Familial isolated aniridia associated with a translocation involving chromosomes 11 and 22 [t(11;22)(p13;q12.2)]

Summary Isolated aniridia segregated as an autosomal dominant trait in a family with 11 affected members spanning five generations. Four of the eight individuals studied had aniridia associated with glaucoma and cataracts. Cytogenetic studies revealed an apparently balanced reciprocal translocation between chromosomes 11 and 22 [t(11;22)(p13;q12.2)], while four unaffected relatives had normal karyotypes. There is no evidence of Wilms tumor or genitourinary abnormalities in any members of the family. Restriction enzyme analysis of the human catalase gene revealed no abnormalities in the individuals with the translocation. A summary of phenotypic abnormalities in 61 cases associated with aniridia is presented, as well as a comparison of breakpoints in 44 cases of 11p deletion. These data indicate that single breaks at 11p13 are associated with isolated aniridia, while deletion of 11p13 results in aniridia combined with Wilms tumor, genitourinary abnormalities, and/or mental retardation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Genetic mechanisms of tumor-specific loss of 11p DNA sequences in Wilms tumor.

Wilms tumor, a common childhood renal tumor, occurs in both a heritable and a nonheritable form. The heritable form may occasionally be attributed to a chromosome deletion at 11p13, and tumors from patients with normal constitutional chromosomes often show deletion or rearrangement of 11p13. It has been suggested that a germinal or somatic mutation may occur on one chromosome 11 and predispose to Wilms tumor and that a subsequent somatic genetic event on the normal homologue at 11p13 may permit tumor development. To study the frequency and mechanism of such tumor-specific genetic events, we have examined the karyotype and chromosome 11 genotype of normal and tumor tissues from 13 childhood renal tumor patients with different histologic tumor types and associated clinical conditions. Tumors of eight of the 12 Wilms tumor patients, including all viable tumors examined directly, show molecular evidence of loss of 11p DNA sequences by somatic recombination (four cases), chromosome loss (two cases), and recombination (two cases) or chromosome loss and duplication. One malignant rhabdoid tumor in a patient heterozygous for multiple 11p markers did not show any tumor-specific 11p alteration. These findings confirm the critical role of 11p sequences in Wilms tumor development and reveal that mitotic recombination may be the most frequent mechanism by which tumors develop.     

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.     

Children with idiopathic hemihypertrophy and beckwith-wiedemann syndrome have different constitutional epigenotypes associated with wilms tumor

Idiopathic hemihypertrophy (IH) is a congenital overgrowth syndrome associated with an increased risk of embryonal cancers in childhood. A related developmental disorder is Beckwith-Wiedemann syndrome (BWS), which increases risk for embryonal cancers, including Wilms tumor. Constitutional epigenetic alterations associated with BWS have been well characterized and include epigenetic alterations of imprinted genes on 11p15. The frequency of hypermethylation of H19 in children with IH and Wilms tumor, 20% (3/15), was significantly lower than the frequency in children with BWS and Wilms tumor, 79% (11/14; P = .0028). These results indicate that children with IH and Wilms tumor have different constitutional epigenotypes from those of children with BWS and Wilms tumor.     

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.     

Definition of the limits of the Wilms tumor locus on human chromosome 11p13

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.     

Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia

Aniridia is a severe eye disease characterized by iris hypoplasia; both sporadic cases and familial cases with an autosomal dominant inheritance exist. Mutations in the PAX6 gene have been shown to be the genetic cause of the disease. Some of the sporadic cases are caused by large chromosomal deletions, some of which also include the Wilms tumor gene (WAGR syndrome), resulting in an increased risk of developing Wilms tumor. Based on the unique registration of both cancer and aniridia cases in Denmark, we have made the most accurate risk estimate to date for Wilms tumor in sporadic aniridia. We have found that patients with sporadic aniridia have a relative risk of 67 (confidence interval: 8.1-241) of developing Wilms tumor. Among patients investigated for mutations, Wilms tumor developed in only two patients out of 5 with the Wilms tumor gene (WT1) deleted. None of the patients with smaller chromosomal deletions or intragenic mutations were found to develop Wilms tumor. Our observations suggest a smaller risk for Wilms tumor than previous estimates, and that tumor development requires deletion of WT1. We report a strategy for the mutational analysis of aniridia cases resulting in the detection of mutations in 68% of sporadic cases and 89% of familial cases. We also report four novel mutations in PAX6, and furthermore, we have discovered a new alternatively spliced form of PAX6.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

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 as part of a WAGR syndrome in a girl whose brother presented hypospadias

Aniridia can arise as part of the WAGR syndrome (Wilms tumour. aniridia, genitourinary anomalies, and mental retardation), due to a deletion or chromosomal region 11p13. We report a girl with a complete WAGR syndrome, whose brother presented hypospadias. Cytogenetic, FISH and molecular studies showed a deletion in one chromosome 11 of the patient. No cytogenetic rearrangement or deletion affecting the genes included in this region (PAX6 and WT1) were observed in her brother and parents. This excludes a higher risk than that of the general population for developing Wilms tumour in the brother and supports that the presence of WAGR syndrome in the patient and hypospadias in her brother is a chance association. We conclude that the identification and definition of the deletions in the WAGR region, which include the WT1 locus are important in order to identify a high tumour risk in infant patients with aniridia including those without other WAGR anomalies.     

Use of catalase polymorphisms in the study of sporadic aniridia

Summary Catalase is known to map at chromosome 11p13. It is one of the closest known markers to the WAGR locus. Restriction fragment length polymorphisms (RFLP) of the catalase gene may be invaluable for studying rearrangements in somatic tumours, linkage in cases of familial Wilms tumour, and the relationship between sporadic and familial aniridia. We describe a catalase RFLP with two different enzymes and use these polymorphisms to exclude deletion of the catalase gene in patients with sporadic aniridia, including one who is known to have a deletion and another suspected of having a deletion.