Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization.

Fluorescence in situ hybridization (FISH) with biotin-labeled probes mapping to 11p13 has been used for the molecular analysis of deletions of the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) locus. We have detected a submicroscopic 11p13 deletion in a child with inherited aniridia who subsequently presented with Wilms tumor in a horseshoe kidney, only revealed at surgery. The mother, who has aniridia, was also found to carry a deletion including both the aniridia candidate gene (AN2) and the Wilms tumor predisposition gene (WT1). This is therefore a rare case of an inherited WAGR deletion. Wilms tumor has so far only been associated with sporadic de novo aniridia cases. We have shown that a cosmid probe for a candidate aniridia gene, homologous to the mouse Pax-6 gene, is deleted in cell lines from aniridia patients with previously characterized deletions at 11p13, while another cosmid marker mapping between two aniridia-associated translocation breakpoints (and hence a second candidate marker) is present on both chromosomes. These results support the Pax-6 homologue as a strong candidate for the AN2 gene. FISH with cosmid probes has proved to be a fast and reliable technique for the molecular analysis of deletions. It can be used with limited amounts of material and has strong potential for clinical applications.     

High-resolution studies in patients with aniridia-Wilms tumor association,Wilms tumor or related congenital abnormalities

Summary We attempted to determine wheter all cases of AWTA (anirida-Wilms tumor association) or any of the following groups of patients show 11p deletion: cases of Wilms tumor with congenital abnormalities other than aniridia, those without any congenital abnormalities, tumor itself in cases of Wilsm tumor without constitutional 11p deletion and cases of aniridia or hemihypertrophy without Wilms tumor. We studied a total of 29 index patients including five cases of AWTA, four cases of Wilms tumor with various congenital abnormalities, 16 cases of Wilms tumor without other abnormalities, three cases of aniridia in one of which Wilms tumor developed later and a case of hemihypertrophy.In all five cases of AWTA and in a case of aniridia who later developed Wilms tumor, 11p deletion involving the p13 band was detected. The mother of the latter also showed an identical 11p deletion. The common segment of deletion was the middle part of the p13. Two possible hypotheses on the mechanism through which Wilms tumor might develop were evaluated, based on the distribution of break points. All other cases, including five with tumor culture, showed a normal karyotype.     

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.     

The gene for catalase is assigned between the antigen loci MIC4 and MIC11

Genetic analysis of the cells of a WAGR patient (W, predisposition to Wilms tumor; A, aniridia; G, genitourinary abnormalities; R, mental retardation), bearing a partial deletion of band 11p13, was performed with biochemical and antigenic 11p markers by using gene dosage, somatic hybridization, molecular hybridization, and indirect immunofluorescence techniques. These studies allowed the regional assignment of the gene for catalase, which is linked to the Wilms tumor locus, between MIC4 and MIC11, two loci encoding for membrane antigens previously mapped to band 11p13.     

The distal region of 11p13 and associated genetic diseases.

The distal region of human chromosome band 11p13 is believed to contain a cluster of genes involved in the development of the eye, kidney, urogenital tract, and possibly the nervous system. Genetic abnormalities of this region can lead to Wilms tumor, aniridia, urogenital abnormalities, and mental retardation (WAGR syndrome). Using 11 DNA markers covering the entire distal region of 11p13, including the WAGR region, we have carried out molecular studies on 58 patients with one or more features of this syndrome and patients with other diseases or structural cytogenetic abnormalities associated with 11p13. Cytogenetic analyses were performed in all cases. In 12 patients we were able to demonstrate deletions of this region. In 2 patients balanced translocations and in 2 additional patients duplications of this region were characterized. In total, 5 chromosomal breakpoints within 11p13 were identified. One of these breakpoints maps within the smallest region of overlap of WAGR deletions. Moreover, we were unable to demonstrate constitutional deletions in a candidate sequence for the Wilms tumor gene or any other marker in 2 patients with aniridia and urogenital abnormalities, 4 patients with Wilms tumor and urogenital abnormalities, 5 patients with bilateral Wilms tumors, and 3 familial Wilms tumor cases. We suggest that the molecular techniques used here (heterozygosity testing for polymorphic markers mapping between AN2 and WT1 and deletion analysis by dosage, cytogenetic analysis, or in situ hybridization) can be employed to identify sporadic aniridia patients with and without increased tumor risk.     

