A novel WT1 mutation in a 46,XY boy with congenital bilateral cryptorchidism, nystagmus and Wilms tumor.

The WT1 gene plays a crucial role in urogenital and gonadal development. Germline WT1 alterations have been described in a wide spectrum of pathological conditions, including kidney diseases, genital abnormalities and Wilms tumor (WT), frequently occurring in combination. We report on a novel WT1 nonsense mutation (c.1105C>T), introducing a premature stop codon in exon 8 (p.Q369X), in a young XY male patient who presented with bilateral cryptorchidism, nystagmus, mild proteinuria and WT, but no sign of severe nephropathy. Although the majority of congenital urogenital abnormalities are not due to constitutional defects of the WT1 gene, our findings provide a rational for considering WT1 mutational analysis as one of the screening options in newborns with congenital defects of the urogenital tract due to the associated high risk of WT.

Cyclin D1 expression analysis in familial breast cancers may discriminate BRCAX from BRCA2-linked cases.

Most familial breast cancers arise in patients who tested negative for germline mutations in BRCA1 and BRCA2 genes (also referred to as BRCAX cases). Several studies aimed to define histopathological and molecular profiles characteristic of BRCA1, BRCA2 and BRCAX tumors have been performed. Major pathological and immunohistochemical differences have been reported in BRCA1 cancers compared to the other two groups, whereas less difference has been observed between BRCA2 and BRCAX cases. The aim of this study was to investigate the ability of selected tumor markers to discriminate BRCAX breast cancers from cancers arising in carriers of mutations in BRCA genes, and their usefulness in selecting familial cases in whom testing for such mutations is more likely to result uninformative. We carried out a morphological and immunohistochemical analysis on 22 BRCA1, 16 BRCA2 and 33 BRCAX familial breast cancers. Age at first diagnosis, histological type and grade, and immunostaining for estrogen receptor (ER), progesterone receptor (PR), p53, HER2/Neu, E-cadherin and cyclin D1 were investigated. The occurrence of somatic mutations of the TP53 gene was also verified. BRCA1 tumors resulted clearly distinguishable from BRCAX cases, occurring at a younger age, being more frequently of higher grade, negative for ER, PR and cyclin D1 expression and positive for p53 alterations. The predictive value of age at diagnosis, histological grade and PR expression was confirmed in a multivariable analysis. When comparing BRCA2 with BRCAX tumors, the only parameter that differed was cyclin D1, which was significantly overexpressed in BRCA2 cases both in the univariable and the multivariable analyses. If confirmed by further studies, our observations indicate that the investigation of cyclin D1 expression in familial breast cancer cases could be used, in conjunction with the analysis of other tumor markers preferentially associated with BRCA1 or BRCA2 tumors, to prioritize hereditary cases for mutation testing in BRCA genes.

Molecular evidence of the independent origin of multiple Wilms tumors in a case of WAGR syndrome.

BACKGROUND: This study investigated the genetic events leading to tumorigenesis in a patient affected with WAGR syndrome who developed multiple distinct Wilms tumors (WTs). PROCEDURE AND RESULTS: At 1 year of age, the child developed two synchronous bilateral WTs that were resected by partial nephrectomy. Histologically, these tumors were fetal rhabdomyomatous nephroblastomas. Immunohistochemical study revealed the absence of nuclear expression of WT1 protein, while beta-catenin protein was expressed at nuclear level by the large majority of tumor cells. Molecular investigations of WT1 gene and exon 3 of beta-catenin (CTNNB1) gene detected no mutations. At 4 years of age, 28 months after the chemotherapy completion, a third WT was diagnosed in the left kidney, and surgically removed before any further chemotherapy. Nine months after surgery, a metastasis was detected in the left lung. Both the third renal tumor and the lung metastasis showed a blastema-predominant morphology. Immunohistochemistry confirmed the lack of expression of WT1 protein, while beta-catenin protein was expressed at nuclear level by the large majority of tumor cells. Molecular analysis of the third renal tumor and the lung metastasis revealed a 4 bp deletion in exon 7 of WT1 gene, leading to a frameshift of the reading frame and to a premature stop of the translation (c.925_928delACTC, p.T309LfsX71); no mutations in the exon 3 of the beta-catenin gene were documented. CONCLUSIONS: These data demonstrate that multiple WTs can arise as a consequence of different genetic events in a patient with genetic predisposition, such as WAGR syndrome.

Retina-derived POU domain factor 1 coordinates expression of genes relevant to renal and neuronal development.

Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. RPF-1 showed nuclear localization, elevated stability, and transactivation of promoters featuring POU consensus sites, and led to reduced cell proliferation and in vivo tumor growth. By addressing the whole transcriptome regulated by its induction, we could detect a gross alteration of gene expression that is consistent with promoter occupancy predicted by genome-wide Chip-chip analysis. Comparison of bound regulatory regions with differentially expressed genes allowed identification of 217 candidate targets. Enrichment of divergent octamers in predicted regulatory regions revealed promiscuous binding to bipartite POUS and POUH consensus half-sites with intervening spacers. Gel-shift competition assay confirmed the specificity of RPF-1 binding to consensus motifs, and demonstrated that the Ser-rich region upstream of the POU domain is indispensable to achieve DNA-binding. Promoter-reporter activity addressing a few target genes indicated a dependence by RPF-1 on transcriptional response. In agreement with its expression in developing kidney and nervous system, the induced transcriptome appears to indicate a function for this protein in early renal differentiation and neuronal cell fate, providing a resource for understanding its role in the processes thereby regulated.

The murine Pou6f2 gene is temporally and spatially regulated during kidney embryogenesis and its human homolog is overexpressed in a subset of Wilms tumors.

We have previously suggested the transcription factor gene POU6F2 as a novel tumor suppressor involved in Wilms tumor (WT) predisposition. Since WT arises from pluripotent embryonic renal precursors, in this study we analyzed the expression of the murine homolog Pou6f2 during kidney embryogenesis and compared it to that of Wt1, the homolog of WT1, a known WT related gene involved in mesenchyme to epithelium conversion. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) performed for Pou6f2 on kidney specimens from embryos, pups, and adult mice, showed that the Pou6f2 mRNA was more abundant in the earliest analyzed phase of kidney organogenesis (E13) than in more advanced fetal stages and in adult animal. In situ RT-PCR demonstrated that Pou6f2 expression parallels the centripetal differentiation of renal morphogenesis. In addition, in E18 kidney, most structures exhibiting Pou6f2 expression stained positively in immunohistochemistry for the Wt1 protein. Finally, quantitative real-time RT-PCR revealed an overexpression (>/=80 times) of POU6F2 compared with normal kidney in 5 of 22 (23%) WTs. The finding of a highly regulated temporal and spatial Pou6f2 expression during renal organogenesis, of its coexpression with Wt1 and of POU6F2 overexpression in a subset of WTs are consistent with a role of POU6F2 in kidney development and provide further support to its involvement in WT.