families with CAKUT or CAKUT in VACTERL (Table 1,Figure 1k, l and m,Supplementary Figure S1 and S3 online). In individual A3051-21 with a left-sided multicystic dysplastic kidney, we found a maternally inherited protein-truncating frameshift mutation (c.127_137dup, p.R46fs*75). This mutation abrogates the N-terminal mitochondrial targeting sequence of TRAP1, which makes this a null allele. The second allele was a missense mutation (c.1324G>A, p.E442K) that segregated from the father. Download .jpg (.15 MB) Help with files Supplementary Figure 3 In individual A4884-21 with CAKUT in VACTERL, including right renal agenesis, vertebral malformations, anal atresia with a rectoperineal fistula, atrial septum defect type II, esophageal atresia, and abnormal position of the thumbs (Table 1 and Supplementary Figure S4 online), we detected compound heterozygous missense mutations in TRAP1 located in the ATPase domain (c.757A>G, p.I253V) and in the HSP90 domain (c.1573C>T, p.L525F; Figure 1l). Download .jpg (.12 MB) Help with files Supplementary Figure 4 In individual EA1717 with CAKUT in VACTERL, including pyelectasis, left VUR, a complex anorectal malformation including anal atresia and persistent cloaca, esophageal atresia, cardiac defects, limb defects, and persistent left vena cava superior (Table 1), we detected compound heterozygous missense mutations that are both located in the HSP90 domain of TRAP1 (c.1330T>A, p.Y444N and c.1663G>A, p.V555I). To exclude the presence of recessive mutations in controls, we sequenced the TRAP1 coding sequence in 800 individuals with the distinct renal phenotype of nephronophthisis. We detected the TRAP1 allele I253V seven times (MAF 0.87%), T444N twice (MAF 0.25%), and R469H twice (MAF 0.025%), all of them as single heterozygous alleles. TRAP1 R46Sfs*75, E442K, L525F, and V555I were absent from our control cohort. Furthermore, no other possibly deleterious variants were present in a homozygous or compound heterozygous state in 800 individuals with nephronophthisis. To determine whether TRAP1 has a function during kidney development, we analyzed Trap1 expression in developing kidney in mouse embryos 13.5 dpc. Trap1 seemed to be expressed at this stage in renal vesicles according to Trap1 transcription assays publically available through the Gudmap project. By in situ hybridization (ISH) in E13.5 mouse embryos, we found Trap1 to be strongly expressed in the kidney, adrenal gland, and gonad. Trap1 expression specifically localized to renal epithelia (Figure 2a′). To characterize TRAP1 localization in adult kidney, we performed immunofluorescence stainings in rat using a monoclonal TRAP1 antibody in conjunction with established renal markers (Figure 3). TRAP1 is present most prominently in peanut-lectin-marked proximal tubules in the renal cortex (Figure 3a and b). In renal medulla, we detected TRAP1 in peanut-lectin-negative tubular segments and in NKCC-marked (Na+K+2Cl- co-transporter) thick ascending limbs of Henle's loop (Figure 3c and d). TRAP1 colocalizes with mitochondrial marker MTCO1 in renal cortex and medulla. In this study, we identified by WER and high-throughput mutation analysis five unrelated families with CAKUT or CAKUT in VACTERL association with recessive mutations in TRAP1. Two siblings with CAKUT had a homozygous missense mutation (R469H), which segregated from a common ancestor of their parents. A girl with VACTERL association had the identical homozygous mutation due to maternal isodisomy of chromosome 16 p-ter and q-ter. In a cohort of 675 individuals with CAKUT and 300 individuals with classic VACTERL association, we identified 3 additional individuals carrying compound heterozygous mutations in TRAP1. Homozygous or compound heterozygous deleterious variants were absent from 800 control individuals. By ISH and immunofluorescence, we showed that Trap1 is expressed in early mouse renal epithelia, whereas the Trap1 protein is present only in defined segments of developed nephrons in rat. In 6500 individuals recorded in the Exome Variant Server server, there are several non-synonymous variants present in TRAP1, including heterozygous truncating variants in 11 individuals. However, deleterious alleles in recessive disease genes, unlike in dominant disease genes, do not underlie direct negative selection through evolution. Consequently, the presence of rare deleterious variants in recessive disease genes in a large cohort is an expected finding. The allele TRAP1 Y444N, detected as a compound heterozygous mutation in an individual with CAKUT in VACTERL, is present homozygously in a single individual of the ESP cohort of 6500 healthy Americans. However, in the context of CAKUT, this does not necessarily mean that the variant is nonpathogenic. CAKUT frequently remain completely asymptomatic. For instance, a double kidney or unilateral renal agenesis typically are an 'accidental finding' in renal ultrasound. The fact that the homozygous mutation TRAP1 R469H was found in an individual with CAKUT and an individual with VACTERL association is surprising. However, in CAKUT and in VACTERL association, intra-familial phenotypic variability is very common.19.Solomon B.D. Pineda-Alvarez D.E. Raam M.S. et al.Evidence for inheritance in patients with VACTERL association.Hum Genet. 