Mutation in Denys-Drash Syndrome WT1 Inherited

Inherited WT1 Mutation in Denys-Drash Syndrome Max J. Coppes, Gerrit Jan Liefers, Mariko Higuchi, et al. Cancer Res 1992;52:6125-6128. Published online November 1, 1992.

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Advances in Brief

Inherited WTJ Mutation in Denys-Drash Syndrome' 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 Patients with the Denys-Drash syndrome (Wilms' tumor, genital anomalies, and nephropathy) have been demonstrated to carry de now constitutional

mutations

We suggest that parents of individuals with the DDS be evalu ated for the presence of the WTJ mutation. Materials and Methods

in WTJ, the Wilms' tumor gene at chromosome

I 1p13. We report three new cases, two carrying a previously described

WTI exon 9 mutation and one with a novel WTJ exon 8 mutation. However, unlike patients in previous reports, one of our three patients inherited the affected allele from his phenotypically unaffected father. This observation indicates that the WTI exon 9 mutation affecting 394Argdemonstrated in over one-half of the patients with the Denys Drash syndrome may exhibit incomplete penetrance. Consequently, fa milial studies in patients affected by this syndrome are recommended.

Patients. The clinical and cytogenetic features of three patients with DDS are summarized in Table 1. D7, the father of patient D5, is at present a healthy 42-year-old male. Physical examination did not reveal any abnormalities; in particular he has bilaterally descended testes of normal

malformations,

and mental

retardation

(the WAGR syndrome) demonstrated chromosome 1lpl 3 germ-line deletions, providing the first indication that this chromosome segment is involved in Wilms' tumorigenesis (1). Molecular analysis of the homozygously deleted region chro

@

In

to his son, patient

and several family members

of patient

D5 were

isolated from peripheral leukocytes as described previously (11). Isola

genitourinary

1 lpl3

penis; he does not have hypospadias.

Genomic DNA Preparation. Constitutional DNA from all three pa tients and their parents

Wilms' tumor, or nephroblastoma, is a renal malignancy that affects approximately 1 in 10,000 children under 16 years of age. Cytogenetic observations in patients with Wilms' tumor,

mosome

and a normal

D5.

Introduction

aniridia,

volume

1985, he donated his left kidney for transplantation

in two Wilms'

tumors

tion of the constitutional

DNAs of patient

D5 and his father (D7) were

repeated following the initial results in order to confirm the analyses. PCR Amplification. All @VT1 exonic sequences from genomic DNA

were amplified by PCR, and the PCR products were analyzed for SSCP (I 2) in order to detect possible deletions, insertions, or point mutations. In the analysis of WTJ exon 8 we used primers (A-2)8 and (S-2)8, and in the analysis

of

@VT1exon 9 primers

(A-2)9 and (S-2)9 which have

been described previously (13). PCR was carried out in a GeneAmp

led, in 1990, to the

independent identification of J'Vfl, a Wilms' tumor suppressor gene, by three groups (2â€”4).Occasionally, the presence of Wilms' tumor in a patient is associated with genital anomalies and nephropathy: the Denys-Drash syndrome (5, 6). The din ical overlap between this syndrome and the WAGR syndrome suggested that the could also arise as a consequence of a deletion of one or more genes localized at chromosome I I p13

PCR system 9600 (Perkin-Elmer) with a final volume of 100 @l, con taming 250 mmol/liter each ofdATP, dGTP, and dTTP; 20 mmol/liter ofdCTP; I mCi I32PIdCTP;200â€”800 ng ofDNA; 50 pmol ofeach PCR primer,

2.5 units

of Taq polymerase;

and

10 MI of Taq polymerase

reaction buffer. SSCP. Following PCR a 5-al aliquot of the amplified product was diluted

with 40 Ml of 0.1% sodium

dodecyl

sulfate-lO

m@i EDTA;

2 @il

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 (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 wassuggestedthat the

Tris base-0.09

DDS follows a dominant

room temperature for 4â€”6 h, at 30 W at 4C for 4â€”6 h, and at 5 W at room temperature for 16 h. The gels were dried and exposed to XAR-5 film (Kodak) for 16â€”96h without an intensifying screen at room temperature.

mode of inheritance with high pene

trance and that it is extremely unlikely that parents of individ uals with the DDS carry the mutations found in the affected children (8). Subsequent analysis of four additional families seemed to confirm this hypothesis (10). We report here the presence of the same 14'Ti exon 9 mutation in a phenotypically abnormal male and his phenotypically unaffected father. This finding demonstrates that U'Tl mutations causing the DDS can indeed be inherited and need not be expressed phenotypically.

