Gessler, Manfred

Jakubiczka, Sybille

Konig, Anja

Schlösser, Hans W.

Wieacker, Peter

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© 1993 Oxford University Press Human Molecular Genetics, 1993, Vol. 2, No. 11 1967-1968 Further evidence that imbalance of WT1 isoforms may be involved in Denys- Drash syndrome Anja König, Sybllle Jakublczka1, Peter Wleacker1, Hans W.Schlösser and Mantred Gessler• lnsbtut für H~ Phl5pp&UilMti'SIIat, Bahnhofslr~e 7A, 0-35037 Marburg, 11nstJM für Humangenetik. Medizinische Hoc:hschule Hannover. 0-3000 Hannover 61 and 2Frauenldiruk der MediZII'lischen Hochschule Hannover. Oslstadtl<rankeus, D-3000 Hannover. Germany Received Juty 7, 1993; Revised and Accepted August 13, 1993 Denys-Drash syndrome (DOS) is a rare developmental disorder of the genifo..uriruuy system characterized by severe nephropathy, genital abnormalities and a predisposition to Wilms' tumor (Denys, 1967, Drash, 1970). All components of the syndrome appear to be caused by dominant mutations of the Wll Wilms' tumor gene (Pelletieret al., 1991). WTl has first been identified as a tumor suppressor gene that is deleted or inactivated in a certain fraction of Wilms' tumors (Call et al. , 1990; Gessler et al., 1990). The gene encodes a DNA-binding zinc finger protein tbat can regulate transcription of other genes (Rauscher et al., 1990). The majority of WTJ mutations detected in DOS patients are highly specific point mutations that alter DNA contact sites within the zinc finger domain of the WI'l protein (Pelletier et al. , 1991). More recently, some cases were described where the WTJ mutations would result in a truncated WTJ proteinthat is missing all or part of the zinc finger domain (Baird et al., 1992; Little et al. , 1993; Ogawa et al. , 1993). These observations favor the interpretation of DOS mutations as dominant negative mutations where a shortened WTJ polypeptide competes with wild-type a) b) G AT C SSCP sense c) 5- I protein produced from the remaining nonnal WTJ allele for effector sites. A single case, patient C.S., described by Bruening et al. (1992), however, suggested that even alterations in the balance between altematively spliced forms of WTJ may be sufficient to generate DOS. There are two sites of alternative splicing in the WTJ gene (Haber et al., 1991; Gessler et al., 1992). The first, leading to the presence or absence of exon 5 has not been associated with a functional correlate. The second site is found at the end of exon 9 where two splice donor sites, located within 9 basepairs, can be used, resulting in the insertion of 3 amino acids ( + KTS) between zinc fingers 3 and 4 with concomitant changes in the DNA binding characteristics of the resulting zinc fingers (Rauscher et al., 1990; Bickmore et al., 1992). The relative ratio of the different WTJ isoforms appears constant in aU cases examined with the longer products being more abundant. Here, we describe a new case of DDS, patient J.A., where a mutation in one of the splice donor sites of exon 9, identical to the one described in patient C.S., has been found supporting the concept that the balance of WTJ isofonns is critical for normal 3' e- II I. I . - ' I. I 1 a g/a · 1/c c- g \ ~: 5' antisense ')I .) Flpre 1. a) SSCP analysis of exon 9 in patient J.A. (laDe I) and two control DNAs (2, 3). PCR amplifk:ation from wbit.e blood cell ONA and denawration of radiolabeled products were done as described (Poulat er aJ., 1993). Sampies were separated on a 6«Ai potyacrylamide gd at 30 Wand 4°C, foUowed by dJying and autaradio&raphy. Tbc arrow l1l8Jb the location of the ncw band detccted in patient J.A. b) For dircct scquenoe analysis exon 9 was IUDJ>!ificd from genomic ONA and purified using Magie DNA Cleanup (Prornega). 1be 248 bp fragmentwas sequenced from both ends by Taq cycle sequencing using 32P-labeUed primers (fmol kit, Promcp). Tbe double band G/A (sense strand) and T/C (antisense strand) was not seeu in sc.qucnces from oontrol ONAs. c) scquenoe of the uou/intron junction. Boch a!temative splice sites (I and li) are shown with thc mutation site marbd by an asterisk. • To whom correspondc:nce should bc addressed 1968 Human Molecu/ar Genetics, 1993, Vol. 2, No. 11 development. Patient J.A. was suspected to have DOSbasedon clinical criteria. J.A. is a 13 year old female child with inconspicuous family history. Renal insufficiency was observed from the age of 2 years on. Chronic glomerulonephritis made dialysis necessary from the age of 8 years on and Jed to renal transplantation at 11.5 