Familial aniridia and translocation t(4;11)(q22;p13) without Wilms' tumor

A family with dominantly inherited aniridia in three generations is presented. All three patients had an apparently balanced chromosome translocation t(4;11)(q22;p13). The patients were otherwise clinically normal and without signs of Wilms' tumor; their erythrocyte catalase activities were within the normal range. We suggest that in this family aniridia is caused either by a submicroscopic deletion at the translocation breakpoint 11p13 or by a position effect on the same chromosome segment. Furthermore, the loci for aniridia and Wilms' tumor susceptibility are separate. It follows that the WAGR complex is caused by a mutation of more than one gene located at 11p13. The theoretical implications of a presumably defective allele causing a mendelian dominant phenotype are discussed.     

Aniridia-Wilms' tumor association: evidence for specific deletion of 11p13.

A 7-year-old boy with aniridia, Wilms' tumor, and mental retardation, previously reported as having an interstitial deletion of the short arm of chromosome 8 resulting from a t(8p+;11q-) translocation (Ladda et al., 1974), has been restudied using high-resolution trypsin-Giemsa banding of prometaphase chromsomes. The results revealed a complex rearrangement with four break points in 8p, 11p, and 11q, leading to a net loss of an interstitial segment of 11p (region p1407 yields p1304) but not of 8p. His red blood cells contained normal activities of glutathione reductase (gene on 8p) and lactate dehydrogeanse A (gene on 11p12), indicating a gene dosage consistent with the chromosomal findings. The revised interpretation of this case agrees with seven others reported as having aniridia and interstitial 11p deletions in establishing the distal half of band 11p13 as the site of gene(s) which lead to aniridia and predispose to Wilms' tumor if present in a hemizygous state. Possible relationships between heterozygous deletion of specific chromosomal bands 11p13 and 13q14 and the autosomal dominant disorders aniridia, Wilms' tumor, and retinoblastoma, respectively, are discussed.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilms'' tumor cells.

Children with associated Wilms' tumor, aniridia, genitourinary malformations, and mental retardation (WAGR syndrome) frequently have a cytogenetically visible germ line deletion of chromosomal band 11p13. In accordance with the Knudson hypothesis of two-hit carcinogenesis, the absence of this chromosomal band suggests that loss of both alleles of a gene at 11p13 causes Wilms' tumor. Consistent with this model, chromosomes from sporadically occurring Wilms' tumor cells frequently show loss of allelic heterozygosity at polymorphic 11p15 loci, and therefore it has been assumed that allelic loss extends proximally to include 11p13. We report here that in samples from five sporadic Wilms' tumors, allelic loss occurred distal to the WAGR locus on 11p13. In cells from one tumor, mitotic recombination occurred distal to the gamma-globin gene on 11p15.5. Thus, allelic loss in sporadic Wilms' tumor cells may involve a second locus on 11p.     

Evidence for WT1 as a Wilms tumor (WT) gene: intragenic germinal deletion in bilateral WT.

The inactivation of two alleles at a locus on the short arm of chromosome 11 (band 11p13) has been suggested to be critical steps in the development of Wilms tumor (WT), a childhood kidney tumor. Two similar candidate WT cDNA clones (WT33 and LK15) have recently been identified on the basis of both their expression in fetal kidney and their location within the smallest region of overlap of somatic 11p13 deletions in some tumors. These homozygous deletions, however, are large and potentially affect more than one gene. Using a cDNA probe to the candidate gene, we have analyzed DNA from both normal and tumor tissue from WT patients, in an effort to detect rearrangements at this locus. We report here a patient with bilateral WT who is heterozygous for a small (less than 11 kb) germinal deletion within this candidate gene. DNA from both tumors is homozygous for this intragenic deletion allele, which, by RNA-PRC sequence analysis, is predicted to encode a protein truncated by 180 amino acids. These data support the identification of this locus as an 11p13 WT gene (WT1) and provide direct molecular data supporting the two-hit mutational model for WT.     

A common region of loss of heterozygosity in Wilms' tumor and embryonal rhabdomyosarcoma distal to the D11S988 locus on chromosome 11p15.5

The development of Wilms' tumor has been associated with two genetic loci on chromosome 11: WTI in 11p13 and WT2 in 11p15.5. Here, we have used loss of heterozygosity (LOH) in Wilms' tumors to narrow the WT2 locus distal to the D11S988 locus. A similar region was apparent for the clinically associated tumor, embryonal rhabdomyosarcoma. We have also demonstrated that a constitutional chromosome translocation breakpoint associated with Beckwith-Wiedemann syndrome and an acquired somatic chromosome translocation breakpoint in a rhabdoid tumor each occur in the same chromosomal interval as the smallest region of LOH in Wilms' tumors and embryonal rhabdomyosarcoma. Finally, we report the first Wilms' tumor without a cytogenetic deletion that shows targeted LOH for 11p15 and 11p13 while maintaining germline status for 11p14.     

