Biomedical subjects

A E Reeve

Publications and source records attributed to A E Reeve.

At least 37 records · Page 2Linked to original sources

PubMed1996

Role of genomic imprinting in Wilms' tumour and overgrowth disorders.

Activation of the silent maternal IGF2 allele has recently been found in approximately half of Wilms' tumour (WTs) examined. This process of imprint relaxation leads to biallelic expression of IGF2 and it has been suggested that this is a key event in the onset of some WTs. Although it has previously been proposed that the 11p15 chromosome region contains a growth-promoting gene and a tumour suppressor gene, the simplest explanation is that increased expression of the IGF2 gene is responsible for somatic overgrowth in the BWS and predisposition to tumours. This model explains overgrowth in BWS cases with unbalanced translocations with paternal dup(11p), and cases with balanced maternal translocations which are physically close to the IGF2 gene. Maternal translocations are envisaged to disrupt the maternal IGF2 imprint by a mechanism similar to the position-effect variegation mechanism in Drosophila. Relaxation of IGF2 imprinting has also been detected in several patients with the BWS syndrome and a patient with gigantism and Wilms' tumour. Recent gene disruption experiments have shown that inactivation of the mouse h19 gene leads to biallelic lgf2 expression and extensive proportional overgrowth. This mouse model has parallels with the BWS and WT where it has been found that biallelic IGF2 expression is accompanied by an epigenetic modification of the H19 gene. From these data it is possible to speculate that an epigenetic modification of the H19 gene may be the primary event leading to the relaxation of IGF2 imprinting.

Alleles↗

PubMed1996

Somatic overgrowth associated with overexpression of insulin-like growth factor II.

Overexpression of the normally imprinted fetal insulin-like growth factor II (IGF2) has been implicated in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). We have detected constitutional relaxation of imprinting of IGF2 in four children with somatic overgrowth who do not show diagnostic features of BWS. Three children showed constitutional abnormalities of H19 methylation. All four children showed nephromegaly and two developed Wilms' tumors. Gene methylation is known to be associated with gene silencing, and three children showed constitutional abnormalities of H19 gene methylation. Disruption of H19 methylation, and concomitant relaxation of IGF2 imprinting, provides another mechanism that can increase IGF2 expression in children with overgrowth. The accumulated data on normal and pathologic IGF2 expression are now sufficient to define an entity, "IGF2 overgrowth disorder," of which BWS may be one extreme manifestation. These findings have broad implications for the characterization of idiopathic overgrowth.

Base Sequence↗

PubMed1996

Equivalent parental distribution of frequently lost alleles and biallelic expression of the H19 gene in human testicular germ cell tumors.

Epigenetic alterations such as genomic imprinting might play an important role in human tumorigenesis, in addition to specific genetic alterations. To clarify the role of genetic and/or epigenetic alterations in the tumorigenesis of testicular germ cell tumors (GCTs), we analyzed 40 primary and 3 metastatic testicular GCTs with regard to specific chromosomal losses and their parental origin. A high incidence of loss of heterozygosity (LOH) was demonstrated on chromosomes 1p, 3p, 11p, and 17p: 9/19 (47%), 18/39 (46%), 13/40 (33%) and 20/36 (56%), respectively. However, there was no correlation between the frequency of LOH on any chromosome and clinicopathological features. Regarding the parental origin of the lost allele at these chromosomes, preferential loss was not demonstrated in this study. To clarify the imprinting status in GCTs, we analyzed the allele-specific expression of the H19 gene, which is paternally imprinted on chromosome 11p. All of 11 tumors without LOH at this locus showed biallelic expression of H19. Based on previous work demonstrating the biallelic expression of H19 in primordial germ cells and spermatogonia in the mouse germ line, these results suggest that the biallelic expression of H19 in testicular GCTs reflects the characteristics of the original germ cells in which the imprinting marking has been erased and not established, rather than loss of imprinting during tumorigenesis. It is also possible that a failure to re-establish the imprinting might be an initial event which leads to testicular GCTs.

Alleles↗

PubMed1995

Altered specificity of IGF2 promoter imprinting during fetal development and onset of Wilms tumour.

