Biallelic DICER1 mutations occur in Wilms tumours.

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.

Constitutional relaxation of insulin-like growth factor II gene imprinting associated with Wilms' tumour and gigantism.

We have examined the imprinting of the insulin-like growth factor II gene (IGF2) in ten normal kidney samples from children with renal embryonal neoplasms. In kidney samples from nine children with normal growth profiles, IGF2 mRNA was transcribed monoallelically, consistent with normal imprinting of the gene. But in one child who had generalized somatic overgrowth, IGF2 was transcribed from both alleles in her kidney, peripheral blood leukocytes and Wilms' tumour. These findings suggest that a defect in genomic imprinting can occur constitutionally, leading to growth abnormalities and predisposition to Wilms' tumour.

Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.

Paired box (PAX) genes play a critical role in human development and disease. The PAX2 gene is expressed in primitive cells of the kidney, ureter, eye, ear and central nervous system. We have conducted a mutational analysis of PAX2 in a family with optic nerve colobomas, renal hypoplasia, mild proteinuria and vesicoureteral reflux. We report a single nucleotide deletion in exon five, causing a frame-shift of the PAX2 coding region in the octapeptide domain. The phenotype resulting from the PAX2 mutation in this family was very similar to abnormalities that have been reported in Krd mutant mice. These data suggest that PAX2 is required for normal kidney and eye development.

A colony-stimulating factor 1 (CSF-1) receptor/platelet-derived growth factor-beta receptor gene fusion confers CSF-1 independence and tumorigenicity on a c-myc-immortalized monocyte cell line.

Monocytes and macrophages express the receptor for the hematopoietic growth factor colony-stimulating factor 1 (CSF-1) and require this factor for growth in culture. A murine monocyte tumor cell line that lacks the usual requirement for CSF-1 was isolated. On the basis of the similarity of the structures of the CSF-1 and platelet-derived growth factor (PDGF) receptors and because monocytes normally secrete PDGF, we analyzed the tumor cell line for anomalous expression of the PDGF-R beta gene. Two different cDNAs that each contain sequences corresponding to the complete coding sequence of PDGF-R beta fused (in frame) to the amino-terminal half of the CSF-1 receptor were isolated. Introduction of these PDGF-R beta-related cDNAs into two partially transformed, CSF-1-dependent monocyte cell lines resulted in autonomous growth and cell transformation. These monocyte cell lines exhibit a novel form of growth factor receptor activation that can lead to oncogenic growth in collaboration with the c-myc oncogene.

An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Insulin-like growth factor II and WT1 transcript localization in human fetal kidney and Wilms' tumor.

In situ hybridization was used to examine, in parallel, the localization of insulin-like growth factor II (Igf2) and WT1 transcripts in normal fetal kidney and Wilms' tumor. The expression of Igf2 and WT1 transcripts in the fetal kidney is almost complementary in both the epithelial and stromal cell lineages derived from the undifferentiated metanephrogenic blastema. The patterns of transcription of Igf2 in three Wilms' tumors appeared to be perturbed as compared to the normal fetal kidney. In these tumors Igf2 transcripts were detected in structures that are developmentally equivalent to the renal vesicle, which in the normal kidney do not contain Igf2 transcripts. These results suggest that Wilms' tumors arise from an alteration in the regulation of Igf2 mRNA synthesis.

A third Wilms' tumor locus on chromosome 16q.

