Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer.

Many mammalian genes contain overlapping antisense RNAs, but the functions and mechanisms of action of these transcripts are mostly unknown. WT1 is a well-characterized developmental gene that is mutated in Wilms' tumor (WT) and acute myeloid leukaemia (AML) and has an antisense transcript (WT1-AS), which we have previously found to regulate WT1 protein levels. In this study, we show that WT1-AS is present in multiple spliceoforms that are usually expressed in parallel with WT1 RNA in human and mouse tissues. We demonstrate that the expression of WT1-AS correlates with methylation of the antisense regulatory region (ARR) in WT1 intron 1, displaying imprinted monoallelic expression in normal kidney and loss of imprinting in WT. However, we find no evidence for imprinting of mouse Wt1-as. WT1-AS transcripts are exported into the cytoplasm and form heteroduplexes with WT1 mRNA in the overlapping region in WT1 exon 1. In AML, there is often abnormal splicing of WT1-AS, which may play a role in the development of this malignancy. These results show that WT1 encodes conserved antisense RNAs that may have an important regulatory role in WT1 expression via RNA:RNA interactions, and which can become deregulated by a variety of mechanisms in cancer.

Wt1 is not essential for hematopoiesis in the mouse.

WT1 has been implicated in human leukemia and hematopoiesis, but its role in stem cell differentiation is not yet fully defined. We show that Wt1-null murine fetal liver cells are capable of reconstituting functional hematopoiesis following transplantation into irradiated recipients. There was also no significant difference between the in vitro colony-forming ability of wild-type and Wt1-null cells. Using a reporter gene assay in a transgenic mouse system, expression from the WT1 promoter was detectable in adult bone marrow, but undetectable in subsets of different hematopoietic cells. We conclude that Wt1 is not essential for murine hematopoiesis and that there may be significant differences in its role between mouse and man.

A WT1 exon 1 mutation in a child diagnosed with Denys-Drash syndrome.

Denys-Drash syndrome (DDS) is characterized by nephropathy, genital abnormalities, and predisposition to Wilms tumor. DDS is associated with constitutional WT1 mutations, the majority being missense mutations in the zinc-finger region. A dominant-negative mode of action of the mutant DDS proteins is thought to explain the more severe genitourinary phenotype seen in DDS compared with children with complete deletion of one WT1 allele. We present a phenotypically female child who presented with bilateral Wilms tumor at 8 months of age. She was found to have an XY karyotype and diagnosed with DDS. In the constitutional DNA of this child we found a previously unreported mutation in exon 1 of WT1. This de novo mutation, delT in codon 40, is predicted to produce a termination signal in codon 90 (F40fsX90). This frameshift mutation results in a severely truncated protein, which would remove both the zinc-finger DNA-binding domain and the majority of the N-terminal regulatory domain, including regions previously shown in vitro to be necessary for inhibition of WT1 transcriptional activity. Our results provide important physiological evidence that the first 40 amino acids of WT1 are capable of functionally important interactions, presumably through their ability to self-associate with full-length WT1.

Frequency and heritability of WT1 mutations in nonsyndromic Wilms' tumor patients: a UK Children's Cancer Study Group Study.

PURPOSE: Constitutional WT1 mutations in patients with Wilms' tumor (WT) have specifically been associated with genitourinary abnormalities, such as cryptorchidism and hypospadias. We sought to ascertain the frequency and heritability of constitutional WT1 mutations in nonsyndromic WT patients. PATIENTS AND METHODS: Constitutional DNA from 282 patients treated at seven United Kingdom Children's Cancer Study Group centers was screened for WT1 mutations using heteroduplex analysis. Bidirectional sequencing was used to confirm the mutation and to analyze the corresponding parental DNA samples. RESULTS: Five different constitutional WT1 mutations were identified in six children. Mutations in four patients were confirmed to be de novo, and all five mutations are predicted to produce truncated protein. The WT1 mutation group had a young median age at diagnosis of 13.8 months, compared with 34.9 months in the group in whom no WT1 mutations were found; four were female and two were male; and all tumors were of favorable histology. The three tumors with known histologic subtype were stromal-predominant. Contrary to expectation, four of six mutations occurred in children with unilateral tumors without any associated genitourinary abnormality. CONCLUSION: Constitutional WT1 mutations occur with a low frequency (2.1%; 95% CI, 0.8% to 4.6%) in nonsyndromic WT patients. Most mutations occurred in children with unilateral WT without associated genitourinary abnormalities, creating difficulties in identifying individuals with germline mutations on phenotype alone. Two factors that may indicate that an individual is carrying a germline WT1 mutation are an early age of onset and stromal-predominant histology of the WT.