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Identification of a new WT1 mutation in a sporadic Wilms’ tumour
Biochimica et Biophysica Acta 1407 (1998) 109^113
Identi¢cation of a new WT1 mutation in a sporadic Wilms' tumour Ana C. Santos a , Maria G. Boavida b , Ad|¨lia Costa c , Leonor Osorio-Almeida a
a;
*
Laborato¨rio de Gene¨tica Molecular, Faculdade de Cieªncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal b Departamento de Gene¨tica Humana, Instituto Nacional de Sau¨de Dr. Ricardo Jorge, 1699 Lisboa Codex, Portugal c Servic,o de Patologia, Hospital de Santa Maria, 1699 Lisboa Codex, Portugal Received 3 March 1998; revised 27 April 1998; accepted 29 April 1998
Abstract A new mutation in WT1 is described in a sporadic unilateral Wilms' tumour consisting of a 17 bp duplication in exon 7 generating a stop codon. The second allele is either partially deleted or presents the same alteration. LOH analysis at 11p15.5 and at the 16q13-16q24.3 regions indicated retention of heterozygosity in the tumour DNA for the markers analysed. The results are consistent with Knudson's hypothesis and confirm that loss of function of WT1 contributes to the development of at least some Wilms' tumours. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Wilms' tumor; WT1
1. Introduction WT1 is one of the genes involved in Wilms' tumour [1,2], a childhood malignancy of the kidney occurring in a frequency of 1 in 10 000 live births [3]. The gene is mainly expressed during the development of the kidney and in genitourinary structures [4]. It encodes a protein with four zinc ¢nger motifs which act both as a transcriptional suppressor and as an activator of growth related genes [5]. WT1 mutations in Wilms' tumours are a rare event, only detectable in 10^15% of the Wilms' tumours [6]. Of these, at least 69% [7] were shown to carry inactivation of the second allele, following the classic two-hit hypothesis [8]. Approx. 1^2% of Wilms' tumour patients have an associated AGR (Aniridia, Genitourinary anomalies and mental Re-
tardation) syndrome and demonstrate intragenic mutations in the remaining WT1 allele in the tumour [7]. In Wilms' tumours associated with Denys-Drash syndrome which account for less than 1% of all tumours, WT1 homozygosity/hemizygosity occurs in almost all cases [9]. Therefore, it can be concluded that complete inactivation of WT1 occurs in WT1 associated Wilms' tumours. Deletions and point mutations are among the most frequent inactivating mutations of WT1. Tandem insertions seem to be a more rare type of mutation and, to our knowledge, only reported in four cases [10^13]. Here, we describe a Wilms' tumour patient with a somatic mutation consisting of a tandem insertion of 17 base pairs in exon 7 generating a stop codon. This ¢nding provides further evidence for the involvement of complete inactivation of WT1 in the subset of Wilms' tumours.