A deletion map of the WAGR region on chromosome 11.

The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.     

Familial predisposition to Wilms tumor does not segregate with the WT1 gene.

Wilms tumor (WT) is one of the more common childhood cancers. A small fraction of WT occurs in association with aniridia, genitourinary abnormalities and mental retardation, the WAGR syndrome, and these cases often are accompanied by a constitutional deletion of all or part of band 11p13. Recently a WT susceptibility gene (WT1), localized to 11p13, has been isolated and shown to be inactivated in some sporadic WTs. In the present study, a highly informative CA repeat polymorphism within the gene was studied in a family with six affected members in three generations. Predisposition to WT in this large family did not segregate with this polymorphism. Furthermore, linkage analysis indicated exclusion of WT predisposition from 11p15. These results provide definitive evidence that familial predisposition to WT can be mediated by a gene other than WT1.     

Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with aniridia

Seventy-seven patients with aniridia, referred for cytogenetic analysis predominantly to assess Wilms tumor risk, were studied by fluorescence in situ hybridization (FISH), through use of a panel of cosmids encompassing the aniridia-associated PAX6 gene, the Wilms tumor predisposition gene WT1, and flanking markers, in distal chromosome 11p13. Thirty patients were found to be chromosomally abnormal. Cytogenetically visible interstitial deletions involving 11p13 were found in 13 patients, 11 of which included WT1. A further 13 patients had cryptic deletions detectable only by FISH, 3 of which included WT1. Six of these, with deletions <500 kb, share a similar proximal breakpoint within a cosmid containing the last 10 exons of PAX6 and part of the neighboring gene, ELP4. Two of these six patients were mosaic for the deletion. The remaining four had chromosomal rearrangements: an unbalanced translocation, t(11;13), with a deletion including the WAGR (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) region, and three balanced rearrangements with what appear to be position effect breakpoints 3' of PAX6: (a) a t(7;11) with the 11p13 breakpoint approximately 30 kb downstream of PAX6, (b) a dir ins(12;11) with a breakpoint >50 kb from PAX6, and (c) an inv(11)(p13q13) with a breakpoint >75 kb downstream of PAX6. The proportion and spectrum of chromosome anomalies in familial (4/14, or 28.5%) and sporadic (26/63, or 41%) cases are not significantly different. An unexpectedly high frequency of chromosomal rearrangements is associated with both sporadic and familial aniridia in this cohort.     

Molecular mapping and cloning of the breakpoints of a chromosome 11p14.1-p13 deletion associated with the AGR syndrome

Chromosome 11p13 is frequently rearranged in individuals with the WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) or parts of this syndrome. To map the cytogenetic aberrations molecularly, we screened DNA from cell lines with known WAGR-related chromosome abnormalities for rearrangements with pulsed field gel (PFG) analysis using probes deleted from one chromosome 11 homolog of a WAGR patient. The first alteration was detected in a cell line from an individual with aniridia, genitourinary anomalies, mental retardation, and a deletion described as 11p14.1-p13. We have located one breakpoint close to probe HU11-164B and we have cloned both breakpoint sites as well as the junctional fragment. The breakpoints subdivide current intervals on the genetic map, and the probes for both sides will serve as important additional markers for a long-range restriction map of this region. Further characterization and sequencing of the breakpoints may yield insight into the mechanisms by which these deletions occur.     

Molecular definition of de novo and genetically transmitted WAGR-associated rearrangements of 11p13

We describe a family in whom the phenotypically normal father carries a balanced insertional translocation, ins(14;11)(q23;p12p14). This individual fathered three mentally retarded children, two with a del(11)(p13) and one with a dup(11)(p13). Two other cases of a de novo del(11)(p13) are also described. All four del(11)(p13) cases presented with WAGR, a complex syndrome associated with a predisposition to Wilms' tumor (WT), aniridia (A), genitourinary abnormalities (G), and mental retardation (R). Using an approach combining karyotype analysis, determination of the gene copy number, and RFLP studies employing five 11p13 DNA markers, we were able to define the chromosomal rearrangement involved in each case. Analysis of these WAGR deletions provides further subdivision of band p13 on chromosome 11.     