Loss of heterozygosity studies have been used to identify chromosomal regions which are frequently deleted and thus indicate areas which may harbor tumor suppressor genes. As a result, both the WT1 gene located in chromosome 11p13 and an unidentified gene(s) within chromosome 11p15 have been implicated in Wilms' tumorigenesis. Cytogenetic and linkage studies suggest that additional non-chromosome 11 sites are involved in Wilms' tumor. Because these sites may also involve loss of heterozygosity, loci on 33 autosomal arms were screened for allele loss in a series of Wilms' tumors. We found that in addition to loss on chromosome 11p (11 of 25 informative tumors) there was significant loss on chromosome 16q (9 of 45 informative tumors), while the total frequency of allele loss excluding these loci was low (9 of 426 total informative loci). These data indicate that losses of both chromosome 11p and 16q alleles are nonrandom events and suggest that 16q is the location of a third tumor suppressor gene underlying Wilms' tumorigenesis. The parental origin of the lost chromosome 16q allele was determined in eight sporadic tumors. Alleles of paternal and of maternal origin were each lost in four sporadic tumors indicating that, unlike chromosome 11p, alleles of either parental origin are lost on 16q.

Exclusion of the Wilms tumour gene (WT1) promoter as a site of frequent mutation in Wilms tumour.

WT1 is a tumour suppressor gene expressed in a specific temporal and spatial pattern in the developing kidney. Up to 15% of Wilms tumours have point mutations in the WT1 gene coding sequence. We have now investigated whether mutations in the WT1 promoter could be associated with loss of control WT1 expression and subsequent Wilms tumour formation. Using single-strand conformational polymorphism (SSCP) analysis we analysed 39 sporadic Wilms tumours for WT1 promoter mutations. We found six linked common sequence polymorphisms and two unlinked less frequent polymorphisms which allowed us to identify four tumours with loss of heterozygosity but none with point mutations, small deletions, insertions or rearrangements. We therefore conclude that WT1 promoter mutations are unlikely to play an important role in Wilms tumorigenesis.

Examination of the role of CSF-1 independence in myc retrovirus induced monocyte tumorigenesis.

These studies were initiated as an attempt to estimate the number and nature of genetic changes that are required in addition to c-myc deregulation during monocyte tumorigenesis, and to determine whether the oncogenic changes that can be created in vitro resemble the actual changes that occur in vivo. We found that superinfecting myc-immortalized monocytes with a colony stimulating factor-1 (CSF-1) expressing retrovirus strongly promoted tumorigenesis, whereas granulocyte/macrophage-CSF (GM-CSF) and v-fms retroviruses, or the spontaneous acquisition of CSF-1 independence did so only moderately. In addition myc-infected monocytes isolated from mice at a stage prior to tumor formation are more tumorigenic than in vitro myc-immortalized monocytes, but they were still largely CSF-1 dependent, and were not as tumorigenic as reinnoculated tumor cells. In the simplest model only two oncogenic activations are required for monocyte/macrophage transformation, immortalization of the cells with c-myc and deregulation of the CSF-1 gene. However, not all mechanisms that result in loss of CSF-1 dependence lead to full tumorigenicity, suggesting that in vivo tumorigenesis may involve multiple secondary events including growth factor independence.

Cloning of novel Wilms tumor gene (WT1) cDNAs; evidence for antisense transcription of WT1.

WT1 is a tumor suppressor gene that has been implicated in Wilms tumor, and is expressed in cells of mesodermal origin. The Wit-1 gene is located approximately 2 kb from the WT1 gene, and is expressed coordinately with WT1. WT1 and Wit-1 are bi-directionally transcribed from the same promoter region. We have screened a human fetal kidney cDNA library to identify novel WT1 cDNA clones. Here we report the cloning of cDNA clones which span part of intron 1 of WT1, exon 1, upstream sequences between WT1 and Wit-1 and part of the Wit-1 gene. Northern blot and RNAase protection analysis using subcloned fragments of the cDNAs corresponding to regions from within intron 1 of WT1 suggest that a 7-10 Kb RNA is expressed in human fetal kidney, which overlaps with WT1 and is transcribed in the same direction as Wit-1.

Differential regulation of the human Wilms tumour suppressor gene (WT1) promoter by two isoforms of PAX2.

