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.     

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.     

A panel of irradiation-reduced hybrids selectively retaining human chromosome 11p13: their structure and use to purify the WAGR gene complex

The irradiation-fusion technique offers a means to isolate intact subchromosomal fragments of one mammalian species in the genetic background of another. Irradiation-reduced somatic cell hybrids can be used to construct detailed genetic and physical maps of individual chromosome bands and to systematically clone genes responsible for hereditary diseases on the basis of their chromosomal position. To assess this strategy, we constructed a panel of hybrids that selectively retain the portion of human chromosome band 11p13 that includes genes responsible for Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (constituting the WAGR syndrome). A hamster-human hybrid containing the short arm of chromosome 11 as its only human DNA (J1-11) was gamma-irradiated and fused to a Chinese hamster cell line (CHO-K1). We selected secondary hybrid clones that express MIC1 but not MER2, cell-surface antigens encoded by bands 11p13 and 11p15, respectively. These clones were characterized cytogenetically by in situ hybridization with human repetitive DNA and were tested for their retention of 56 DNA, isozyme, and antigen markers whose order on chromosome 11p is known. These cell lines appear to carry single, coherent segments of 11p spanning MIC1, which range in size from 3000 kb to more than 50,000 kb and which are generally stable in the absence of selection. In addition to the selected region of 11p13, two cell lines carry extra fragments of the human centromere and two harbor small, unstable segments of 11p15. As a first step to determine the size and molecular organization of the WAGR gene complex, we analyzed a subset of reduced hybrids by pulsed-field gel electrophoresis. A small group of NotI restriction fragments comprising the WAGR complex was detected in Southern blots with a cloned Alu repetitive probe. One of the cell lines (GH3A) was found to carry a stable approximately 3000-kb segment of 11p13 as its only human DNA. The segment encompasses MIC1, a recurrent translocation breakpoint in acute T-cell leukemia (TCL2), and most or all of the WAGR gene complex, but does not include the close flanking markers D11S16 and delta J. This hybrid forms an ideal source of molecular clones for the developmentally fascinating genes underlying the WAGR syndrome.     

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.     

Ferritin H gene polymorphism in idiopathic hemochromatosis

Summary We have analysed karyotypes and DNA from three patients with aniridia (congenital absence of irises) and Wilms' tumour. All three had constitutional deletions from the short arm of chromosome 11. The minimum region of overlap of the deletion involves a small region of band 11p13 presumed to contain the genetic loci responsible for both phenotypic abnormalities. Using cells from these patients, somatic cell hybrids with transformed mouse cells have been prepared. Individual subclones retaining either the deletion-11 chromosome or the normal chromosome 11, in addition to a variety of other human chromosomes, have been identified. The relative position of these breakpoints have been determined and the panel of hybrids has been used to map randomly-isolated 11p13 DNA sequences. The characterisation of these deletions has provided a useful panel of hybrids for random mapping strategies designed to identify the Wilms' and aniridia genes.     

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.     

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.     

Characterization of a panel of somatic cell hybrids for subregional mapping along 11p and within band 11p13

Summary The short arm of chromosome 11 carries genes involved in malformation syndromes, including the aniridia/genitourinary abnormalities/mental retardation (WAGR) syndrome and the Beckwith-Wiedemann syndrome, both of which are associated with an increased risk of childhood malignancy. Evidence comes from constitutional chromosomal aberrations and from losses of heterozygosity, limited to tumor cells, involving regions 11p13 and 11p15. In order to map the genes involved more precisely, we have fused a mouse cell line with cell lines from patients with constitutional deletions or translocations. Characterization of somatic cell hybrids with 11p-specific DNA markers has allowed us to subdivide the short arm into 11 subregions, 7 of which belong to band 11p13. We have thus defined the smallest region of overlap for the Wilms' tumor locus bracketed by the closest proximal and distal breakpoints in two of these hybrids. The region associated with the Beckwith-Wiedemann syndrome spans the region flanked by two 11p15.5 markers, HRAS1 and HBB. These hybrids also represent useful tools for mapping new markers to this region of the human genome.     

The genes for the highly homologous Ca(2+)-binding proteins oncomodulin and parvalbumin are not linked in the human genome.

The chromosomal loci of the human parvalbumin and oncomodulin single-copy genes that encode structurally and evolutionarily closely related Ca(2+)-binding proteins were determined by somatic cell hybrid analysis. Southern blot analysis of genomic DNA from 25 human-hamster somatic cell hybrids showed that the human gene for oncomodulin resides on chromosome 7. Analysis of human-mouse hybrids selectively retaining human chromosome 7 or a portion of it allowed specific assignment of the gene locus to the p11-p13 region of chromosome 7 known to be mutated or deleted in patients with the Greig cephalopolysyndactyly syndrome. By gene dosage analysis on Southern blots, we showed that the gene for human parvalbumin maps distally to the cat eye syndrome marker D22S9 on chromosome 22q. Using somatic cell hybrids containing parts of human chromosome 22, the parvalbumin gene was sublocalized to the region 22q12-q13.1. This region contains a linkage group that maps to mouse chromosome 15, region E, and includes the SIS, ARSA, and DIA 1 genes. Our findings are consistent with the recent localization of the mouse parvalbumin gene to this region by two independent methods (C. H. Zühlke et al., 1989, Genet. Res. 54:37-43; S. Adolph et al., 1989, Cytogenet. Cell Genet. 52:177-179).     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

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.     

The potassium channel gene HK1 maps to human chromosome 11p14.1, close to the FSHB gene

Transiently activating (A-type) potassium (K) channels are important regulators of action potential and action potential firing frequencies. HK1 designates the first human cDNA that is highly homologous to the rat RCK4 cDNA that codes for an A-type K-channel. The HK1 channel is expressed in heart. By somatic cell hybrid analysis, the HK1 gene has been assigned to human chromosome 11p13-p14, the WAGR deletion region (Wilms tumor, aniridia, genito-urinary abnormalities and mental retardation). Subsequent pulsed field gel (PFG) analysis and comparison with the well-established PFG map of this region localized the gene to 11p14, 200–600kb telomeric to the FSHB gene.     

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.     

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.     

Isolation and regional localisation of DNA sequences from a human chromosome 11-specific cosmid library

Summary A cosmid library has been prepared in the lorist-B vector from a mouse/human somatic cell hybrid containing region 11q23-11pter as the only human component. This chromosome region is stably maintained in the hybrid as a result of translocation onto one copy of mouse chromosome 13. Individual cosmids containing human DNA were isolated by their ability to hybridise with total human DNA, digested with either HindIII or EcoRI, and 33 individual unique sequences were identified. These fragments were then isolated and subcloned into the bluescribe plasmid vector. Regional localisation of these unique sequences was achieved using a panel of somatic cell hybrids containing different overlapping deletions of chromosome 11. The majority of the 33 mapped sequences derived from the long arm of chromosome 11. Two clones were located within the 11p13–p14 region, which is associated with a predisposition to Wilms' tumour. These probes supplement those already mapped to this chromosome and will assist in the generation of a detailed chromosome 11 linkage map.     

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.     

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.     

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.