Sequences which flank an 11p deletion observed in an hepatocellular carcinoma map to 11p13

Summary There is considerable interest in the 11p13 region because of its involvement in Wilms tumor, sporadic aniridia, and other congenital abnormalities. Cloned DNA sequences from this region might be useful in understanding the chromosomal abnormalities which lead to such disorders. However, few such markers exist. Using somatic cell hybrids which contain defined 11p deletions, two cloned DNA sequences which flank a deletion generated in an hepatocellular carcinoma (as a consequence of hepatitis B virus integration) were mapped to 11p13. Thus both ends of the deletion observed in an hepatocellular carcinoma are within 11p13.     

CpG islands surround a DNA segment located between translocation breakpoints associated with genitourinary dysplasia and aniridia

We have isolated a DNA segment absent from all the constitutionally deleted chromosomes 11 of our patients with Wilms tumor. This marker separates two balanced translocations that break in band 11p13: the distal one associated with aniridia (AN2), and the proximal one with genitourinary dysplasia (GUD). The GUD breakpoint maps within the smallest region of overlap (SRO) for the Wilms tumor (WT) gene locus, thus strengthening the previous suggestion of an association between Wilms tumor and other abnormalities of the genitourinary system. The 11p13 translocation breakpoint associated with T-cell acute lymphatic leukemia (T-ALL) is centromeric to the SRO and separated from the WT locus by at least one known gene. This region of the human genome (11p13) is rich in CpG islands that potentially identify genes, some of which may be involved in the various phenotypes associated with the WAGR syndrome. This is consistent with the proposition that the majority of human genes are in G-negative bands.     

Familial Wiedemann-Beckwith syndrome and a second Wilms tumor locus both map to 11p15.5.

Wilms tumor of the kidney occurs with increased frequency in association with two clinically and cytogenetically distinct congenital syndromes, the Wiedemann-Beckwith syndrome (WBS) and the triad of aniridia, genitourinary anomalies, and mental retardation (WAGR). Constitutional deletions in the latter situation and similar alterations in sporadic Wilms tumors have implicated the chromosomal 11p13 region in neoplastic development. In contrast, some sporadic cases of WBS have been reported to have a constitutional duplication of chromosome 11p15. In order to resolve this seeming paradox, we have analyzed a family segregating WBS for linkage to DNA markers mapped to chromosome 11p. Consonant with the cytogenetic alterations in sporadic WBS cases, we obtained evidence for tight linkage of the mutation causing the syndrome to markers located at 11p15.5. Also consistent with this localization, we identified a subset of Wilms tumors, not associated with WBS, which have attained somatic homozygosity through mitotic recombination, with the smallest shared region of overlap being distal to the beta-globin complex at 11p15.5. These data provide evidence that familial WBS likely results from a defect at the same genetic locus as does its sporadic counterpart. Further, the data suggest there is another locus, distinct from that involved in the WAGR syndrome, which plays a role in the association of Wilms tumor with WBS.     

The E7-associated cell-surface antigen: a marker for the 11p13 chromosomal deletion associated with aniridia-Wilms tumor.

Unbalanced interstitial deletions of the p13 region of human chromosome 11 have been associated with congenital hypoplasia or aplasia of the iris, mental retardation, ambiguous genitalia, and predisposition to Wilms tumor of the kidney. Utilizing somatic cell hybrids containing either the normal or abnormal chromosome 11 from a child with Wilms tumor and aniridia, we previously mapped the E7 cell-surface antigen to the 11p1300-to-11p15.1 region. To localize even further the site of this antigen on chromosome arm 11p, we have produced somatic cell hybrids from the fibroblasts of a second child with Wilms tumor and aniridia and a different deletion of 11p [46,XY, del (11)(pter----p14.1::p11.2----qter)]. Furthermore, the normal and deleted chromosome 11 could also be distinguished on the basis of a restriction fragment length polymorphism for the beta-globin gene. Hybrid cells containing the deleted chromosome were not killed in the presence of complement and the E7 monoclonal antibody (which recognizes E7 cell surface antigen), while hybrid cells containing the patient's normal chromosome 11 were killed. Thus, expression of the E7-associated cell-surface antigen can be mapped to the 11p13 region, and it appears to be a potential marker of the chromosome abnormality associated with aniridia-Wilms tumor.     