M boric acid-2.5

msi EDTA

running

buffer at 30 W at

Sequencing. The mechanisms used for direct sequencing of bio tinylated PCR products have recently been described (10, 14). Briefly,

PCR amplification with one primer biotinylated at the 5' end and one nonbiotinylated primer was performed when the PCR product was

to be used for sequencing. The final volume remained 100 pl, I mCi [32PIdCTPwas omitted, the concentration of the nucleotides became 250 mmol/liter for each deoxynucleotide triphosphate, and the amount

Received9/17/82; accepted9/28/92. The costs ofpublication of this article weredefrayedin part by the paymentof page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This

work

was supported

by the Dc Bartolo

Funds(M.

requests

for reprints

should

be addressed,

utilizing

J. C., B. R. G. W.) and at Department

of Cancer

Biologj@ NN1-06, The ClevelandClinic Foundation, 9500 Euclid Avenue,Cleve land, OH 44195. 3 The

abbreviations

used

are:

DDS,

Denys-Drash

syndrome;

PCR,

aliquot

of the biotinylated

PCR product

on streptavidin-coated

mag

netic Dynabeads (Dynal, Merseyside, United Kingdom) was performed

the Ohio Department of Health (A. B. Z.). 2 To whom

of primer used was decreased to 5â€”15pmol. Adsorption of a 5O-pl a magnetic

particle

concentrator

(Dynal)

as recommended

by

the manu-facturer. Following denaturation in 100 p1of 0.15 MNaOH, biotinylated and nonbiotinylated strands were separated on the mag netic particle

concentrator

and both strands

were sequenced

using a

Sequenase (United States Biochemical) kit according to the instructions

polymerase

of the manufacturer.

chain reaction; SSCP, single strand conformational polymorphisms. 6125


INHERITED WI! MUTATIONS IN DDS syndromePresentExternalPatientage Table IClinical and cytogeneticfindingsfor mutationDl7FemaleNormal (yr)genitaliaInternal bilateral+394Arg-TrpD513AmbiguousNormal

threepatients withDenys-Drash

tumorNephropathyWJ'I

reproductive organsKaryotypeWilms'

ovaries Normal uterus46,XX+, testes

structures46,XY394Arg-TrpDeceasedFemaleStreak Residual mtillerian gonads46,XY+377His-Tyr

Dl0 a Kidneys I, Kidneys

removed removed

at age 18 months. at age 19 months.

sequence of the protein domains that are critical for DNA se quence recognition by zinc finger proteins. The JVfJ exon 9

Results PCR/SSCP

analysis used to search for deletions,

insertions,

or base pair mutations within the @YI'1exons demonstrated (Fig. 1) a mobility shift in exon 9 (encoding zinc finger 3) of one allele from patients Dl and D5, and in exon 8 (encoding zinc finger 2) of one allele from patient DIO. Constitutional DNA from the parents of Dl (D2 and D3) and D10 (D8 and D9) demonstrated a normal (homozygous) SSCP pattern (Fig. 1). A homozygous SSCP pattern was also found for the mother (D6), three paternal aunts (51D7, S2D7, S3D7), and brother (BD5) of D5, but the father (D7) of D5 showed a heterozygous SSCP pattern similar to the one demonstrated for his son (Fig. 1). The heterozygous SSCP pattern found in the father was confirmed on a second blood sample taken 3 months after the first one. In addition, very numerous tandem repeat studies were performed on samples from D5, D6, and D7 and were found to be consis tent with the presumed paternity (data not shown). DNA of a (the only) paternal uncle of D5 could not be obtained. Sequence analysis of constitutional DNA revealed a I I29@to T transition converting amino acid residue 377His to Tyr in patient DlO (Fig. 2A), and a â€˜ â€˜80C to T transition converting amino acid residue 394Arg to Trp in patients Dl and D5 (Fig. 2B). Sequence analysis ofD7 revealed the same â€˜ â€˜80C toT transition found in patient D5 (Fig. 2B). The described muta tions were confirmed by sequence analysis of at least five addi tional samples from any given specimen.