* Corresponding author. Fax: +351 (1) 2948530. 0925-4439 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 9 8 ) 0 0 0 3 2 - 5
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2. Materials and methods 2.1. Patient AGM, a male child aged 5 years, with normal genitalia, developed a sporadic Wilms' tumour of the right kidney at the age of 5 years. Histological analysis of the surgically resected sample revealed a capsulated tumour without evidence of nuclear anaplasia, containing stroma and epithelial elements such as tubules, microcysts and glomerular bodies. No blastemal tissue was observed, probably due to pre-operative chemotherapy treatment (S. Fleming, pers. commun.). 2.2. Molecular analysis Simultaneous DNA and RNA extraction was carried out from fresh tumour or from para¤n embedded tissue, according to Santos and Oso¨rio Almeida [14]. DNA was obtained from peripheral blood lymphocytes of the patient and his parents, following described methods [15,16]. The search for WT1 mutations was carried out using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of exons 2^10 of tumour DNA as previously described [10,17]. Exon 1 was analysed as described by Hu¡ et al. [18]. Direct genomic sequencing of lymphocyte and tumour DNA was performed after ampli¢cation, using Dynabeads (Dynal, Merseyside, UK), essentially as described by Hogg et al. [17]. PCR covering exons 7^9 of WT1 was carried out in tumour DNA using the gene Amp XL PCR kit (Perkin Elmer) and primers described by Baird et al. [10]. The following conditions were used: 500 ng of DNA, 150 pmole of each primer, 1.6 mM Mg2 , 40 mM dNTP blend in a 50 Wl reaction volume. Ampli¢cation was for 35 cycles at 95³C for 1 min, 58³C for 1 min and 72³C for 5 min. Cycling was preceded by 7 min at 95³C, 1 min at 58³C and followed by 10 min at 72³C. The PCR products were analysed in 1% agarose gel electrophoresis. Tumour mRNA analysis was performed by RTPCR (Pharmacia bulk ¢rst strand cDNA kit) with primers for exons 1, 2, 6, 8 and 10 [10] and for
exon 4 (primers cD 5U4: 5P-GAC AAT TTA TAC CAA ATG ACA TCC CC-3P and cD 3U4: 5P-CCC TTT AAG GTG GCT CCT AAG TTC-3P), exon 7 (primers cD 5U7: 5P-GAT GTG CGA CGT GTG CCT GGA G-3P and cD 3U7: 5P-CAG TGT GCT TCC TGC TGT GCA A TC-3P), exon 9 (primer cD 3U9: 5P-CTG TAT GAG TCC TGG TGT GGG TC-3P) and 3P-UTR (primers localized 934 bp away from the gene, see [19]). Ampli¢cation of exons 1^2, 2^6, 6^8 and 8^10 was carried out according to Baird et al. [10]. Conditions for ampli¢cation of exons 4^7, 4^9 and 7^9 were the following: 1/30 of the ¢rst strand cDNA product were added to the PCR reaction mixture (10 mM Tris-HCl pH 8.8, 50 mM KCl, 1.5 mM Mg Cl2 , 0.1% Triton X-100, Taq DNA polymerase, Pharmacia), and subjected to 45 cycles of ampli¢cation 3 min at 95³C, 1.5 min at 62³C and 6 min at 72³C. Cycling was preceded by 7 min at 95³C, 2 min at 62³C and followed by 10 min at 72³C. All PCR products were analysed in 4% agarose gel electrophoresis. Ampli¢cation of exon 4^3P-UTR region was with the gene Amp XL PCR kit in the conditions described above, but with 1/10 of the ¢rst strand cDNA product. Electrophoresis was in 1% agarose gel. In parallel, PCR was performed on tumour cDNA for the OCRL (oculocerebrorenal) gene with primers which amplify several exons from the middle part of the gene, as a control for the presence of cDNA in the sample [20]. LOH screening was performed at genomic regions suggested to harbour additional loci implicated in Wilms' tumorigenesis, namely 11p15.5 and 16q1316q24.3 [21,22]. LOH analysis for 11p15 was performed with primers for IGF2 [23] and for GQ- and AQ-globin [24]. LOH analysis for 16q was performed by PCR using described primers for microsatellites [25]. These included D16S408, D16S514, D16S496, D16S512, D16S515, D16S516, D16S507, D16S402, D16S520, D16S413. PCR was performed in a reaction volume of 25 Wl including [K-32 P]dCTP. The following bu¡ers were used: 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl2 , 0.1% Triton X-100 for primers D16S514, D16S496, D16S512, D16S515, D16S516, D16S507, D16S511, D16S520, D16S413; 20 mM Tris-HCl, pH 8.55, 16 mM (NH4 )2 SO4 , 150 Wg/ml BSA, 2.5 mM MgCl2 for primer D16S408; 20 mM Tris-HCl, pH 8.5, 16 mM (NH4 )2 SO4 , 150 Wg/ml
