Inherited WTJ Mutation in Denys-Drash Syndrome'

(CANCERRESEARCH52, 6125-6128, NovemberI, 19921 Advances in Brief

Max J. Coppes,2 Gerrit Jan Liefers, Mariko Higuchi, Arthur B. Zinn, J. Williamson Balfe, and Bryan R. G. Williams Department ofCancer Biology, Research Institute, The Cleveland Clinic Foundation (M. J. C., G. J. L, M. H., B. R. G. W.J, Cleveland, Ohio 44195, Departments of Pediatrics and Genetics, Case Western Reserve University (A. B. Z.J, Cleveland, Ohio 44106 and Division ofNephrology, Department ofPediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada MSG 1X8 (J. W. B.J

Abstract We suggest that parents of individuals with the DDS be evalu ated for the presence of the WTJ mutation. Patients with the Denys-Drash syndrome (Wilms' tumor, genital anomalies, and nephropathy) have been demonstrated to carry de now Materials and Methods constitutional mutations in WTJ, the Wilms' tumor gene at chromosome I 1p13. We report three new cases, two carrying a previously described Patients. The clinical and cytogenetic features of three patients with WTI exon 9 mutation and one with a novel WTJ exon 8 mutation. DDS are summarized in Table 1. D7, the father of patient D5, is at However, unlike patients in previous reports, one of our three patients present a healthy 42-year-old male. Physical examination did not reveal inherited the affected allele from his phenotypically unaffected father. any abnormalities; in particular he has bilaterally descended testes of This observation indicates that the WTI exon 9 mutation affecting normal volume and a normal penis; he does not have hypospadias. In 394Argdemonstrated in over one-half of the patients with the Denys 1985, he donated his left kidney for transplantation to his son, patient Drash syndrome may exhibit incomplete penetrance. Consequently, fa D5. milial studies in patients affected by this syndrome are recommended. Genomic DNA Preparation. Constitutional DNA from all three pa Introduction tients and their parents and several family members of patient D5 were isolated from peripheral leukocytes as described previously (11). Isola Wilms' tumor, or nephroblastoma, is a renal malignancy that tion of the constitutional DNAs of patient D5 and his father (D7) were affects approximately 1 in 10,000 children under 16 years of repeated following the initial results in order to confirm the analyses. age. Cytogenetic observations in patients with Wilms' tumor, PCR Amplification. All @VT1exonicsequences from genomic DNA aniridia, genitourinary malformations, and mental retardation were amplified by PCR, and the PCR products were analyzed for SSCP (the WAGR syndrome) demonstrated chromosome 1lpl 3 (I 2) in order to detect possibledeletions, insertions, or point mutations. In the analysis of WTJ exon 8 we used primers (A-2)8 and (S-2)8, and germ-line deletions, providing the first indication that this in the analysis of @VT1exon 9 primers (A-2)9 and (S-2)9 which have chromosome segment is involved in Wilms' tumorigenesis (1). been described previously (13). PCR was carried out in a GeneAmp Molecular analysis of the homozygously deleted region chro PCR system 9600 (Perkin-Elmer) with a final volume of 100 @l,con mosome 1lpl3 in two Wilms' tumors led, in 1990, to the taming 250 mmol/liter each ofdATP, dGTP, and dTTP; 20 mmol/liter independent identification of J'Vfl, a Wilms' tumor suppressor ofdCTP; I mCi I32PIdCTP;200â€”800ngofDNA; 50 pmol ofeach PCR gene, by three groups (2â€”4).Occasionally, the presence of primer, 2.5 units of Taq polymerase; and 10 MI of Taq