J Med Genet 1993 30: 767-772 767 Mutational screening of the Wilms's tumour J Med Genet: first published as 10.1136/jmg.30.9.767 on 1 September 1993. Downloaded from gene, WT1, in males with genital abnormalities Paul A Clarkson, Helen R Davies, Denise M Williams, Rakesh Chaudhary, Ieuan A Hughes, Mark N Patterson Abstract gene on chromosome 11 band p 3.34 Wilms's Several lines of evidence suggest that tumour is a childhood malignancy of the kid- the Wilms's tumour susceptibility gene, ney, and in several tumours intragenic muta- WT1, has an important role in genital as tions of the WT1 gene have been found, thus well as kidney development. WT1 is confirming that WT1 is one of the genes expressed in developing kidney and geni- involved in the development of Wilms's tu- tal tissues. Furthermore, mutations in mour."7 The WT1 gene comprises 10 exons WT1 have been detected in patients which encode a protein with amino acid se- with the Denys-Drash syndrome (DDS), quence motifs characteristic of a transcription which is characterised by nephropathy, factor.34 The four 3' exons each encode zinc genital abnormalities, and Wilms's tu- finger structures involved in DNA binding, mour. It is possible that WT1 mutations and the WT1 protein has been shown to bind may cause genital abnormalities in the specifically to the DNA sequence recognised absence of kidney dysfunction. We tested by the early growth response gene, EGRI.8 this hypothesis by screening the WT1 Furthermore, in transient transfection assays gene for mutation in 12 46,XY patients WT1 functions as a transcriptional repressor with various forms of genital abnormal- of genes containing the EGRI target se- ity. Using single strand conformation quence.9 A subset ofWilms's tumours is there- polymorphism (SSCP) we did not detect fore thought to arise from loss of function of any WT1 mutations in these patients. WT1, leading to the deregulated expression of However, in addition to the 12 patients, growth promoting genes and the uncontrolled three DDS patients were also analysed growth of embryonic kidney cells. using SSCP, and in all three cases hetero- The evidence that implicates WT 1 in genital zygous WT1 mutations were found which as well as kidney development comes from would be predicted to disrupt the DNA expression studies of WT1 and from genetic binding activity of WT1 protein. These data.'0 WT1 is expressed in fetal kidney, geni- results support the notion that DDS re- tal ridge, and fetal gonad." 12 The precise cell sults from a dominant WT1 mutation. specific pattern of expression has led to the http://jmg.bmj.com/ However, WT1 mutations are unlikely suggestion that WT1 is involved in mesenchy- to be a common cause of male genital mal-epithelial transitions in embryonic kidney abnormalities when these are not and genital structures." More recently, WT1 associated with kidney abnormalities. mutations have been identified in subjects with (J Med Genet 1993;30:767-72) Denys-Drash syndrome (DDS)."'>6 This rare condition is characterised by a specific nephro- Abnormalities of the genital system resulting pathy, often accompanied by gonadal/genital on October 5, 2021 by guest. Protected copyright. in an intersex phenotype are known to arise abnormalities or Wilms's tumour or both.'7 through a number of genetic mechanisms.' The syndrome affects both males and females. When the affected subject has a normal male The genital phenotype is very variable but karyotype (46,XY) the condition is described most often presents as male pseudohermaph- as male pseudohermaphroditism. Clinical mal- roditism, with micropenis, cryptorchidism, or formations include micropenis (or clitorome- perineal hypospadias. True hermaphroditism galy), cryptorchidism (undescended testes), has also been reported in DDS.'8 In almost all and hypospadias (a misplaced urethral open- cases studied, DDS subjects carry a heterozy- ing). Such phenotypes can arise from andro- gous mutation in WT1 which is predicted to Department of Paediatrics, gen biosynthetic defects, for example, 17f- affect the ability of the WT1 protein to bind University of hydroxysteroid dehydrogenase and 5a-reduc- DNA. Constitutional mutations in WT 1 were Cambridge, tase deficiency. Alternatively, there may be a also reported in two subjects with Wilms's Addenbrooke's defect in the response to as Hospital, Hills Road, hormonal signals, is tumour, hypospadias, and cryptorchidism, but Cambridge CB2 2QQ, seen in the androgen insensitivity syndrome, without nephropathy.'9 These observations UK. caused by mutations in the androgen receptor suggest that the genital abnormality in the P A Clarkson gene.2 In many cases neither hormonal syn- H R Davies WAGR syndrome (Wilms's tumour, aniridia, D M Williams thesis nor the response appears