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The Wilms tumor suppressor gene 1 (WT1) encodes a transcription factor playing a major role during early embryonal development of the kidneys and the urogenital system in mammals. It is highly expressed in the early developing kidney and the gonads (1,2). WT1 is located on chromosome 11p13, consists of 10 exons, covers 50 kb and belongs to the zinc finger family of transcription factors (3,4). It was detected by positional cloning from the genomic DNA of patients with WAGR syndrome (Wilms tumor, Aniridia, Genitourinary malformations, mental Retardation) carrying deletions of the 11p13 region. Since then, apart from deletions of the WT1 gene in WAGR syndrome and tumor-specific alterations in Wilms tumors, germline mutations of the WT1 gene were detected causing early Wilms tumors in patients with Denys Drash syndrome (DDS), another syndrome with inborn urogenital malformations consisting of male pseudohermaphroditism, nephropathy and Wilms tumor (5,6). Until now almost 150 cases of DDS have been described, and mutations of the WT1 gene have been detected in 48 to date genetically analyzed cases (7–9). In three analyzed cases no mutations in the coding sequence of the WT1 gene were found (10,11).

Additionally, germline mutations of WT1 were found so far only in six children, with severe urogenital malformations and no other signs of DDS or WAGR syndrome, who developed Wilms tumor at an early age (12–15). This study reports the diagnosis in a boy with severe hypospadias and bilateral cryptorchidism, who was born 1 year before cloning of the WT1 gene and developed a bilateral large Wilms tumor at the age of 15 months, which required extensive surgery and chemotherapy for treatment.

THE PATIENT

The patient was born in 1989 as the second child after an uneventful pregnancy (1 y before the cloning of the WT1 gene). He presented at birth with severe hypospadias and bilateral cryptorchidism (Fig. 1). The karyotype was normal male (46, XY). Penis size was normal, the scrotum was normally rugated and no hyperpigmentation was visible. Defects of testosterone synthesis, androgen-receptor defects and congenital adrenal hyperplasia were ruled out by biochemical and molecular genetic studies. Sonography of the kidneys revealed a duplication of the left kidney and left ureter (Fig. 2). A mild vesicoureteral reflux was treated with low-dose antibiotics.

Figure 1
figure 1

External genitalia of the patient.

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Figure 2
figure 2

Internal genitalia of the patient.

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At the age of 15 months during a routine control by the general pediatrician, abdominal extension was observed and a giant abdominal mass (18 cm × 18 cm) reaching from the upper left to the middle abdomen was palpated. Subsequently bilateral Wilms tumor of stromal-predominant histology was diagnosed. For treatment three courses of chemotherapy (1. Actinomycin, Vincristin, 2. Cyclophosphamide, Adriamycin, Vincristin, Actinomycin, 3. VP 16, Carboplatin, Ifosfamid, Adriamycin, Vincristin) and left-sided nephrectomy was needed.

During surgery a thorough examination of the internal genitalia revealed both testes being attached to the frontal lower poles of the kidneys, and rudiments of a uterus and vagina were seen. The left testis was removed and the right testis was brought and fixed in the scrotum.

At the age of 6 and 7 y, surgical corrections of the urogenital malformation were performed. The rudimentary vagina was excised and the right endocrine nonfunctional testis, as proven by negative HCG test, was removed. Until now (7 y after therapy), no relapse of Wilms tumor and no further signs of DDS or WAGR syndrome were detectable.

METHODS AND RESULTS

Analysis of the WT1 gene was performed as previously described (12). The patient's genomic DNA was isolated from peripheral leukocytes, and subsequently PCR-SSCP analysis of all 10 exons of the WT1 gene was performed. An aberrant banding pattern was observed in exon 8. Sequencing revealed a C to T transition in one allele at position 1084 resulting in a stop codon (362Arg to Stop). During translation this mutation leads to a truncated protein missing the last three zink fingers which are necessary for DNA binding (Fig. 3) (16).

Figure 3
figure 3

(A) Sequence analysis of the patient and a healthy control showing the heterozygous point mutation (1084 C->T) of the patient. (B) WT1 gene and protein structure including the mutation of the patient, which is leading to a truncated protein (25).

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DISCUSSION

WAGR syndrome and DDS are rare syndromes with association of urogenital malformations and Wilms tumor due to deletions and mutations of the WT1 gene. WAGR syndrome has been observed in 2% of Wilms tumor cases. Hypospadias and cryptorchidism were reported in up to 4.4% in children with Wilms tumor (17–19). In a recent study in 4% of patients with urogenital anomalies and Wilms tumor, WT1 mutations were detected (20). The boy described in this study so far is one of the very rare published cases with severe urogenital malformations and early developing Wilms tumor due to a mutation in the WT1 gene without signs of DDS or WAGR syndrome.

All patients with germline mutations of the WT1 gene are only heterozygous for the mutated allele. Mice, homozygously deleted for WT1, fail to develop kidneys or gonads, verifying a critical role for WT1 in urogenital development (21). There are three major types of mutations: 1. missense mutations altering amino acids that directly interact with the DNA target, 2. substitutions of amino acids involved in zinc complexing, and 3. nonsense mutations leading to the removal of at least two zinc fingers. As shown in transfection assays, WT1 mutations can act in a dominant negative fashion with loss of normal DNA binding of WT1 through loss or conformational change of the DNA binding zinc finger region and thereby decreased transcriptional suppression activity (16,22). It has been suggested that in most cases with a heterozygous WT1 mutation, a second hit leading to loss of heterozygosity seems to be important for tumor genesis, because almost all Wilms tumors in those patients are homozygous for the mutated allele (12,23,24).

From this study and our current knowledge, we have to conclude that molecular genetic studies of the WT1 gene are mandatory in the work up of newborns with severe urogenital anomalies. As children with severe urogenital malformations due to a heterozygous mutation in the WT1 gene are at high risk for the development of a Wilms tumor at an early age, efforts should be taken to identify those children immediately after birth to follow them closely. In children with severe hypospadias and cryptorchidism after exclusion of other chromosomal, endocrine, or androgen-receptor-mediated disorders, either ultrasound controls of the kidneys in brief intervals (4 weekly) or molecular analysis of the WT1 gene must be performed.

As mutational screening of the WT1 gene by means of PCR-SSCP analysis or direct sequencing have become easy tools of investigation, we favor these methods to identify children being at high risk for development of a Wilms tumor. This will result in an earlier diagnosis and treatment of this malignant disease. Moreover in the future frequent ultrasound controls of the kidneys probably can be limited to individuals with mutations of the WT1 gene, in the remaining patients without WT1 mutations ultrasound monitoring may be reduced to longer (e.g. 3-6 months) intervals. This approach has the advantage of a better cost effectiveness by reduction of the otherwise necessary frequent control visits in a large number of patients.