polymerase Wilms' tumor in a patient is associated with genital anomalies reaction buffer. and nephropathy: the Denys-Drash syndrome (5, 6). The din SSCP. Following PCR a 5-al aliquot of the amplified product was ical overlap between this syndrome and the WAGR syndrome diluted with 40 Ml of 0.1% sodium dodecyl sulfate-lO m@i EDTA; 2 @il @ suggested that the could also arise as a consequence of a of this mixture were added to 2 Mlof Sequenase stop mix [95% forma mide-20 mM EDTA-0.05% bromophenol blue-0.05% xylene cyanol deletion of one or more genes localized at chromosome I I p13 (United States Biochemical Corporation, Cleveland, OH). The DNA (7).Recently,constitutionalWTJmutationshaveindeedbeen samples were denatured at 95'C for 3 mm and immediately placed demonstrated in 23 of 25 reported individuals with DDS, pro on ice before being run on 6% polyacrylamide nondenaturing gels viding evidence of a direct role for @Vf1in this syndrome (8â€” containing 10% glycerol. Electrophoresis was performed in 0.09 M 10).Basedon two familiesevaluated,it wassuggestedthatthe Tris base-0.09 M boric acid-2.5 msi EDTA running buffer at 30 W at DDS follows a dominant mode of inheritance with high pene room temperature for 4â€”6h,at 30 W at 4C for 4â€”6h,and at 5 W at trance and that it is extremely unlikely that parents of individ room temperature for 16 h. The gels were dried and exposed to XAR-5 uals with the DDS carry the mutations found in the affected film (Kodak) for 16â€”96hwithout an intensifying screen at room children (8). Subsequent analysis of four additional families temperature. seemed to confirm this hypothesis (10). We report here the Sequencing. The mechanisms used for direct sequencing of bio tinylated PCR products have recently been described (10, 14). Briefly, presence of the same 14'Ti exon 9 mutation in a phenotypically PCR amplification with one primer biotinylated at the 5' end and abnormal male and his phenotypically unaffected father. This one nonbiotinylated primer was performed when the PCR product was finding demonstrates that U'Tl mutations causing the DDS can to be used for sequencing. The final volume remained 100 pl, I mCi indeed be inherited and need not be expressed phenotypically. [32PIdCTPwas omitted, the concentration of the nucleotides became 250 mmol/liter for each deoxynucleotide triphosphate, and the amount Received9/17/82; accepted9/28/92. of primer used was decreased to 5â€”15pmol.Adsorption of a 5O-pl The costs ofpublication of this articleweredefrayedin part by the paymentof aliquot of the biotinylated PCR product on streptavidin-coated mag page charges. This article must therefore be hereby marked advertisement in accord netic Dynabeads (Dynal, Merseyside, United Kingdom) was performed ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Dc Bartolo Funds(M. J. C., B. R. G. W.) and utilizing a magnetic particle concentrator (Dynal) as recommended by the Ohio Department of Health (A. B. Z.). the manu-facturer. Following denaturation in 100 p1of 0.15 MNaOH, 2 To whom requests for reprints should be addressed, at Department of Cancer biotinylated and nonbiotinylated strands were separated on the mag Biologj@NN1-06,The ClevelandClinic Foundation, 9500 EuclidAvenue,Cleve netic particle concentrator and both strands were sequenced using a land, OH 44195. 3 The abbreviations used are: DDS, Denys-Drash syndrome; PCR, polymerase Sequenase (United States Biochemical) kit according to the instructions chain reaction; SSCP, single strand conformational polymorphisms. of the manufacturer. 6125