The specificity of IGF2 promoter imprinting was examined in embryonal tissues and Wilms tumour. In several fetal tissues of approximately 12 weeks gestation, IGF2 was found to be monoallelically expressed from all IGF2 promoters i.e. P1, P2, P3 and P4. However, in tissues of slightly older gestation age (15-17 weeks) relaxation of imprinting at the P1 promoter was evident, although the P2-P4 promoters remained imprinted. These data indicate that early in embryogenesis a population of cells exists in which all IGF2 promoters are imprinted, but that as development proceeds the imprinting of the P1 promoter is relaxed. The pattern of IGF2 promoter imprinting was also analysed in Wilms tumour. In some tumours, the pattern of promoter imprinting was identical to that found in early fetal kidney, indicating that this tumour originates within early embryonic kidney tissue. In contrast, in tumours in which relaxation of imprinting had occurred, imprinting relaxation affected all IGF2 promoters. This aberrant pattern of promoter imprinting, which was not detected in fetal kidney, provides further evidence that pathological relaxation of IGF2 imprinting is involved in the genesis of Wilms tumour.

Adult↗

PubMed1995

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.

Base Sequence↗

PubMed1995

Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.

In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.

Alleles↗

PubMed1995

Genetic mosaicism at the insulin locus in liver associated with childhood hepatoblastoma.

Hepatoblastoma is commonly associated with loss of heterozygosity (LOH) involving chromosome region 11p15.5. This region may contain an as yet unidentified tumor suppressor gene relevant to this and other tumors associated with the Beckwith-Wiedemann syndrome. Using the insulin (INS) locus as a marker for this region we have identified two of three patients with hepatoblastoma whose livers demonstrated genetic mosaicism. One tumor arose from the clone demonstrating LOH in the liver while the other arose from the clone without LOH.

Alleles↗

PubMed1995

Comparative in situ hybridization analysis of PAX2, PAX8, and WT1 gene transcription in human fetal kidney and Wilms' tumors.

Wilms' tumor (WT) is a childhood renal neoplasm with histological features resembling fetal kidney development. Two members of the paired box family of genes, PAX2 and PAX8, are expressed in WT and are potentially involved in its induction. A zinc finger gene, WT1, which is involved in WT induction, encodes a DNA binding protein, and like PAX2 and PAX8 proteins is a transcription factor with an important role in kidney development. We have compared the expression patterns of PAX2, PAX8, and WT1 in fetal kidney and WTs by in situ hybridization. The PAX2, PAX8, and WT1 genes were transcribed in the condensed mesenchyme and early stages of epithelial differentiation in fetal kidney. WT1 gene transcription was observed in the glomeruli of fetal kidney until a later stage in development than PAX genes. In WTs all three genes were expressed in the condensed blastema, but WT1 expression was not detectable in the epithelial structures in two WTs. No evidence of attenuation of PAX gene expression was found in WT. These results suggest that in some WTs the expression of WT1 is attenuated in structures that continued to express PAX genes. It is unlikely that both PAX2 and PAX8 genes would be mutated in WT. However, failure of PAX gene expression to attenuate in WTs may result from mutations involved in the onset of the tumor.

DNA-Binding Proteins↗

PubMed1994

Mosaic and polymorphic imprinting of the WT1 gene in humans.

We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.

Alleles↗

PubMed1994

Alternative messenger RNA forms and open reading frames within an additional conserved region of the human PAX-2 gene.

PAX-2 is a member of a family of genes containing a highly conserved paired box domain. The paired box domain encodes a DNA binding motif, indicating that PAX proteins may function as transcriptional regulators, participating in a hierarchical network of gene regulation during embryogenesis. In this report, we provide evidence that there is an additional conserved region near the predicted COOH-terminus of PAX-2, PAX-5, and PAX-8. We also describe alternative splicing of a conserved 83-nucleotide segment in the 3' coding sequence of the PAX-2 gene. PAX-2 transcripts that contain the 83-nucleotide insertion encode a putative protein with an alternative COOH-terminus. The presence of an additional conserved region and alternative splicing within the predicted COOH-terminal region of several PAX genes has implications for the evolutionary origins and for the DNA binding site specificity of PAX-2, PAX-5, and PAX-8.

Alternative Splicing↗

PubMed1994

Expression of WT1 protein in fetal kidneys and Wilms tumors.