defective, sug- genitourinary abnormalities, mental retard- R Chaudhary gesting that a mutation in a gene which func- ation), which is associated with constitutional I A Hughes tions in another aspect of genital development deletion of 13 M N Patterson IIp is caused by deletion of the may be the cause of the phenotype. WT1 gene itself. Constitutional WT1 muta- Correspondence to A recent addition to the genes currently tions therefore appear to act as dominant mu- Dr Patterson. known to be involved in genital development is tations and are associated with nephropathy or Received 29 January 1993. Revised version accepted WT1, which was originally isolated as a candi- Wilms's tumour or both, in the presence or 30 April 1993. date for the Wilms's tumour predisposition absence of genital abnormalities. 768 Clarkson, Davies, Williams, Chaudhary, Hughes, Patterson The purpose of this study was to determine DENYS-DRASH SYNDROME PATIENTS whether WT1 mutations cause genital abnor- Three DDS patients were included in this malities in the absence of nephropathy or analysis, since mutations in WTI were J Med Genet: first published as 10.1136/jmg.30.9.767 on 1 September 1993. Downloaded from Wilms's tumour. As part of a previous study, expected. Subject S12 was born with perineal the androgen receptor gene was screened for hypospadias and cryptorchidism, suggestive of mutation in a group of 46,XY patients with an undervirilised male. The karyotype was various genital abnormalities compatible with 46,XY and ultrasound showed normal male a diagnosis of androgen insensitivity syn- internal genital anatomy and no Mullerian drome.20 In the majority of patients with phe- structures. There was a normal response to a notypes such as cryptorchidism, micropenis, three day hCG stimulation test. The initial and hypospadias, mutations in the androgen diagnosis in this patient was partial androgen receptor gene were not found. It is therefore insensitivity syndrome (PAIS). At 9 months of possible that other genetic defects account for age, however, S 12 presented with Wilms's the phenotypes in these patients. Given the tumour and diffuse mesangial sclerosis, typical association between WT1 mutations and the of DDS. This patient was investigated in an genital abnormalities in DDS, we screened independent study.23 Patient R6 (also 46,XY) WT 1 for mutation in a cohort of 46,XY was born with the appearance of virilised patients with various genital abnormalities, female external genitalia (clitoromegaly, in- but without any kidney dysfunction. complete fusion of the labia, and rugose labio- scrotal folds). Gonadal histology showed nor- mal infantile testes, and there was a normal Patients testosterone response to hCG. Pelvic ultra- PATIENTS WITH GENITAL ABNORMALITIES sound showed no Mullerian structures and a The phenotypes of the patients studied are diagnosis of PAIS was again made. At 5 summarised in table 1. Each had a normal months of age she presented in established 46,XY karyotype. All non-DDS patients were renal failure. A renal biopsy showed diffuse also analysed for androgen binding activity, mesangial sclerosis, and a diagnosis of DDS and qualitatively normal binding was seen. was made. While awaiting renal transplantation, Two patients (P1 and P4) had low levels of she developed peritonitis and overwhelming binding, but since this phenomenon has been sepsis as a complication of her dialysis and died observed in Denys-Drash syndrome, these at 61 months of age. Patient CAM114 (46,XY) patients were included.2" The androgen recep- was born with normal external female genitalia tor gene had also been screened for mutation and presented at 6 months of age with nephro- by PCR-SSCP in some of the patients, and in tic syndrome. Histological examination of a each case no evidence for mutation was found. renal biopsy showed diffuse mesangial sclero- The majority of the patients presented with sis and a diagnosis of DD S was made.24 severe undervirilisation (either predominantly CAMi 14 was found at laparotomy to have male with perineal hypospadias, crypt- internal female genital structures. This patient was also investigated in an independent study orchidism, and micropenis, or predominantly http://jmg.bmj.com/ female with clitoromegaly). The majority of (corresponding to patient LB).'5 patients also had apparently normal testes by histology or the hCG stimulation test which provides a measure of the ability of the testes Materials and methods to synthesise androgen in response to gonado- ANDROGEN BINDING ASSAY trophin.22 We also included one patient with Fibroblast cell lines were established for all true hermaphroditism (F14). This patient also patients from genital skin biopsies, obtained at presented with other congenital abnormalities,