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BSA, 1.0 mM MgCl2 for primer D16S518; 50 mM KCl, 10 mM Tris-HCl, pH 9, 0.1% Triton X-100, 2.5 mM MgCl2 for primer D16S402. The PCR products were analysed on a denaturing (7 M urea) 6% polyacrylamide gel. The gel was dried and autoradiographed at 380³C. 3. Results 3.1. Cytogenetic and FISH analysis Cytogenetic analysis of peripheral blood lymphocytes and of primary tumour cell cultures revealed a normal 46,XY karyotype. FISH analysis on cell suspensions prepared from para¤n embedded tumour sample with the alphoid probe (pL11A, see [26]), showed that both 11 chromosomes were present in the tumour cells. 3.2. Molecular analysis Individual PCR products of exons 2^10 of WT1 in the tumour DNA indicated a larger than expected fragment in exon 7. SSCP analysis of exon 7 showed a mobility shift in the banding pattern (Fig. 1). Di-
Fig. 2. Illustration of part of the sequence of intron 6-exon 7 of WT1 from blood and tumour DNA of the patient, showing the 17 bp insertion in the tumour DNA. Insertion begins at * and is a direct repeat of the underlined sequence. Flanking direct repeats are signaled in boxes. Inverted repeats are limited by v. Capital letters, exon; lowercase letters, intron.
rect sequence analysis of this exon revealed a 17 bp insertion consisting of a tandem duplication of the sequence GTGTGCCTGGAGTAGCC, after the 27th nucleotide of exon 7 (Fig. 2). The insertion creates a one base pair frameshift resulting in the generation of a stop codon 12 nucleotides downstream (Fig. 2). SSCP and sequencing patterns of exon 7 showed the exclusive presence of the mutated allele (Figs. 1 and 3). Sequencing of exon 7 in lymphocyte DNA revealed only the wild type allele (Fig. 3). No transcript was detected in tumour cDNA ampli¢ed from exons 1^2, 2^6, 4^7, 4^9, 7^9, 8^10 and from exon 4^3P-UTR. However, mRNA was present, since the OCRL cDNA could be ampli¢ed from the same sample (not shown). Search for LOH in 11p15.5 and 16q indicated retention of heterozygosity in the tumour DNA. 4. Discussion
Fig. 1. PCR-SSCP analysis of WT1 exon 7 in tumour DNA from the patient (lane 1), compared with ¢ve other tumour samples (lanes 2^6). Lanes 2, 3, 5 and 6 reveal normal patterns. All bands in lane 1 are upshifted, suggesting an insertion.
In the present study, a somatic mutation consisting of a 17 bp duplication in exon 7 of WT1, generating a stop codon, was found in a sporadic unilateral Wilms' tumour. Only the mutant allele was detected in SSCP and DNA sequencing analysis. In situ hy-
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direct repeats present in the surrounding DNA [18]. In the tumour DNA, we observe small tetranucleotide £anking direct repeats (Fig. 2). An inverted repeat is also observed (Fig. 2), possibly leading to the formation of a hairpin loop. However, the free energy of 33.8 kcal/mole involved in the loop formation (DNAsis 5.00 version, 1987) seems rather weak, leaving a slipped mispairing mechanism as the most likely one to explain this insertion. In the present case, in which inactivation of both alleles of WT1 took place, retention of heterozygosity in known critical regions for Wilms' tumour, 11p15.5 and 16q13-24.3, was veri¢ed for the markers examined. Although this may suggest that inactivation of both copies of WT1 would be su¤cient for Wilms' tumorigenesis, additional search for LOH in critical regions in other Wilms' tumours with WT1 inactivation is needed to con¢rm this hypothesis. Fig. 3. Sequencing gel showing exon 7 nucleotide sequences from blood and tumour DNA of the patient. The 17 bp duplication is indicated by the arrows in the tumour DNA. Insertion begins at *.