syndromePresentExternalPatientage Table IClinical and cytogeneticfindingsfor threepatients withDenys-Drash

mutationDl7FemaleNormal(yr)genitaliaInternal reproductive organsKaryotypeWilms' tumorNephropathyWJ'I bilateral+394Arg-TrpD513AmbiguousNormal ovaries Normal uterus46,XX+, testes structures46,XY394Arg-TrpDeceasedFemaleStreakResidual mtillerian Dl0 gonads46,XY+377His-Tyr a Kidneys removed at age 18 months. I, Kidneys removed at age 19 months.

Results sequence of the protein domains that are critical for DNA se quence recognition by zinc finger proteins. The JVfJ exon 9 PCR/SSCP analysis used to search for deletions, insertions, or base pair mutations within the @YI'1exons demonstrated mutations found in patients Dl and D5 affect amino acid res (Fig. 1) a mobility shift in exon 9 (encoding zinc finger 3) of one idue 394Arg, which has been implicated in hydrogen bonding allele from patients Dl and D5, and in exon 8 (encoding zinc interactions with a guanidine residue in the cognate DNA finger 2) of one allele from patient DIO. Constitutional DNA recognition sequence (15). This mutation has now been re from the parents of Dl (D2 and D3) and D10 (D8 and D9) ported in 14 of 25 (@55%) individuals with the DDS. The WTJ demonstrated a normal (homozygous) SSCP pattern (Fig. 1). A exon 8 mutation found in patient D1O affects amino acid resi homozygous SSCP pattern was also found for the mother (D6), due 377His and disrupts one of the four zinc binding ligands of three paternal aunts (51D7, S2D7, S3D7), and brother (BD5) zinc finger 2; this mutation has not been described previously. of D5, but the father (D7) of D5 showed a heterozygous SSCP In addition to these two @Vf1missense mutations, an exon 9 pattern similar to the one demonstrated for his son (Fig. 1). The missense mutation affecting 396Asp has been reported previ heterozygous SSCP pattern found in the father was confirmed ously in three patients, an exon 8 missense mutation affecting on a second blood sample taken 3 months after the first one. In 366Mg in two patient, an exon 7 missense mutation affecting addition, very numerous tandem repeat studies were performed 330Cys in one patient, an insertion ofG at position 821 in exon on samples from D5, D6, and D7 and were found to be consis 6 resulting in the immediate generation of a stop codon in one tent with the presumed paternity (data not shown). DNA of a patient, and finally, an intron 9 mutation, which prevents splic (the only) paternal uncle of D5 could not be obtained. ing at one of the alternative splice donor sites of exon 9, in Sequence analysis of constitutional DNA revealed a II29@to another patient (8â€”10).The JVI'l zinc finger region, encoded by T transition converting amino acid residue 377His to Tyr in exons 7â€”10,and especially exon 9, seems to be a â€œhotspotâ€•for patient DlO (Fig. 2A), and a â€˜â€˜80CtoT transition converting constitutional mutations causing the DDS. amino acid residue 394Arg to Trp in patients Dl and D5 The identification of constitutional WTJ zinc finger muta (Fig. 2B). Sequence analysis ofD7 revealed the same â€˜â€˜80CtoT tions in patients with the DDS, as well as the described WTJ transition found in patient D5 (Fig. 2B). The described muta exon 9 mutation in a phenotypically normal father of a child tions were confirmed by sequence analysis of at least five addi with this syndrome, raises the question of the origin of the tional samples from any given specimen. mutations. A recessive mode of inheritance, resulting from the Discussion inheritance of a mutant JVFJ allele from each parent, seems unlikely given the fact that in the 23 patients reported with a We report three patients with the DDS who were found to constitutional WTJ mutation, only one abnormal allele has have constitutional JYFJ mutations which alter the amino acid been observed in each patient (8â€”10).Although false negative results have been reported for SSCP analysis, it is considered a very sensitive assay (8). It seems unlikely that the SSCP assay would have failed to reveal a second mutation in all 23 patients analyzed. The data are more consistent with an autosomal @ :p:@l dominant mode of inheritance for this disorder. Because all cases reported to date have occurred sporadically, the WTJ allelic penetrance has been assumed to be very high (8), leading @is.. i.-. .â€”@ ,aâ€”1ul@ to the suggestion that it would be extremely unlikely for a parent of a child with the DDS to carry a nonpenetrant muta N tion (8). This hypothesis has been supported by the analysis of EXONS EXON9 constitutional DNA from both parents of six of the previously Fig. I. PCR/SSCP analysis of: W1'l exon 8 in patient D10, her mother (D8), reported cases (8, 10). None of the parents carried the mutant her father (D9), and an unaffectedcontrol (C);and of 1+71exon 9 in patient Dl, allele of their affected child. However, our results demonstrate her mother (D2), her father (D3), an unaffected control, and patient D5, his mother(D6), father(D7), brother(BD5), paternal grandparents(MD7 and FD7), that a phenotypically unaffected parent can have and transmit a and three paternal aunts (SID7, S2D7, and S3D7). The PCR/SSCP analysiswas J3PfJ exon 9 mutation to his affected son. performed as described in â€œMaterialsandMethods;â€•the primer sets used for PCR ofexon 8 [(A-2)8and (S-2)81,andexon 9 ((A-2)9and (A-2)91havebeenpublished The presence of the WTJ exon 9 missense germline mutation previously (13). The WJ'l exon 8 and 9 PCR products ofthe unaffected controls in an unaffected parent suggests either that W7'I mutations (C) demonstrate the wild type mobility (homozygous) pattern for both exons. The exhibit incomplete penetrance [in contrast to what was previ @VJlexon 8 PCR products of D8 and D9 are normal, whereas the PCR product of DlO shows two bands. The WT1exon 9 products of D2, D3, S1D7, S2D7, ously assumed (8)], possibly related to somatic mosaicism (16) S3D7, FD7, MD7, D6, and BD5 show a normal SSCP mobility pattern, whereas or genomic imprinting (17). Insofar as the latter possibility is the D5 and D7 products show a heterozygouspattern. Nondenatured samples were used to delineate the position of migration of the double stranded DNA concerned, the recent demonstration of equivalent expression products for both exons (data not shown). of paternally and maternally inherited WTJ alleles in Wilms' 6126