Harvey-ras allele deletion detected by in situ hybridization to Wilms' tumor chromosomes

Summary We have examined the chromosomes from a case of sporadic Wilms' tumor using in situ hybridization to determine whether the Ha-ras (c-Ha-ras 1) oncogene had been deleted as the result of a reciprocal chromosomal translocation between the short arm of chromosome 11 (breakpoint 11p13) and the long arm of chromosome 12 (breakpoint 12q13). Neither the derivative 11 nor derivative 12 chromosome hybridized significantly to the Ha-ras probe, which indicated that this cellular oncogene was deleted as a consequence of the translocation. This conclusion is supported by a Southern blot analysis which demonstrates loss of a Harvey-ras allele. These results support the view that the Ha-ras oncogene may be functionally involved in Wilms' tumor development.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

Molecular analysis of aniridia patients for deletions involving the Wilms' tumor gene

A human aniridia candidate (AN) gene on chromosome 11p13 has been cloned and characterized. The AN gene is the second cloned gene of the contiguous genes syndrome WAGR (Wilms' tumor, aniridia, genitourinary malformations, mental retardation) on chromosome 11p13, WT1 being the first gene cloned. Knowledge about the position of the AN and WT1 genes on the map of 11p13 makes the risk assessment for Wilms' tumor development in AN patients possible. In this study, we analyzed familial and sporadic aniridia patients for deletions in 11p13 by cytogenetic analyses, in situ hybridization, and pulsed field gel electrophoresis (PFGE). Cytogenetically visible deletions were found in 3/11 sporadic AN cases and in one AN/WT patient, and submicroscopic deletions were identified in two sporadic AN/WT patients and in 1/9 AN families. The exact extent of the deletions was determined with PFGE and, as a result, we could delineate the risk for Wilms' tumor development. Future analyses of specific deletion endpoints in individual AN cases with the 11p13 deletion should result in a more precise risk assessment for these patients.     

Chromosome 17p Deletion in Human Medulloblastoma: A Missing Checkpoint in the Hedgehog Pathway

Although deregulation of Hedgehog signalling is considered to play a crucial oncogenic role and commonly occurrs in medulloblastoma, genetic lesions in components of this pathway are observed in a minority of cases. The recent identification of a novel putative tumor suppressor (RENKCTD11) on chromosome 17p13.2, a region most frequently lost in human medulloblastoma, highlights the role of allelic deletion of the gene in this brain malignancy, leading to the loss of growth inhibitory activity via suppression of Gli-dependent activation of Hedgehog target genes. The presence on 17p13 of another tumor suppressor gene (p53) whose inactivation cooperates with Hedgehog pathway for medulloblastoma formation, suggests that 17p deletion unveils haploinsufficiency conditions leading to abrogation of either direct and indirect checkpoints of Hedgehog signalling in cancer.     

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles     

A null allele of esterase D is a marker for genetic events in retinoblastoma formation

Summary The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14.11 is required for tumorigenesis. Observations reported by Benedict and colleagues in one case of bilateral retinoblastoma, LA-RB 69, provided early evidence in favor of this hypothesis. By examining levels of esterase D, an enzyme also mapping to 13q14.11, it was previously inferred that one chromosome 13 in this patient's somatic cells contained a submicroscopic deletion of the Rb and esterase D loci and that this chromosome was retained in her tumor while the normal chromosome 13 was lost. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, we have found that (1) low but detectable quantities of esterase D protein and enzymatic activity are present in tumor cells from LA-RB 69; (2) fibroblast from this patient contain two copies of the esterase D gene, indicated by heterozygosity at an ApaI polymorphic site within this gene; and (3) tumor cells from the same patient are homozygous at this site, indicating loss and reduplication of the esterase D locus. These results demonstrate that one of the two esterase D alleles in this patient acted as a null or silent allele — that is, was present in the genome with markedly decreased protein expression. This mutant allele acted as a marker for tumor-associated loss of chromosome 13 heterozygosity, in concordance with previous proposals.     

Smallest region of overlap in Wilms tumor deletions uniquely implicates an 11p13 zinc finger gene as the disease locus.

The development of Wilms tumor (WT) has been associated with the inactivation of a "tumor suppressor" locus in human chromosome 11 band p13. Several WTs that exhibit homozygous deletions of an 11p13 candidate WT gene in its entirety have been reported. We report here a partial deletion of the candidate gene which, upon comparison with other documented homozygous deletions, permitted a precise definition of the critical genomic target in Wilms tumor. The smallest region of overlap between these deletions is a 16-kb segment of DNA encompassing the 5' exon(s) of an 11p13 gene coding for a zinc finger protein, together with an associated CpG island. This finding supports the notion that the candidate gene in question corresponds to the 11p13 WT1 Wilms tumor locus.     