bridization revealed the presence of both chromosomes 11. The presence of a constitutional or a somatic deletion encompassing exon 7, undetected by our methods on the basis of PCR analysis, cannot be excluded. The limited quantities of blood and tumour DNA precluded its analysis by Southern blotting. RNA analysis failed to reveal any transcripts from tumour cells. The failure to detect any abnormal transcripts in the tumour tissue may be due to mRNA instability. It is known that stop codons may cause not only early termination of translation and exon skipping [27], but also rapid mRNA decay [28]. The fact that a tandem insertion was detected in exon 7 of WT1 deserves further comment. To our knowledge, only four tandem insertional mutations have been described in Wilms' tumour and two of them in or near exon 7 [10,13]. On the other hand, tandem insertions in exon 7 have been reported as a preferential event among WT1 mutations in a series of patients with leukaemia [29].Therefore, exon 7 may be particularly prone to this type of mutation. Like in other genes [30,31], most insertions or deletions found in WT1 have been related to inverted or
Acknowledgements This work was supported by a grant from Junta Nacional de Investigac,a¬o Cient|¨¢ca e Tecnolo¨gica (JNICT 845/SAU/92). We would like to thank Dr. John K. Cowell, Department of Neurosciences, Research Institute, Cleveland Clinic Foundation, Cleveland, OH, for helpful discussion, to Dr. David Deszo for the gift of the OCRL (oculocerebrorenal) cDNA probe, to Jose¨ Manuel Furtado for tumour culture, and to Margarida Montoito for preparation of the manuscript.
References [1] K.M. Call, T. Glaser, C.Y. Ito, A.J. Buckler, J. Pelletier, D.A. Haber, E.A. Rose, A. Kral, H. Yeger, W.H. Lewis, C. Jones, D.E. Housman, Cell 60 (1990) 509^520. [2] M. Gessler, A. Poustka, W. Cavenee, R.L. Weve, S.H. Orkin, G. Bruns, Nature 343 (1990) 774^778. [3] E. Matsunaga, Hum. Genet. 57 (1981) 231^246. [4] K. Pritchard-Jones, S. Fleming, D. Davidson, W. Bickmore, D. Porteous, C. Gosden, J. Bard, A. Buckler, J. Pelletier, D. Housman, V. Van Heyningen, N. Hastie, Nature 346 (1990) 194^197. [5] Z.Y. Wang, Q.Q. Qui, T.F. Deuel, J. Biol. Chem. 268 (1993) 9172^9175. [6] N.D. Hastie, Annu. Rev. Genet. 28 (1994) 523^558. [7] M. Little, C. Wells, Hum. Mut. 9 (1997) 209^225.
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A.C. Santos et al. / Biochimica et Biophysica Acta 1407 (1998) 109^113 [8] A.G. Knudson, L.C. Strong, J. Natl. Cancer Inst. 48, (2) (1972) 313^324. [9] M. Little, K. Williamson, M. Mannens, A. Kelsey, C. Gosden, N. Hastie, V. van Heyningen, Hum. Mol. Genet. 2 (1993) 259^264. [10] P.N. Baird, N. Groves, D.A. Haber, D.E. Housman, J.K. Cowell, Oncogene 7 (1992) 2141^2149. [11] R. Varanasi, N. Bardeesy, M. Ghahremani, M.J. Petruzzi, N. Nowak, M.A. Adam, P. Grundy, T.B. Shows, J. Pelletier, Proc. Natl. Acad. Sci. USA 91 (1994) 3554^3558. [12] S. Park, A. Bernard, K.E. Bove, D.A. Sens, D.J. HazenMartin, A.J. Garvin, D.A. Haber, Nature Genet. 5 (1993) 363^367. [13] A.C. Santos, L. Oso¨rio-Almeida, P.N. Baird, J.M. Silva, M.G. Boavida, J.K. Cowell, Hum. Genet. 92 (1993) 83^86. [14] A.C. Santos, L. Oso¨rio-Almeida, Trends Genet. 9 (1993) 231. [15] Gross-Bellard, P. Oudet, P. Chambon, Eur. J. Biochem. 36 (1973) 32^38. [16] S.A. Miller, D.D. Dykes, H.F. Polesky, Nucleic Acids Res. 16 (1988) 1215. [17] A. Hogg, Z. Onadim, P.N. Baird, J.K. Cowell, Oncogene 7 (1992) 1445^1451. [18] V. Hu¡, N. Ja¡e, G.F. Saunders, L.C. Strong, F. Villalba, E.C. Ruteshouser, Am. J. Hum. Genet. 56 (1995) 84^90. [19] M.H. Little, J. Prosser, A. Condie, P.J. Smith, V. van Heyningen, N.D. Hastie, Proc. Natl. Acad. Sci. USA 89 (1992) 4791^4795.