Catalase levels in patients with aniridia and/or Wilms' tumor: utility and limitations

The gene for red blood cell (RBC) catalase has recently been mapped to 11p13, and a gene dosage effect has been demonstrated for individuals with triplication or deletion of that region. Deletion of the 11p13 band has also been associated with aniridia, with and without Wilm's tumor. We studied the RBC catalase levels in individuals without detectable chromosomal abnormalities but with aniridia, Wilm's tumor, and the combination of aniridia and Wilms' tumor, to determine whether catalase levels might provide evidence for a submicroscopic chromosomal deletion in the 11p13 region. All karyotypically normal patients were found to have normal catalase levels.     

A highly polymorphic locus cloned from the breakpoint of a chromosome 11p13 deletion associated with the WAGR syndrome

Children with constitutional deletions of chromosome 11p13 suffer from aniridia, genitourinary malformations, and mental retardation and are predisposed to develop bilateral Wilms tumor (the WAGR syndrome). The critical region for these defects has been narrowed to a segment of band 11p13 between the catalase and the beta-follicle-stimulating hormone genes. In this report, we have cloned the endpoints from a WAGR patient whose large cytogenetic deletion, del(11)(p14.3::p13), does not include the catalase gene. The deletion was characterized using DNA polymorphisms and found to originate in the paternally derived chromosome 11. The distal endpoint was identified as a rearrangement of locus D11S21 in conventional Southern blots of the patient's genomic DNA, but was not detected in leukocyte DNA from either parent or in sperm DNA from the father. The proximal endpoint was isolated by cloning the junction fragment and was mapped in relation to other markers and breakpoints. It defines a new locus in 11p13-delta J, which is close to the Wilms tumor gene and the breakpoint cluster region (TCL2) of the frequent t(11;14)(p13;q11) translocation in acute T-cell leukemia. An unusual concentration of base pair substitutions was discovered at delta J, in which 9 of 44 restriction sites tested (greater than 20%) vary in the population. This property makes delta J one of the most polymorphic loci on chromosome 11 and may reflect an underlying instability that contributed to the original mutation. The breakpoint extends the genetic map of this region and provides a useful marker for linkage studies and the analysis of allelic segregation in tumor cells.     

A tumor chromosome rearrangement further defines the 11p13 Wilms tumor locus.

A sporadic Wilms tumor, WT-21, with an (11;14)-(p13;q23) reciprocal translocation has been identified. The translocation is found in tumor cells, but not in the patients' circulating lymphocytes. Molecular analysis of somatic cell hybrids segregating the derivative translocation chromosomes reveals a submicroscopic interstitial deletion at the translocation breakpoint, as well as a cytologically undetectable interstitial deletion in the nontranslocation chromosome 11, resulting in a homozygous deletion in 11p13. Pulsed-field gel analysis of tumor DNA indicates that the two deletions are indistinguishable, and the homozygously deleted region is less than 875 kb. The homozygously deleted regions of three other sporadic Wilms tumors overlap with the deleted region in WT-21, and the candidate cDNA clone for the 11p13 Wilms tumor gene described by Call et al. (Cell 60, 509-520, 1990) is included in the deleted region. These findings strengthen previous conclusions regarding the obligate location for the 11p13 WT locus and support the suggestion that the Wilms tumor gene has been cloned.     

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.     

Molecular analysis of a reciprocal translocation t(5;11)(q11;p13) in a WAGR patient

Summary Most patients with the complex association aniridia — predisposition to Wilms' tumor (WAGR syndrome) present with a de novo constitutional deletion of band 11p13. We report a patient with WAGR syndrome and a reciprocal translocation between chromosomes 5 and 11 t(5;11)(q11;p13). High resolution banding cytogenetic analysis and molecular characterization using 11p13 DNA markers showed a tiny deletion encompassing the gene for CAT but sparing the gene for FSHB. This suggests that syndromes associated with apparently balanced translocations may be due to undetectable loss of material at the breakpoint(s) rather than to breakage in the gene itself.