Significant reduction of WT1 gene expression,possibly due to epigenetic alteration in Wilms' tumor

WT1 at 11p13 is a tumor suppressor gene, an aberration of which causes Wilms' tumor (WT). Since WT1 expression is reduced in a certain proportion of WTs and its mutation is found only in 10-20% of WTs, we examined WT1 gene silencing due to epigenetic alteration in a total of 22 WTs. WT1 expression was significantly reduced in half of WTs without any mutation in the WT1 gene itself, suggesting that the reduction of expression was possibly epigenetic. We found promoter hypermethylation in one WT with loss of heterozygosity (LOH) and showed that promoter methylation reduced reporter gene activity by a reporter assay. These data suggested that methylation was an epigenetic mechanism leading to WT1 silencing and that the expression-reduced allele by hypermethylation combined with LOH was consistent with the revised two-hit model. In addition, as the beta-catenin mutation is frequently associated with the WT1 mutation, the association of WT1 silencing with the beta-catenin mutation was also investigated. beta-catenin mutated in only one WT without WT1 silencing, suggesting that the beta-catenin mutation was not associated with the reduction of WT1 expression.     

Chromosome 5 allele loss at the glucocorticoid receptor locus in human colorectal carcinomas

Matched normal/tumor DNA pairs from sporadic colon carcinoma patients were examined for chromosome 5 allele loss using a probe for a functional gene (glucocorticoid receptor = GRL) locus. This locus maps (5q11-q13) close to one of two alternative sites recently reported for a constitutional deletion in a familial adenomatous polyposis (FAP) patient. Tumor-specific allele loss of at least 27% at GRL supports the hypothesis that both hereditary and sporadic forms of colon cancer result from mutations of the same gene. The proximity of the GRL locus to the region of 5q affected in FAP and the observed tumor-specific allele loss at this locus suggest that further research is needed regarding whether genetic alterations in the glucocorticoid receptor may be associated with colon carcinogenesis.     

Mitotic deletions of 11p15.5 in two different tumors indicate that the CALCA locus is distal to the PTH locus

We have compared the constitutional and tumor genotypes in two patients with Wilms tumor and adrenocortical carcinoma. The allelic distribution of chromosome 11-specific markers spanning chromosome 11 from pter to qter (HRAS1-HBB-[CALCA/PTH]-FSHB-CAT-APOA1) and an approach combining RFLP analysis and gene copy number determination showed that a mitotic deletion had occurred in both tumors. The loss of one copy of the gene for alpha-calcitonin-gene-related polypeptide (CALCA), together with that of a more distal marker (HRAS1 or HBB), indicates that CALCA is distal to the gene for parathyroid hormone (PTH), which was not deleted in either tumor. These results suggest that mitotic deletion mapping may be as useful as meiotic deletion or recombination mapping in ordering closely linked markers, such as CALCA and PTH, for which other approaches, including physical mapping and multipoint linkage analysis, have failed to accurately identify the gene order.     

The QM gene is X-linked and therefore not involved in suppression of tumorigenesis in Wilms' tumor

Summary Inactivation of one or more tumor-suppressor genes on the short arm of chromosome 11 is thought to play a role in the etiology of Wilms' tumor. A candidate gene, QM, was recently isolated by subtractive hybridization between a tumorigenic cell line (deleted for part of 11p) and a non-tumorigenic cell line (the tumorigenic cell line carrying an extra t(X;11)copy). We show here with an exon-specific polymerase chain reaction that the genomic homolog of the QM cDNA is located in the G6PD-color vision genes region in Xq28. No homologous sequences could be detected on 11p. Our experiments indicate that the QM gene is not involved in the suppression of Wilms' tumor.     

Genomic structure, chromosomal location, and mutation analysis of the human CDC14A gene.

Human CDC14A is a dual-specificity phosphatase that shares sequence similarity with the recently identified tumor suppressor, MMAC1/PTEN/TEP1. By radiation hybrid mapping, we localized CDC14A to chromosome band 1p21, a region that has been shown to exhibit loss of heterozygosity in highly differentiated breast carcinoma and malignant mesothelioma. We have mapped the exon-intron structure of CDC14A gene and found an in-frame ATG at 14 codons upstream of the previously reported start site (GenBank Accession No. AF000367). In screening a panel of 136 cDNAs from tumor cell lines for coding mutations, we have identified a 48-bp in-frame deletion in the cDNA of the breast carcinoma cell line, MDA-MB-436. This deletion is the result of an acceptor splice site mutation (AG to AT) in intron 12 that causes the skipping of exon 13 in the gene. Loss of expression of the wildtype allele in the same breast cell line supports the possibility that CDC14A may be a tumor suppressor gene that is targeted for inactivation during tumorigenesis.