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[20] O. Attree, I.M. Olivos, I. Okabe, L.C. Bailey, D.L. Nelson, R.A. Lewis, R. Mc Innes, L. Nussbaum, Nature 358 (1992) 239^242. [21] V. Hu¡, G.F. Saunders, Biochim. Biophys. Acta 1155 (1993) 295^306. [22] E. Austruy, S. Candon, I. Henry, G. Gyapay, M.F. Tournade, M. Mannens, D. Callen, C. Junien, C. Jeanpierre, Genes Chromosomes Cancer 14 (1995) 285^294. [23] S. Rainier, L.A. Johonson, C.J. Dobry, A.J. Ping, P.E. Grundy, A.P. Feinberg, Nature 362 (1993) 747^749. [24] A.E. Kulosik, J. Lyon, E. Kohne, H. Glizer, C.R. Bartram, E. Kleihauer, Br. J. Haematol. 70 (1988) 455^458. [25] G. Gyapay, J. Morisse, A. Vignal, C. Dig, Fizames, P. Millasseau, S. Marc, G. Bernardi, M. Lathrop, J. Weissenbach, Nat. Genet. 7 (1994) 246^249. [26] J.S. Waye, G.M. Greig, H.F. Willard, Hum. Genet. 77 (1987) 151^156. [27] H.C. Dietz, D. Valle, C.A. Francomano, R.J. Kendzioc, R.E. Pyeritz, G.R. Cutting, Science 259 (1993) 680^683. [28] L.E. Maquat, RNA 1 (1995) 453^465. [29] L. King-Underwood, J. Renshaw, K. Pritchard-Jones, Blood 87, (6) (1996) 2171^2179. [30] E.R. Fearon, K.R. Cho, J.M. Nigro, S.E. Kern, J.W. Simons, J.M. Ruppert, S.R. Hamilton, A.C. Preisinger, G. Thomas, K.W. Kinzler, B. Vogelstein, Science 247 (1990) 49^56. [31] M. Krawczak, N. Cooper, Hum. Genet. 86 (1991) 425^441.
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Identi¢cation of a new WT1 mutation in a sporadic Wilms' tumour Ana C. Santos a , Maria G. Boavida b , Ad|¨lia Costa c , Leonor Osorio-Almeida a
a;
*
Laborato¨rio de Gene¨tica Molecular, Faculdade de Cieªncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal b Departamento de Gene¨tica Humana, Instituto Nacional de Sau¨de Dr. Ricardo Jorge, 1699 Lisboa Codex, Portugal c Servic,o de Patologia, Hospital de Santa Maria, 1699 Lisboa Codex, Portugal Received 3 March 1998; revised 27 April 1998; accepted 29 April 1998
Abstract A new mutation in WT1 is described in a sporadic unilateral Wilms' tumour consisting of a 17 bp duplication in exon 7 generating a stop codon. The second allele is either partially deleted or presents the same alteration. LOH analysis at 11p15.5 and at the 16q13-16q24.3 regions indicated retention of heterozygosity in the tumour DNA for the markers analysed. The results are consistent with Knudson's hypothesis and confirm that loss of function of WT1 contributes to the development of at least some Wilms' tumours. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Wilms' tumor; WT1
1. Introduction WT1 is one of the genes involved in Wilms' tumour [1,2], a childhood malignancy of the kidney occurring in a frequency of 1 in 10 000 live births [3]. The gene is mainly expressed during the development of the kidney and in genitourinary structures [4]. It encodes a protein with four zinc ¢nger motifs which act both as a transcriptional suppressor and as an activator of growth related genes [5]. WT1 mutations in Wilms' tumours are a rare event, only detectable in 10^15% of the Wilms' tumours [6]. Of these, at least 69% [7] were shown to carry inactivation of the second allele, following the classic two-hit hypothesis [8]. Approx. 1^2% of Wilms' tumour patients have an associated AGR (Aniridia, Genitourinary anomalies and mental Re-