PAX2 is a member of the paired box family of genes with an important role in kidney, genital tract and eye development. Another gene essential for kidney and genital tract development is the Wilms tumour gene, WT1. PAX2 and WT1 encode transcription factors expressed during fetal kidney development in patterns that overlap both spatially and temporally. The overlap of PAX2 and WT1 expression in fetal kidney prompted us to determine whether PAX2 regulates the WT1 gene. To investigate this possibility, the WT1 promoter and a series of WT1 promoter deletion fragments were cloned into a luciferase reporter vector, and used in co-transfection experiments with PAX2 expression constructs. PAX2 transactivated the WT1 promoter up to 35-fold in CHO-K1 cells, and from four- to sevenfold in 293 cells. Two regions of the WT1 promoter were required in the same promoter construct for efficient transactivation by PAX2 in CHO-K1 cells, and purified recombinant PAX2 protein was found to bind to two sites in the WT1 promoter, at -205/-230 and +377/+402. Removal of WT1 promoter sequences containing the -205/-230, or +377/+402 binding sites abolished transactivation of the WT1 promoter by PAX2 in CHO-K1 cells, and had a differential effect on transactivation of the WT1 promoter in 293 cells, depending on the PAX2 isoform used. A fragment containing the -205/-230 site alone could be transactivated by PAX2. These findings suggest that PAX2 is a tissue-specific modulator of WT1 expression, and is involved in cell growth control via WT1.

PAX2 mutations in renal-coloboma syndrome: mutational hotspot and germline mosaicism.

The renal-coloboma syndrome (RCS, MIM 120330) is an autosomal dominant disorder caused by PAX2 gene mutations. We screened the entire coding sequence of the PAX2 gene for mutations in nine patients with RCS. We found five heterozygous PAX2 gene mutations: a dinucleotide insertion (2G) at position 619 in one sporadic RCS case, a single nucleotide insertion (619 + G) in three unrelated cases, and a single nucleotide deletion in a familial case. In this familial case, three affected sibs showed a striking ocular phenotypic variability. Each of the sibs carried a 619insG mutation, whilst unaffected parents did not, suggesting the presence of germline mosaicism. Interestingly, the 619insG mutation has been previously reported in several patients and is also responsible for the Pax21Neu mouse mutant, an animal model of human RCS. This study confirms the critical role of the PAX2 gene in human renal and ocular development. In addition, it emphasises the high variability of ocular defects associated with PAX2 mutations ranging from subtle optic disc anomalies to microphthalmia. Finally, the presence of PAX2 germline mosaicism highlights the difficulties associated with genetic counselling for PAX2 mutations.

Genes encoding the platelet-derived growth factor (PDGF) receptor and colony-stimulating factor 1 (CSF-1) receptor are physically associated in mice as in humans.

The BALB/c mouse DNA was analyzed by field-inversion gel electrophoresis to determine the orientation and distance between the beta-platelet-derived growth factor receptor-encoding gene (Pdgfr) and the colony-stimulating factor 1 receptor-encoding gene (Csfmr). It was found that the 5' portion of the Pdgfr gene was cleaved by the enzyme ClaI into two fragments. The 425-kb fragment hybridized with a 3' Pdgfr and a 5' Csfmr probe. This result shows that the Csfmr gene is 3' relative to the Pdgfr gene, and suggests that the Pdgfr and Csfmr genes are physically linked.

The genetics of primary vesico-ureteric reflux.

INTRODUCTION: Vesico-ureteric reflux (VUR), reflux of urine from the bladder into the ureter and towards the kidney, is an important cause of end-stage renal failure in both children and adults. Primary VUR is considered to be a result of a disruption of the normal anti-reflux mechanism of the ureterovesical junction (UVJ). VUR is common, occurring in approximately 1% to 2% of newborns in Caucasian populations. The aetiology of VUR is thought to involve a substantial genetic component, supported by the observation that VUR frequently occurs in multiple members of the same family. The purpose of this article is to review the literature supporting a genetic cause of VUR, and to draw together observations and make suggestions regarding differential diagnosis of VUR, which might help in future studies on the genetic aetiology of VUR. RESULTS: A common theme arising was the notion that VUR may be caused by multiple genes in the population. However, any one individual with VUR may carry a single dominant mutant allele. Overall, progress has been made in mapping putative VUR loci in both humans and mice, although the mode(s) of inheritance and the exact nature of the underlying defect are still poorly understood. CONCLUSIONS: It is likely that over the next few years VUR genes will be mapped and, once identified, the challenge will be to understand how changes in the expression of these genes lead to the underlying defect in VUR.