mutations

found in patients

tions in patients

We report three patients with the DDS who were found to have constitutional JYFJ mutations which alter the amino acid

with the DDS, as well as the described

WTJ

exon 9 mutation in a phenotypically normal father of a child with this syndrome, raises the question of the origin of the mutations. A recessive mode of inheritance, resulting from the inheritance

Discussion

Dl and D5 affect amino acid res

idue 394Arg, which has been implicated in hydrogen bonding interactions with a guanidine residue in the cognate DNA recognition sequence (15). This mutation has now been re ported in 14 of 25 (@55%) individuals with the DDS. The WTJ exon 8 mutation found in patient D1O affects amino acid resi due 377His and disrupts one of the four zinc binding ligands of zinc finger 2; this mutation has not been described previously. In addition to these two @Vf1missense mutations, an exon 9 missense mutation affecting 396Asp has been reported previ ously in three patients, an exon 8 missense mutation affecting 366Mg in two patient, an exon 7 missense mutation affecting 330Cys in one patient, an insertion ofG at position 821 in exon 6 resulting in the immediate generation of a stop codon in one patient, and finally, an intron 9 mutation, which prevents splic ing at one of the alternative splice donor sites of exon 9, in another patient (8â€”10).The JVI'l zinc finger region, encoded by exons 7â€”10,and especially exon 9, seems to be a â€œhot spotâ€•for constitutional mutations causing the DDS. The identification of constitutional WTJ zinc finger muta

of a mutant

JVFJ

allele

from

each

parent,

seems

unlikely given the fact that in the 23 patients reported with a constitutional WTJ mutation, only one abnormal allele has 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 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 to the suggestion that it would be extremely unlikely for a parent of a child with the DDS to carry a nonpenetrant muta tion (8). This hypothesis has been supported by the analysis of

:p:@l @is..

N

i.-.

.â€”@

EXONS

,aâ€”1ul@

constitutional

EXON9

Fig. I. PCR/SSCP analysis of: W1'l exon 8 in patient D10, her mother (D8),

her father (D9), and an unaffectedcontrol (C); and of 1+71exon 9 in patient Dl, her mother (D2), her father (D3), an unaffected control, and patient D5, his mother(D6),

father(D7), brother(BD5),

paternal grandparents(MD7

and FD7),

DNA from both parents

of six of the previously

reported cases (8, 10). None of the parents carried the mutant allele of their affected child. However, our results demonstrate that a phenotypically unaffected parent can have and transmit a

and three paternal aunts (SID7, S2D7, and S3D7). The PCR/SSCP analysiswas

J3PfJ

performed as described in â€œMaterialsand Methods;â€•the primer sets used for PCR

The presence of the WTJ exon 9 missense germline mutation in an unaffected parent suggests either that W7'I mutations exhibit incomplete penetrance [in contrast to what was previ ously assumed (8)], possibly related to somatic mosaicism (16) or genomic imprinting (17). Insofar as the latter possibility is

ofexon 8 [(A-2)8and (S-2)81,and exon 9 ((A-2)9and (A-2)91havebeenpublished previously (13). The WJ'l exon 8 and 9 PCR products ofthe unaffected controls (C) demonstrate the wild type mobility (homozygous) pattern for both exons. The @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, S3D7, FD7, MD7, D6, and BD5 show a normal SSCP mobility pattern, whereas

the D5 and D7 products show a heterozygous pattern. Nondenatured samples were used to delineate the position of migration of the double stranded DNA products for both exons (data not shown).

exon

concerned,

9 mutation

to his

affected

the recent demonstration

son.

of equivalent

expression

of paternally and maternally inherited WTJ alleles in Wilms' 6126


INHERITED U@I@1 MUTATIONS IN DDS

A

377 His

EXON S

3' TGGACATA@4@JAGA 5' 377Tyr

DlO

Wild Type

3'

3, 1 G G

__________________

I G

A

C

C

A

A

C IA

1 A

A

A

C G

C

A A

A 5,

5,

B

3, C

EXON9

C

WildType A

C

G

T

[email protected]

T

394

G G

Arg

3' CCTGG9@1CCTCTT 5'

C

C C

394 Trp

I

C I I

5, D7

Dl 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 WTJ exon 9 (codingzinc finger 3) in patients Dl, D5, and D7 (the father of patient D5). Sequencingwas performedas descn'bedin â€œMaterials and Methods.â€• The nucleotidenumberingsystemis basedon the codingsequenceonly and considers

the A of the ATG initiatoras +1. The numberingsystemfor the aminoacidsconsidersthe initiatormethionineas +1. (A) @YI1 exon 8 sequenceanalysisof

@

constitutional DNA of patient D1O shows a â€˜ 129C to 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).