years. The girl was growth retarded and showed primary amenorrhea. External genitalia were normal female with endocrinological investigations revealing a hypergonadotrophic hypogonadism. The cytogenetic examination of peripheral blood lymphocytes showed a normal male karyotype. The combination of gonadal dysgenesis and chronic renal failure resulting from glomerular disease suggested the diagnosis of DOS. Because of the increased risk of malignancy gonads were removed. At laparotomy, uterus, Fallopian tubes and streak gonads were observed. Histologie examination showed normal Fallopian rubes, a ductus epididymis and dysgenetic gonadal tissue without typical testicular structures. The analysis of fibrobJast cultures from the streak gonads also revealed a normal male karyotype. The clinical picture with Müllerian structures present suggests that the AMH-producing fetal cells must have degenerated early. On the other band, testosteron-producing Leydig cells must also have been active for some time, as a ductus epid.idymis was present. During initial mo1ecular studies a deletion of the Y chromosome was not detected by PCR-studies employing primer pairs of the foUowing loci: PABY, SRY, ZFY, DYSI9, DYS14, alpha-satellite, DXYS19, and DYZL (data not shown). To search for possible alterations of WTJ in this patient the coding region of the gene was assayed by SSCP analysis (Orita et al. , 1989). Only in exon 9 a slight alteration of the bands could be detected. The incomplete separation ofthe variant band from tbe normal band precluded direct analysis of the altered fragment. Exon 9 was therefore amplified from genomic DNA and sequenced directly from both ends. A single alteration could be detected in repeated sequencing reactions resulting in a G- A transversion in intron 9, five basepairs downstream the splice donor site (Figure 1). This is the same mutation as described in DOSpatient C.S. by Bruening et al. (1992). These authors have shown by transfection of minigene constructs containing WT1 exons 9 and 10 that this alteration abolishes the use of the second alternative splice donor site at exon 9. Thus, the Ionger + KTS isoform of the WT1 protein can only be produced from the remaining nonnal allele, leading to an altered balance between these protein isoforms. The independent finding of the same kind of splicing mutant in a second patient with DOS provieles additional evidence that the precise ratio of different WT1 isoforms may be critical during certain steps of kidney and gonadal development. ACKNOWLEDGEMENT This work was supponed by a grant from the Deutsche Forschungsgemeinschaft (GeS39/3-3). REFERENCES I. Baird,P.N., Santos,A., Groves,N., J~.L. mi Cowell,J.K. (1992) Hum. MoL Gene~. 1, 301-305. 2. Bruening,W., Bardeesy,N., Silverman,B.L., Cohn,R.A., Machin,G.A., Aronson,A.J., Housman,D. and Pdletier,J. (1992) Na111re Gmetics l, 144-148. 3. Call,K.M., Glaser,T., Ito,C.Y., Buclder,A.J., Pelletier,J., Haber,D.A., Rose,E.A., Kral,A., Yeger,H., Lewis,W.H., etaL (1990) ca/60, S09-S20. 4. Denys,P., Malvaux,P., van den Berghe,H., Tanghe,W. and Proesmans,W. (1967) Ardt. Fron. Ped. 24, 729-739. S. Drash.A., Sbetman.F., Hartmann,W.H. and Blizurd,R.M. (1970) J. Ptdiarr. 76, 585-593. 6. Gessler,M., Konig,A. and Bruns,G.A. (1992) Genomics U, 807-813. 7. Gessler,M., Poustka,A., Cavenee,W., Neve,R.L., Orkin,S.H. and Bruns,G.A. {1990) Nanue 343, 774-778. 8. Haber,D.A., Sohn,R.L., Buclder,A.J., Pelletier,J., Call,K.M. and Housman,D.E. (1991) Proc. Natl. Acad. Sei. USA 88, 9618-9622. 9. Uttle,M.H., Williamson,K.A., Mannens,M., Kelsey,A., Gosden,C., Hastie,N.D. and van Heyningen,V. (1993) Hum. MoL Gene'~. l, 2.59-264. 10. Ogawa,O., Eccles,M.R., Yun,K., Mueller,R.F., Holdaway,M.D.D. and Reeve,A.E. {1993) Hum. MoL Genn. l, 203-204. 11. Orita,M., Suzukt,Y., Seldya,T. and Hayashi,K. {1989) Genomfes S, 874-879. 12. Pelletier,J., Bruening,W., Kashtan,C.E., Mauer,S.M., MaruveJ,J.C., Striegel,J.E., Houghton,D.C., Junien,C., Habib,R., Fouscr,L. etal. (1991) .eeu 67, 437-447. 13. Poulal,F., Morin,D., König,A., Bnm,P., Giltay,J., Su1tan,C., Dumas,R., Gessler,M. and Berta,P. (1993) Hum. Gene~. 91, 285-286. 14. Rauscber,F.J., Morris,J.F., Townay,O.E., Cook,D.M. and Curran,T. (1990) Science 250, 1259-1262. 

Further evidence that imbalance of WT1 isoforms may be involved in Denys-Drash syndrome

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Further evidence that imbalance of WT1 isoforms may be involved in Denys-Drash syndrome

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