tardation) syndrome and demonstrate intragenic mutations in the remaining WT1 allele in the tumour [7]. In Wilms' tumours associated with Denys-Drash syndrome which account for less than 1% of all tumours, WT1 homozygosity/hemizygosity occurs in almost all cases [9]. Therefore, it can be concluded that complete inactivation of WT1 occurs in WT1 associated Wilms' tumours. Deletions and point mutations are among the most frequent inactivating mutations of WT1. Tandem insertions seem to be a more rare type of mutation and, to our knowledge, only reported in four cases [10^13]. Here, we describe a Wilms' tumour patient with a somatic mutation consisting of a tandem insertion of 17 base pairs in exon 7 generating a stop codon. This ¢nding provides further evidence for the involvement of complete inactivation of WT1 in the subset of Wilms' tumours.
* Corresponding author. Fax: +351 (1) 2948530. 0925-4439 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 9 8 ) 0 0 0 3 2 - 5
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2. Materials and methods 2.1. Patient AGM, a male child aged 5 years, with normal genitalia, developed a sporadic Wilms' tumour of the right kidney at the age of 5 years. Histological analysis of the surgically resected sample revealed a capsulated tumour without evidence of nuclear anaplasia, containing stroma and epithelial elements such as tubules, microcysts and glomerular bodies. No blastemal tissue was observed, probably due to pre-operative chemotherapy treatment (S. Fleming, pers. commun.). 2.2. Molecular analysis Simultaneous DNA and RNA extraction was carried out from fresh tumour or from para¤n embedded tissue, according to Santos and Oso¨rio Almeida [14]. DNA was obtained from peripheral blood lymphocytes of the patient and his parents, following described methods [15,16]. The search for WT1 mutations was carried out using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of exons 2^10 of tumour DNA as previously described [10,17]. Exon 1 was analysed as described by Hu¡ et al. [18]. Direct genomic sequencing of lymphocyte and tumour DNA was performed after ampli¢cation, using Dynabeads (Dynal, Merseyside, UK), essentially as described by Hogg et al. [17]. PCR covering exons 7^9 of WT1 was carried out in tumour DNA using the gene Amp XL PCR kit (Perkin Elmer) and primers described by Baird et al. [10]. The following conditions were used: 500 ng of DNA, 150 pmole of each primer, 1.6 mM Mg2 , 40 mM dNTP blend in a 50 Wl reaction volume. Ampli¢cation was for 35 cycles at 95³C for 1 min, 58³C for 1 min and 72³C for 5 min. Cycling was preceded by 7 min at 95³C, 1 min at 58³C and followed by 10 min at 72³C. The PCR products were analysed in 1% agarose gel electrophoresis. Tumour mRNA analysis was performed by RTPCR (Pharmacia bulk ¢rst strand cDNA kit) with primers for exons 1, 2, 6, 8 and 10 [10] and for