BACKGROUND: Wilms' tumors are embryonic kidney neoplasms believed to result from a perturbation in the development of the metanephric blastema. A candidate gene (WT1) has been cloned that has been found to be mutated in a number of Wilms' tumors, consistent with its suggested role as a tumor suppressor gene. This gene has been shown to be essential to the normal development of the embryonic kidney. EXPERIMENTAL DESIGN: The aim of the present study was to provide information on the level at which the WT1 gene is regulated. Immunohistochemistry, immunofluorescence, and in situ hybridization was used to examine the localization of WT1 protein and mRNA, respectively. We further used immunofluorescence to examine the WT1 expression in seven Wilms' tumors. RESULTS: In fetal kidneys, WT1 transcripts were detected with increasing levels of hybridization signal in induced blastemal cells, renal vesicles, pre-podocytes of comma- and S-shaped bodies, and podocytes of glomeruli. WT1 protein, detected by an antibody raised against recombinant WT1 fusion protein, was seen in the nuclei of the same cell types mentioned above, and the staining intensity was comparable to the levels of WT1 transcripts. Immunostaining of seven Wilms tumors demonstrated that WT1 protein was expressed only in neoplastic structures whose normal counterparts also expressed WT1 protein. Neither stromal cells nor rhabdomyoblasts contained WT1 protein. CONCLUSIONS: The results show that in fetal kidney, WT1 transcripts and protein are coordinately expressed, and strongly associated with differentiation of metanephric blastemal cells into epithelial cells. Furthermore, the finding that WT1 transcripts and protein are coordinately expressed, suggests that WT1 gene expression is primarily regulated at the level of transcription.

Blotting, Western↗

PubMed1994

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.

Base Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1993

Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour.

Genomic imprinting has been implicated in the onset of several embryonal tumours but the mechanism is not well understood. Maternal chromosome 11p15 loss of heterozygosity and paternal chromosome 11 isodisomy suggest that imprinted genes are involved in the onset of Wilms' tumour and the Beckwith-Wiedemann syndrome. The insulin-like growth factor II (IGF2) gene located at 11p15.5 has been put forward as a candidate gene as it is maternally imprinted (paternally expressed) in the mouse, and is expressed at high levels in Wilms' tumours. We report here that the IGF2 gene is expressed from the paternal allele in human fetal tissue, but that in Wilms' tumour expression can occur biallelically. These results provide, to our knowledge, the first evidence that relaxation of imprinting may play a role in the onset of disease and suggest a new genetic mechanism involved in the development of cancer.

Alleles↗

PubMed1993

Structural analysis of the human insulin-like growth factor-II P3 promoter.

The expression of insulin-like growth factor-II (IGF-II) has been observed previously in many human cancers. The human IGF-II P3 promoter has been shown by others to give rise to abundant 6.0 kb and 2.2 kb fetal transcripts which are expressed in a variety of both paediatric and adult tumours. In order to determine the mechanism by which the P3 promoter is controlled, the promoter was analysed in cell lines using chloramphenicol acetyltransferase (CAT) assay and DNAase I footprinting techniques. The data indicated that P3 is a complex promoter involving at least nine transcription factor binding sites. Furthermore, high levels of 5-methylcytosine detected in the P3 promoter of HeLa genomic DNA suggest that IGF-II gene expression may also be influenced by DNA methylation.

Base Sequence↗

PubMed1993

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.

Alleles↗

PubMed1993

The insulin-like growth factor 1 receptor gene is normally biallelically expressed in human juvenile tissue and tumours.

We have examined insulin-like growth factor 1 receptor (IGF1R) gene expression for evidence of imprinting in 15 informative patients with embryonal tumours. Biallelic expression was observed in all but one sample of normal juvenile kidney and liver, and in 9/10 associated Wilms' tumours, 3/3 hepatoblastomas and 2/2 adrenal tumours. A single patient with Beckwith-Wiedemann Syndrome (BWS) demonstrated monoallelic expression of the maternally derived IGF1R allele in normal kidney, associated Wilms' tumour and in peripheral blood lymphocytes. The observed biallelic expression of the IGF1R gene in all but one patient strongly suggests that the human gene is not normally imprinted.

Adrenal Gland Neoplasms↗

PubMed1993

Human insulin-like growth factor type I and type II receptors are not imprinted.

We have shown that the insulin-like growth factor type I and II receptors are expressed equally from the maternal and paternal alleles in human tissues. The imprinting status of the type I insulin-like growth factor receptor has not been reported while the type II receptor has previously been shown to be maternally expressed in the mouse. That the imprinting of the insulin-like growth factor type II receptor is not conserved between mouse and humans suggests that the physiological role of the IGF2 receptor may differ between these two species.

Alleles↗