A 377 His EXON S 3' TGGACATA@4@JAGA5'

377Tyr

Wild Type DlO 3, 3' ______1 G I G G A C C A A C 1 IA A

A A GC C A A A 5, 5,

WildType

B 3, ACG T EXON9 C

C [email protected] T 394 G Arg G C 3' CCTGG9@1CCTCTT 5' C C 394 Trp I C I I 5, Dl D7

3' A C G I T:;=@ A C G I 3' C C. C @-. C I I (; @. .., (; C C C I C T I 5, Fig. 2. Sequenceanalysisof W7'l exon 8 (codingzinc fmger 2) in patient Dl0 and of WTJexon 9 (codingzinc finger 3) in patients Dl, D5, and D7 (the father of patient D5).Sequencingwasperformedas descn'bedin â€œMaterialsandMethods.â€•Thenucleotidenumberingsystemis basedon the codingsequenceonlyand considers the A of the ATG initiatoras +1. The numberingsystemfor the aminoacidsconsidersthe initiatormethionineas +1. (A) @YI1exon8 sequenceanalysisof @ constitutional DNA of patient D1O shows a â€˜129Cto T point mutation which converts 377His to Tyr. (B) WTJ exon 9 sequence analysis demonstrates a 180( to I transition which converts 394Arg to Trp in patients Dl, D5, and D7 (the father of D5).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. INHERITED WI! MUTATIONS IN DDS tumors and normal tissues heterozygous for the WTJ gene sug W. Associationd'un syndromeanatomo-pathologiquede pseudohermaphro disme masculin, d'une tumeur de Wilms, d'une nÃ©phropathieparenchyma gests that J@Vf1isnot subject to transcriptional imprinting (18). teuse et d'un mosaicismXX/XY. Arch. Fr. Pediatr., 24: 729â€”739,1967. We therefore believe that another cause of incomplete pene 6. Drash, A., Sherman, F., Hartmann, W., and Blizzard,R. M. A. A syndrome trance is the more likely explanation for our findings. Unfor of pseudohermaphroditism, Wilms' tumor, hypertension, and degenerative renal disease. J. Pediatr., 76: 585â€”593,1970. tunately, we have been unable to assess the possibility of so 7. Jadresic, L., Leake, J., Gordon, I., Dillon, M. J., Grant, D. B., Pritchard, J., matic mosaicism in this individual; sperm analysis is not Risdon, R. A., and Barratt, T. M. Clinicopathologic review oftwelve children feasible because D7 has had a vasectomy performed and has with nephropathy, Wilms tumor, and genital abnormalities (Drash syn drome). J. Pediatr., 117: 717â€”725,1990. thus far not consented to providing us with a skin biopsy for 8. Pelletier, J., Bruening, W., Kashtan, C. E., Mauer, S. M., Manivel, J. C., fibroblast culture and analysis. Striegel,J. E., Houghton, D., Junien, C., Habib, R., Fouser, L., Fine, R. N., This report demonstrates that JJ'Tl mutations in patients Silverman, B. L., Haber, D. A., and Housman, D. Germline mutations in the Wilms' tumor suppressor gene are associatedwith abnormal urogenital de with DDS may be inherited from a phenotypically normal par velopment in Denys-Drash syndrome. Cell, 67: 437â€”447,1991. ent, the carrier of the mutation. Accordingly, familial studies of 9. Bruening,W., Bardeesy,N., Silverman,B. L, Cohn, R. A., Machin, G. A., affected patients are indicated. It is expected that such studies Aronson, A. J., Housman, D., and Pelletier, J. Germline intronic and exonic mutations in the Wilms' tumour gene (WJ'l) affecting urogenital develop will improve the genetic counseling provided to individuals af ment. Nature Genet., 1: 144â€”148,1992. fected by this syndrome and their families and enhance our 10. Baird, P. N., Santos, A., Groves, N., Jadresic, L., and Cowell, J. K. Consti understanding ofthe role of @VF1ingenitourinary development tutional mutations in the WT1 gene in patients with Denys-Drash syndrome. Hum. Mol. Genet., 1: 301â€”305,1992. and Wilms' tumorigenesis. 