exon 4 (primers cD 5U4: 5P-GAC AAT TTA TAC CAA ATG ACA TCC CC-3P and cD 3U4: 5P-CCC TTT AAG GTG GCT CCT AAG TTC-3P), exon 7 (primers cD 5U7: 5P-GAT GTG CGA CGT GTG CCT GGA G-3P and cD 3U7: 5P-CAG TGT GCT TCC TGC TGT GCA A TC-3P), exon 9 (primer cD 3U9: 5P-CTG TAT GAG TCC TGG TGT GGG TC-3P) and 3P-UTR (primers localized 934 bp away from the gene, see [19]). Ampli¢cation of exons 1^2, 2^6, 6^8 and 8^10 was carried out according to Baird et al. [10]. Conditions for ampli¢cation of exons 4^7, 4^9 and 7^9 were the following: 1/30 of the ¢rst strand cDNA product were added to the PCR reaction mixture (10 mM Tris-HCl pH 8.8, 50 mM KCl, 1.5 mM Mg Cl2 , 0.1% Triton X-100, Taq DNA polymerase, Pharmacia), and subjected to 45 cycles of ampli¢cation 3 min at 95³C, 1.5 min at 62³C and 6 min at 72³C. Cycling was preceded by 7 min at 95³C, 2 min at 62³C and followed by 10 min at 72³C. All PCR products were analysed in 4% agarose gel electrophoresis. Ampli¢cation of exon 4^3P-UTR region was with the gene Amp XL PCR kit in the conditions described above, but with 1/10 of the ¢rst strand cDNA product. Electrophoresis was in 1% agarose gel. In parallel, PCR was performed on tumour cDNA for the OCRL (oculocerebrorenal) gene with primers which amplify several exons from the middle part of the gene, as a control for the presence of cDNA in the sample [20]. LOH screening was performed at genomic regions suggested to harbour additional loci implicated in Wilms' tumorigenesis, namely 11p15.5 and 16q1316q24.3 [21,22]. LOH analysis for 11p15 was performed with primers for IGF2 [23] and for GQ- and AQ-globin [24]. LOH analysis for 16q was performed by PCR using described primers for microsatellites [25]. These included D16S408, D16S514, D16S496, D16S512, D16S515, D16S516, D16S507, D16S402, D16S520, D16S413. PCR was performed in a reaction volume of 25 Wl including [K-32 P]dCTP. The following bu¡ers were used: 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl2 , 0.1% Triton X-100 for primers D16S514, D16S496, D16S512, D16S515, D16S516, D16S507, D16S511, D16S520, D16S413; 20 mM Tris-HCl, pH 8.55, 16 mM (NH4 )2 SO4 , 150 Wg/ml BSA, 2.5 mM MgCl2 for primer D16S408; 20 mM Tris-HCl, pH 8.5, 16 mM (NH4 )2 SO4 , 150 Wg/ml
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BSA, 1.0 mM MgCl2 for primer D16S518; 50 mM KCl, 10 mM Tris-HCl, pH 9, 0.1% Triton X-100, 2.5 mM MgCl2 for primer D16S402. The PCR products were analysed on a denaturing (7 M urea) 6% polyacrylamide gel. The gel was dried and autoradiographed at 380³C. 3. Results 3.1. Cytogenetic and FISH analysis Cytogenetic analysis of peripheral blood lymphocytes and of primary tumour cell cultures revealed a normal 46,XY karyotype. FISH analysis on cell suspensions prepared from para¤n embedded tumour sample with the alphoid probe (pL11A, see [26]), showed that both 11 chromosomes were present in the tumour cells. 3.2. Molecular analysis Individual PCR products of exons 2^10 of WT1 in the tumour DNA indicated a larger than expected fragment in exon 7. SSCP analysis of exon 7 showed a mobility shift in the banding pattern (Fig. 1). Di-
Fig. 2. Illustration of part of the sequence of intron 6-exon 7 of WT1 from blood and tumour DNA of the patient, showing the 17 bp insertion in the tumour DNA. Insertion begins at * and is a direct repeat of the underlined sequence. Flanking direct repeats are signaled in boxes. Inverted repeats are limited by v. Capital letters, exon; lowercase letters, intron.
