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.     

An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms' tumor

We have recently described the isolation of a candidate for the Wilms' tumor susceptibility gene mapping to band p13 of human chromosome 11. This gene, primarily expressed in fetal kidney, appears to encode a DNA binding protein. We now describe a sporadic, unilateral Wilms' tumor in which one allele of this gene contains a 25 bp deletion spanning an exon-intron junction and leading to aberrant mRNA splicing and loss of one of the four zinc finger consensus domains in the protein. The mutation is absent in the affected individual's germline, consistent with the somatic inactivation of a tumor suppressor gene. In addition to this intragenic deletion affecting one allele, loss of heterozygosity at loci along the entire chromosome 11 points to an earlier chromosomal nondisjunction and reduplication. We conclude that inactivation of this gene, which we call WT1, is part of a series of events leading to the development of Wilms' tumor.     

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.     

A mitotic recombination in Wilms tumor occurs between the parathyroid hormone locus and 11p13

Summary Wilms tumor is believed to occur as the result of two mutations affecting both alleles of a critical gene located within the p13 band of chromosome 11 (Knudson and Strong 1972; Riccardi et al. 1978). Several mechanisms by which these mutations occur have already been determined in retinoblastoma (Cavenee et al. 1983) and Wilms tumor (Koufos et al. 1984; Orkin et al. 1984; Reeve et al. 1984; Fearon et al. 1984a; Eccles et al. 1984). Of the various mechanisms, however, no example of a mitotic recombination was demonstrated in Wilms tumor. An example is presented here which has been detected by the use of restriction fragment length polymorphisms (RFLPs) mapping to chromosome 11p. In addition the data presented are consistent with the mapping location of parathyroid hormone (PTH) being proximal to 11p13.     

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.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

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.     

Constitutional and somatic deletions of two different regions of maternal chromosome 11 in Wilms tumor

Loss of heterozygosity for 11p markers and preferential loss of maternal alleles have been described in Wilms tumor. In this report we describe the molecular characterization of the constitutional and somatic 11p rearrangements in a del(11p13) WAGR patient with Wilms tumor. Both rearrangements led to loss of maternal alleles for two different regions of 11p, namely, 11p13 and 11p14----p15. This result clearly suggests that Knudson's hypothesis of two hits at the same locus does not necessarily apply to Wilms tumor. Moreover, the loss of 11p15 maternal alleles in the tumor is not incompatible with maternal inheritance of predisposition at 11p13. The putative roles of these two loci are discussed.     

Parental origin of de novo constitutional deletions of chromosomal band 11p13.

One-half of all cases of Wilms tumor (WT), a childhood kidney tumor, show loss of heterozygosity at chromosomal band 11p13 loci, suggesting that mutation of one allele and subsequent mutation or loss of the homologous allele are important events in the development of these tumors. The previously reported nonrandom loss of maternal alleles in these tumors implied that the primary mutation occurred on the paternally derived chromosome and that it was "unmasked" by loss of the normal maternal allele. This, in turn, suggests that the paternally derived allele is more mutable than the maternal one. To investigate whether germinal mutations are seen with equal frequency in maternally versus paternally inherited chromosomes, we determined the parental origin of the de novo germinal 11p13 deletions in eight children by typing lymphocyte DNA from these children and from their parents for 11p13 RFLPs. In seven of the eight cases, the de novo deletion was of paternal origin. The one case of maternal origin was unremarkable in terms of the size or extent of the 11p13 deletion, and the child did develop WT. Transmission of 11p13 deletions by both maternal and paternal carriers of balanced translocations has been reported, although maternal inheritance predominates. These data, in addition to the general preponderance of paternally derived, de novo mutations at other loci, suggest that the increased frequency of paternal deletions we observed is due to an increased germinal mutation rate in males.     

Homozygous deletions define a region of 8p23.2 containing a putative tumor suppressor gene

Loss of heterozygosity at microsatellite loci in chromosomal band 8p23.2 is a frequent event in squamous cell carcinomas of the head and neck, suggesting that this region contains a putative tumor suppressor. Allelic loss studies on laryngeal and oral/oropharyngeal tumors have restricted the size of this region to approximately 1 cM. A similar pattern of deletions is also observed in prostatic and ovarian adenocarcinomas. As part of an effort to identify this gene by positional cloning, we developed a physical contig consisting of 12 overlapping bacterial artificial chromosome (BAC) clones spanning this interval. We developed sequence-tagged sites from the ends of these BACs and used them, along with seven microsatellite loci, to detect and map homozygous deletions in four head and neck squamous cancer cell lines. Our mapping analysis further restricted the consensus minimal region of deletion to a <191-kb interval.     

A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region.

We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels. Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors. As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones. Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors.     

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 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.     

11p13 deletions can be more frequent than the PAX6 gene point mutations in Polish patients with aniridia

Aniridia is a rare, bilateral, congenital ocular disorder causing incomplete formation of the iris, usually characterized by iris aplasia/hypoplasia. It can also appear with other ocular anomalies, such as cataracts, glaucoma, corneal pannus, optic nerve hypoplasia, macular hypoplasia, or ectopia lentis. In the majority of cases, it is caused by mutation in the PAX6 gene, but it can also be caused by microdeletions that involve the 11p13 region. Twelve unrelated patients of Polish origin with a clinical diagnosis of aniridia were screened for the presence of microdeletions in the 11p13 region by means of multiplex ligation probe amplification (MLPA). Additionally, the coding regions of the PAX6 gene were sequenced in all probands. MLPA examination revealed different size deletions of the 11p13 region in five patients. In three cases, deletions encompassed the entire PAX6 gene and a few adjacent genes. In one case, a fragment of the PAX6 gene was deleted only. In the final case, the deletion did not include any PAX6 sequence. Our molecular findings provide further evidence of the existence of the distant 3′ regulatory elements in the downstream region of the PAX6 gene, which is known from other studies to influence the level of protein expression. Sequence analysis of the PAX6 gene revealed the three different point mutations in the remaining four patients with aniridia. All the detected mutations were reported earlier. Based on accomplished results, the great diversity of the molecular basis of aniridia was found. It varies from point mutations to different size deletions in the 11p13 region which encompass partly or completely the PAX6 gene or cause a position effect.     

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.     

Further mapping of an ataxia-telangiectasia locus to the chromosome 11q23 region.

We recently mapped the gene for ataxia-telangiectasia group A (ATA) to chromosome 11q22-23 by linkage analysis, using the genetic markers THY1 and pYNB3.12 (D11S144). The most likely order was cent-AT-S144-THY1. The present paper describes further mapping of the AT locus by means of a panel of 10 markers that span approximately 60 cM in the 11q22-23 region centered around S144 and THY1. Location scores indicate that three contiguous subsegments within the [S144-THY1] segment, as well as three contiguous segments telomeric to THY1, are each unlikely to contain the AT locus, while the more centromeric [STMY-S144] segment is most likely to contain the AT locus. These data, together with recent refinements in the linkage and physical maps of 11q22-23, place the AT locus at 11q23.