11. Coppes, M. J., Bonnetta, L., Huang, A., Hoban, P., Chilton-MacNeill,S., Campbell,C. E., Weksberg,R., Yeger,H., Reeve,A. E., and Williams,B. R. Acknowledgments G. Lossofheterozygositymappingin Wilmstumor indicatesthe involvement of three distinct regions and a limited role for non-disjunction or mitotic We thank Dr. John Cowell, Institute of Child Health, London, recombination. Genes Chromosomes Cancer, in press, 1992. United Kingdom, for his advise on using biotinylated primers for direct 12. Orita, M., Suzuki, Y., Sekiya, I., and Hayashi, K. Rapid and sensitive de tection of point mutations and DNA polymorphisms using the polymerase sequencing of PCR products. chain reaction. Genomics, 5: 874â€”879,1989. 13. Haber, D. A., Sohn, R. L., Buckler, A. J., Pelletier, J., Call, K. M., and References Housman, D. E. Alternative splicing and genomic structure of the Wilma tumor gene WTJ. Proc. NatI. Aced. Sci. USA, 88: 9618â€”9622,1991. I. Riccardi, V. M., Sujansky, E., Smith, A. C., and Francke, U. Chromosomal 14. Hogg, A., Onadim, Z., Baird, P., and Cowell, J. K. Detection of heterozygous imbalance in the Aniridia-Wilms' tumor association: I Ip interstitial deletion. mutations in the RB1 gene in retinoblastoma patients using single-strand Pediatrics, 61: 604â€”610,1978. conformation polymorphysm analysis and polymerase chain reaction se 2. Bonetta, L., Kuehn, S. E., Huang, A., Law, D. J., Kalikin, L. M., Koi, M., quencing. Oncogene, 7: 1445â€”1451,1992. Reeve,A. E., Brownstein,B. H., Yeger,H., Williams,B.R. G., and Feinberg, 15. Pavletich, N. P., and Pabo, C. 0. Zinc finger-DNA recognition: crystal struc A. P. Wilmstumorlocus on llpl3 definedbymultipleCpG island-associated ture of a Zif268-DNA complex at 2.1 A. Science (Washington DC), 252: transcripts. Science (Washington DC), 250: 994â€”997,1990. 809â€”817,1991. 3. Call, K. M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D. A., 16. Hall, J. G. Reviewand hypotheses:somatic mosaicism:observationsrelated Rose, E. A., Kral, A., Yeger, H., Lewis, W. H., Jones, C., and Housman, D. to clinicalgenetics.Am. J. Hum. Genet., 43: 355â€”363,1988. E. Isolation and characterization of a zinc finger polypeptide gene at the 17. Hall, J. G. Genomic imprinting: review and relevance to human diseases. human chromosome 11 Wilms tumor locus. Cell, 60: 509â€”520,1990. Am. J. Hum. Genet., 46: 857â€”873,1990. 4. Gessler, M., Poustka, A., Cavenee, W., Neve, R. L., Orkin, S. H., and Bruns, 18. Little, M. H., Dunn, R., Byrne, J. A., Smith, P. J., Pritchard-Jones, K., Van G. A. P. Homozygousdeletion in Wilms tumors of a zinc-fingergene iden Heyningen, V., and Hastie, N. D. Equivalent expression of paternally and tified by chromosome jumping. Nature (Lond.), 343: 774â€”778,1990. maternallyinherited @VT1allelesin normal fetal tissueand Wilma' tumours. 5. Denys, P., Malvaux, P., Van Den Berghe, H., Tangue, W., and Proesmans, Oncogene, 7:635â€”641,1992.

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Max J. Coppes, Gerrit Jan Liefers, Mariko Higuchi, et al.

Cancer Res 1992;52:6125-6128.

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