rect sequence analysis of this exon revealed a 17 bp insertion consisting of a tandem duplication of the sequence GTGTGCCTGGAGTAGCC, after the 27th nucleotide of exon 7 (Fig. 2). The insertion creates a one base pair frameshift resulting in the generation of a stop codon 12 nucleotides downstream (Fig. 2). SSCP and sequencing patterns of exon 7 showed the exclusive presence of the mutated allele (Figs. 1 and 3). Sequencing of exon 7 in lymphocyte DNA revealed only the wild type allele (Fig. 3). No transcript was detected in tumour cDNA ampli¢ed from exons 1^2, 2^6, 4^7, 4^9, 7^9, 8^10 and from exon 4^3P-UTR. However, mRNA was present, since the OCRL cDNA could be ampli¢ed from the same sample (not shown). Search for LOH in 11p15.5 and 16q indicated retention of heterozygosity in the tumour DNA. 4. Discussion
Fig. 1. PCR-SSCP analysis of WT1 exon 7 in tumour DNA from the patient (lane 1), compared with ¢ve other tumour samples (lanes 2^6). Lanes 2, 3, 5 and 6 reveal normal patterns. All bands in lane 1 are upshifted, suggesting an insertion.
In the present study, a somatic mutation consisting of a 17 bp duplication in exon 7 of WT1, generating a stop codon, was found in a sporadic unilateral Wilms' tumour. Only the mutant allele was detected in SSCP and DNA sequencing analysis. In situ hy-
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direct repeats present in the surrounding DNA [18]. In the tumour DNA, we observe small tetranucleotide £anking direct repeats (Fig. 2). An inverted repeat is also observed (Fig. 2), possibly leading to the formation of a hairpin loop. However, the free energy of 33.8 kcal/mole involved in the loop formation (DNAsis 5.00 version, 1987) seems rather weak, leaving a slipped mispairing mechanism as the most likely one to explain this insertion. In the present case, in which inactivation of both alleles of WT1 took place, retention of heterozygosity in known critical regions for Wilms' tumour, 11p15.5 and 16q13-24.3, was veri¢ed for the markers examined. Although this may suggest that inactivation of both copies of WT1 would be su¤cient for Wilms' tumorigenesis, additional search for LOH in critical regions in other Wilms' tumours with WT1 inactivation is needed to con¢rm this hypothesis. Fig. 3. Sequencing gel showing exon 7 nucleotide sequences from blood and tumour DNA of the patient. The 17 bp duplication is indicated by the arrows in the tumour DNA. Insertion begins at *.
bridization revealed the presence of both chromosomes 11. The presence of a constitutional or a somatic deletion encompassing exon 7, undetected by our methods on the basis of PCR analysis, cannot be excluded. The limited quantities of blood and tumour DNA precluded its analysis by Southern blotting. RNA analysis failed to reveal any transcripts from tumour cells. The failure to detect any abnormal transcripts in the tumour tissue may be due to mRNA instability. It is known that stop codons may cause not only early termination of translation and exon skipping [27], but also rapid mRNA decay [28]. The fact that a tandem insertion was detected in exon 7 of WT1 deserves further comment. To our knowledge, only four tandem insertional mutations have been described in Wilms' tumour and two of them in or near exon 7 [10,13]. On the other hand, tandem insertions in exon 7 have been reported as a preferential event among WT1 mutations in a series of patients with leukaemia [29].Therefore, exon 7 may be particularly prone to this type of mutation. Like in other genes [30,31], most insertions or deletions found in WT1 have been related to inverted or
Acknowledgements This work was supported by a grant from Junta Nacional de Investigac,a¬o Cient|¨¢ca e Tecnolo¨gica (JNICT 845/SAU/92). We would like to thank Dr. John K. Cowell, Department of Neurosciences, Research Institute, Cleveland Clinic Foundation, Cleveland, OH, for helpful discussion, to Dr. David Deszo for the gift of the OCRL (oculocerebrorenal) cDNA probe, to Jose¨ Manuel Furtado for tumour culture, and to Margarida Montoito for preparation of the manuscript.
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