6127


INHERITED WI! MUTATIONS IN DDS

tumors and normal tissues heterozygous for the WTJ gene sug gests that J@Vf1is not subject to transcriptional imprinting (18). We therefore believe that another cause of incomplete pene trance is the more likely explanation for our findings. Unfor tunately, we have been unable to assess the possibility of so matic mosaicism in this individual; sperm analysis is not feasible because D7 has had a vasectomy performed and has thus far not consented to providing us with a skin biopsy for fibroblast culture and analysis. This report demonstrates that JJ'Tl mutations in patients with DDS may be inherited from a phenotypically normal par ent, the carrier of the mutation. Accordingly, familial studies of affected patients are indicated. It is expected that such studies will improve the genetic counseling provided to individuals af fected by this syndrome and their families and enhance our understanding ofthe role of @VF1 in genitourinary development and Wilms' tumorigenesis. Acknowledgments We thank Dr. John Cowell, Institute of Child Health, London, United Kingdom, for his advise on using biotinylated primers for direct sequencing

of PCR products.

W. Associationd'un syndromeanatomo-pathologiquede pseudohermaphro disme masculin, d'une tumeur de Wilms, d'une nÃ©phropathieparenchyma

teuse et d'un mosaicismXX/XY. Arch. Fr. Pediatr., 24: 729â€”739, 1967. 6. Drash, A., Sherman, F., Hartmann, W., and Blizzard,R. M. A. A syndrome of pseudohermaphroditism,

Wilms'

tumor, hypertension,

and degenerative

renal disease. J. Pediatr., 76: 585â€”593,1970. 7. Jadresic, L., Leake, J., Gordon, I., Dillon, M. J., Grant, D. B., Pritchard, J., Risdon, R. A., and Barratt, T. M. Clinicopathologic review oftwelve children

with nephropathy, Wilms tumor, and genital abnormalities (Drash syn drome). J. Pediatr., 117: 717â€”725,1990. 8. Pelletier, J., Bruening, W., Kashtan, C. E., Mauer, S. M., Manivel, J. C.,

Striegel,J. E., Houghton, D., Junien, C., Habib, R., Fouser, L., Fine, R. N., Silverman, B. L., Haber, D. A., and Housman, D. Germline mutations in the

Wilms' tumor suppressor gene are associatedwith abnormal urogenital de velopment in Denys-Drash syndrome. Cell, 67: 437â€”447,1991.

9. Bruening,W., Bardeesy,N., Silverman,B. L, Cohn, R. A., Machin, G. A., Aronson, A. J., Housman, D., and Pelletier, J. Germline intronic and exonic mutations in the Wilms' tumour gene (WJ'l) affecting urogenital develop

ment. Nature Genet., 1: 144â€”148, 1992. 10. Baird, P. N., Santos, A., Groves, N., Jadresic, L., and Cowell, J. K. Consti tutional mutations in the WT1 gene in patients with Denys-Drash syndrome.

Hum. Mol. Genet., 1: 301â€”305, 1992. 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. G. Lossofheterozygositymappingin Wilmstumor indicatesthe involvement of three distinct regions and a limited role for non-disjunction or mitotic recombination. Genes Chromosomes Cancer, in press, 1992. 12. Orita, M., Suzuki, Y., Sekiya, I., and Hayashi, K. Rapid and sensitive de tection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5: 874â€”879,1989. 13. Haber, D. A., Sohn, R. L., Buckler, A. J., Pelletier, J., Call, K. M., and

Housman, D. E. Alternative splicing and genomic structure of the Wilma tumor gene WTJ. Proc. NatI. Aced. Sci. USA, 88: 9618â€”9622, 1991.

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