PAX2 is an antiapoptotic molecule with deregulated expression in medulloblastoma.

PAX2 is a paired box transcription factor possessing a fundamental role in the embryogenesis of hindbrain and urinary tract. PAX genes are proto-oncogenes, PAX2 expression may contribute to the pathogenesis of renal cell carcinoma. Because of the expression of PAX2 in the developing hindbrain and its essential role in cerebellar development, it has been hypothesized that PAX2 may also be involved in medulloblastoma tumorigenesis. We investigated the expression pattern of PAX2 and various genes of the neuronal lineage in medulloblastoma and glioma cell lines. We found high expression of PAX2 mRNA and PAX2 protein in medulloblastoma cells and some glioma cell lines independent of their neuronal lineage gene expression signature. Gene suppression of PAX2 decreased the expression of the PAX2 transcriptional target GDNF in Daoy cells and had a profound cytotoxic effect in vitro on Daoy medulloblastoma and T98G glioma cells. Expression of PAX2 was then assessed in two separate medulloblastoma tissue microarrays with a total of 61 patient samples by immunohistochemistry. PAX2 expression was detected in the majority of medulloblastoma samples and correlated with less differentiated histology. Therefore, PAX2 is a biomarker for a more aggressive medulloblastoma phenotype and may represent a novel therapeutic target.

PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein.

The retinoblastoma protein (RB)-E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G(1)/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB-E2F1 pathway.

Congenital polycystic kidney disease in lambs.

AIM: To describe the pathology and inheritance of a congenital polycystic kidney disease (PKD) of sheep. METHODS: Mode of inheritance of PKD was investigated by evaluation of results of the disorder from planned matings in two consecutive years within subsets of a flock that had a high prevalence of PKD in lambs. Gross pathological and histopathological studies were based on tissues derived from this study. Haematoxylin and eosin (H&E)-stained paraffin sections of kidney, liver, extrahepatic biliary and pancreatic ducts, pancreas and epididymis were used to describe the lesions. RESULTS: Twenty-five lambs affected by PKD, of both sexes, were born, numbers in accord with those expected for an autosomal recessive disorder in the population studied. In all cases for which tissues were available, the renal, bile ductal (intrahepatic and extrahepatic), pancreatic and epididymal tissues had widespread dysplastic changes and associated cyst formation. CONCLUSIONS: The findings of renal cysts in conjunction with cysts in other organs are unifying features in many of the human and animal forms of PKD and suggest a related pathogenic and genetic base consistent with an autosomal recessive disorder.

The role of PAX2 in normal and abnormal development of the urinary tract.

The molecular etiology of many urinary tract abnormalities in children remains unknown, but a number of genes with a key role in urogenital development have now been identified. PAX2, one such gene, encodes a transcription factor which is critically required for epithelial differentiation within the urogenital tract. Recent studies suggest that PAX2 mutations lead to urological abnormalities and renal failure, while overexpression of PAX2 in the kidneys of mice causes multifocal microcystic tubular dilatation. In humans persistent PAX2 expression has been identified in multicystic dysplastic kidneys. In this review, recent research on the developmental gene, PAX2, and its involvement in normal and abnormal kidney development is summarized. In addition, an overview of the phenotypes associated with either loss-of-function PAX2 mutations or PAX2 overexpression is presented. A brief summary of factors that are known to regulate PAX2 and genes that may be regulated by PAX2 protein is also included.