2010; 127: 731-733Crossref PubMed Scopus (37) Google Scholar, 20.Solomon B.D. Pineda-Alvarez D.E. Raam M.S. et al.Evidence for inheritance in patients with VACTERL association.Human genetics. 2010; 127: 731-733Crossref PubMed Scopus (61) Google Scholar, 21.Hilger A. Schramm C. Draaken M. et al.Familial occurrence of the VATER/VACTERL association.Pediatr Surg Int. 2012; 28: 725-729Crossref PubMed Scopus (30) Google Scholar Even in a single individual different CAKUT phenotypes may be present, for instance, left renal agenesis and right VUR. The frequencies of individuals with recessive TRAP1 mutations in our cohorts (0.15% in CAKUT, 0.6% in CAKUT with VACTERL) suggest that mutations in TRAP1 are a rare cause of these conditions. Similarly, mutations in two recently identified CAKUT-causing genes, WNT4 and DSTYK, are rare causes of CAKUT.7.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,8.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 These findings in humans, along with numerous CAKUT-mouse models, indicate that CAKUT are a common clinical phenotype arising from a multitude of different single-gene causes. In conclusion, we propose that recessive mutations in TRAP1 are a novel rare cause of isolated CAKUT and the first recessive cause of the VACTERL association. We obtained blood samples and pedigrees following informed consent from individuals with CAKUT and from individuals with VACTERL association. Approval for human subjects research was obtained from the University of Michigan Institutional Review Board and other institutions involved. The diagnosis of CAKUT and VACTERL association was based on published clinical criteria.9.Solomon B.D. VACTERL/VATER association.Orphanet J Rare Dis. 2011; 6: 56Crossref PubMed Scopus (284) Google Scholar We performed homozygosity mapping as described previously.17.Hildebrandt F. Heeringa S.F. Ruschendorf F. et al.A systematic approach to mapping recessive disease genes in individuals from outbred populations.PLoS Genet. 2009; 5: e1000353Crossref PubMed Scopus (133) Google Scholar Exome library preparation and next generation sequencing was conducted using the SeqCap EZ Exome v2 (Nimblegen, Roche NimbleGen, Madison, WI) and Genome Analyzer II (Illumina, San Diego, CA). Subsequent variant detection, filtering, and analysis have been described previously by the authors.14.Chaki M. Airik R. Ghosh A.K. et al.Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.Cell. 2012; 150: 533-548Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar,15.Zhou W. Otto E.A. Cluckey A. et al.FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair.Nat Genet. 2012; 44: 910-915Crossref PubMed Scopus (169) Google Scholar All detected variants were confirmed by Sanger sequencing. Immunofluorescence was performed as previously described by the authors14.Chaki M. Airik R. Ghosh A.K. et al.Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.Cell. 2012; 150: 533-548Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar using a Leica SP5X system (Leica Microsystems GmbH, Wetzlar, Germany) with an upright DM6000 compound microscope (Leica Microsystems GmbH), and images were processed with the Leica AF software suite. The following antibodies were used: TRAP1 (Abcam, Cambridge, MA (TRAP1-6), cat# ab2721), MTCO1 (Abcam cat# ab45918), NKCC2 (LSBio cat# LS-C150446, Seattle, WA), and NCCT (Millipore cat# AB3553, Billerica, MA). Specificity of the anti-TRAP1 antibody for rat TRAP1 was confirmed in immunoblot (Supplementary Figure S5 online). Download .jpg (.06 MB) Help with files Supplementary Figure 5 ISH was conducted on sections of wild-type mouse embryos with an Naval Medical Research Institute background at embryonic day 13.5. Mouse embryos were dissected into ice-cold phosphate-buffered saline, fixed overnight in 4% paraformaldehyde/phosphate-buffered saline, and then processed into paraffin wax. ISH was performed on paraffin sections (5μm) using antisense probes generated by PCR from an E11.0 total embryo complementary DNA library, and specific staining was verified using a sense probe. PCR products contained 3′ T7 and 5′ T3 RNA polymerase binding sites for in vitro transcription, and probes were purified using G-50 sephadex columns (GE Healthcare, Bio-Sciences, Piscataway, NJ). The 779-bp probe for Trap1 spans exons 13–17 (accession: NM_026508.2). ISH was performed according to the protocol from Chotteau-Lelievre et al.22.Chotteau-Lelievre A. Dolle P. Gofflot F. Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes.Methods Mol Biol. 2006; 326: 61-87PubMed Google Scholar with minor modifications, and detection of alkaline phosphatase activity was visualized using BM Purple (Roche Diagnostics, Mannheim, Germany). After staining, slides were quickly dehydrated in 80% and then 100% ethanol, cleared twice for 1 min in xylene (Roth, Carl Roth GmbH, Karlsruhe, Germany), and coverslips were mounted with Entellan mounting medium (Merck, Merck KGaA, Darmstadt, Germany). Photographs were obtained using the AxioVision software (Zeiss, Carl Zeiss, Oberkochen, Germany) with a Zeiss AxioCam and SteREO Discovery.V12 microscope (Carl Zeiss). Three sections from at least two different embryos were analyzed. Next generation sequencing reads alignment and variant detection was done with Genomics Workbench software (CLC Biotech, Cambridge, MA). Mapping parameters were as follows: global alignment, length fraction=0.9, and similarity fraction=0.9. Genetic location is according to the assembly of the Genome Reference Consortium GRCh37. We thank the physicians and families for participating in this study, the German self-help organization for people with anorectal malformations (SoMA eV), and all participating physicians, nurses, research assistants, laboratory analysts, and project members of AGORA (Aetiologic research into Genetic and Occupational/Environmental Risk Factors for Anomalies in Children) for their support in building this biobank. FH is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and a Frederick GL Huetwell Professor. ACH, GCD, Enrika Bartels, ES, DS, SG-D, SM, SH-C, MMN, ML, HMR, and MD are members of the 'Network for the Systematic Investigation of the Molecular Causes, Clinical Implications and Psychosocial Outcome of Congenital Uro-Rectal Malformations' (CURE-Net). This research was supported by grants from the National Institutes of Health (to FH; R01-DK045345 and R01-DK088767), by the March of Dimes Foundation (6FY11-241), by the Division of Intramural Research, by the National Human Genome Research Institute (NHGRI), by the National Institutes of Health and Human Services, by the Bundesministerium für Bildung und Forschung (grant 01GM08107), by the BONFOR program of the University of Bonn (to Enrika Bartels; grant O-149.0099, and to GCD; grant O-149.0096), by Sophia Scientific Research Foundation (to Enrika Bartels; grant SSWO S13/9), by the associazione Italiana per la Ricerca sul Cancro (AIRC) (to FE; grant IG13128), and by the Italian Ministry of Health (to FE; grant GR-2010-2310057). Web Resources: 1000 Genomes Browser, http://browser.1000genomes.org; Ensembl Genome Browser, http://www.ensembl.org; Exome Variant Server, http://evs.gs.washington.edu/EVS; Mutation Taster, http://www.mutationtaster.org; Gudmap (GenitoUrinary Molecular Anatomy Project), http://www.gudmap.org; Online Mendelian Inheritance in Man (OMIM), http://www.omim.org; Polyphen2, http://genetics.bwh.harvard.edu/pph2; Sorting Intolerant From Tolerant (SIFT), http://sift.bii.a-star.edu.sg; The Human Protein Atlas, http://www.proteinatlas.org; UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway. Figure S1. Evolutionary conservation of altered TRAP1 amino acid residues. Figure S2. Haplotype comparison at the TRAP1 locus in individuals carrying the allele TRAP1 R469H. Figure S3. Clinical findings in individuals A3051-21 with CAKUT and individual A4884-21 with VACTERL association. Figure S4. Chromatograms of newly identified mutations in TRAP1. Figure S5. Immunoblot validation of the anti-TRAP1 antibody specificity for rat TRAP1. Table S1. Whole-exome resequencing (WER) statistics and variant filtering workflow of WER in family A3403 with congenital abnormalities of the kidney and urinary tract (CAKUT). Table S2. Whole-exome resequencing statistics and variant filtering workflow in individual A4252-21 with VACTERL. Table S3. Demographics and characterization of 675 individuals with isolated congenital abnormalities of the kidney and urinary tract (CAKUT). Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .doc (.05 MB) Help with doc files Supplementary Information
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