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.     

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.     

Constitutional mosaic t(2;7)(q33;p22) and other rearrangements in a girl with Wilms' tumor

A 2-year-old girl with sporadic unilateral Wilms' tumor (WT) not associated with aniridia was found to have, besides other chromosome abnormalities, a t(2;7)(q33;p22) in 6% of her lymphocytes. A comparison with 7 previous WT cases without aniridia in whom diverse chromosomal aberrations were present, reveals a wide heterogeneity and lead us to tentatively classify such changes as causal, secondary, and casual.     

Resolution of the two loci for autosomal dominant aniridia, AN1 and AN2, to a single locus on chromosome 11p13.

Two distinct loci have been proposed for aniridia; AN1 for autosomal dominant aniridia on chromosome 2p and AN2 for the aniridia in the WAGR contiguous gene syndrome on chromosome 11p13. In this report, the kindred segregating for autosomal dominant aniridia, which suggested linkage to acid phosphatase-1 (ACP1) and led to the assignment of the AN1 locus on chromosome 2p, has been updated and expanded. Linkage analysis between the aniridia phenotype and ACP1 does not support the original linkage results, excluding linkage up to theta = 0.17 with Z = -2. Tests for linkage to other chromosome 2p markers. APOB, D2S71, D2S5, and D2S1, also excluded linkage to aniridia. Markers that have been isolated from the chromosome 11p13 region were then analyzed in this aniridia family. Two RFLPs at the D11S323 locus give significant evidence for linkage. The PvuII polymorphism detected by probe p5S1.6 detects no recombinants, with a maximum lod score of Z = 6.97 at theta = 0.00. The HaeIII polymorphism detected by the probe p5BE1.2 gives a maximum lod score of Z = 2.57 at theta = 0.00. Locus D11S325 gives a lod score of Z = 1.53 at theta = 0.00. These data suggest that a locus for aniridia (AN1) on chromosome 2p has been misassigned and that this autosomal dominant aniridia family is segregating for an aniridia mutation linked to markers in the 11p13 region.     

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.     

Autosomal dominant aniridia linked to the chromosome 11p13 markers catalase and D11S151 in a large Dutch family

In a large pedigree with autosomal dominant aniridia, we found close linkage between the aniridia locus AN2 and the markers catalase (CAT) (zeta = 7.27 at theta = 0.00) and D11S151 (zeta = 3.86 at theta = 0.10) flanking the AN2 locus on 11p13. Positive lod scores were also obtained for the 11p13----11p14 markers D11S16 and FSHB with the linkage group CAT/AN2/D11S151. We conclude that the autosomal dominant aniridia in this family is due to a mutation at the AN2 locus on 11p13. We have excluded linkage (zeta less than -2 at theta less than 0.18) between the aniridia and the chromosome 2p25 marker D2S1 (linked to ACP1).     

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.     

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.     

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.     

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.     

Location of the gene involving the small eye mutation on mouse chromosome 2 suggests homology with human aniridia 2 (AN2)

Using an interspecific backcross, we have mapped the gene involved in the mouse Small eye mutation (SeyMH) relative to six cloned markers on chromosome 2 (Hox-5.1, Cas-1, Fshb, Bmp-2a, and ld) and the agouti locus. The results suggest that the Sey gene maps between Fshb and Cas-1. Human mapping studies have shown that the aniridia (AN2) gene, which is part of the Wilms tumor susceptibility, aniridia, genitourinary abnormalities, and mental retardation (WAGR) complex, is also between FSHB and CAT on human chromosome 11. The conserved linkage of the cloned markers and the similarity of the Sey/+ and AN2/+ phenotypes suggest that the gene involved in the Sey mutation is the mouse homolog of the human AN2 gene.     

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.     

Molecular analysis of chromosome region 11p13 in patients with Drash syndrome

Summary The association of nephropathy, Wilms' tumour and genital abnormalities is known as Drash syndrome. Two of these features are also seen in the WAGR (Wilms' tumour, aniridia, genito-urinary abnormalities, mental retardation) complex, known to be associated with deletions of chromosome region 11p1S. We have carried out karyotypic and molecular studies in 10 Drash patients, 5 males and 5 females. All the males had a 46XY karyotype as did 3/5 of the phenotypic females, the other two having a 46XX karyotype. One of the 46XX females also had a deletion of region 11p13–p12, the only detectable autosomal chromosome abnormality in any of the patients studied. Lymphoblastoid cell lines were prepared from 6 of the Drash patients and were used in dosage studies using a variety of DNA probes from the 11p13 region. There was no evidence of microdeletions in any patient with a normal karyotype. Because of the 46XY karyotype in phenotypic females, selected X and Y chromosome loci were analysed and all found to be normal. Although Drash syndrome is likely to be of genetic origin, there are no readily detected deletions within the 11p13 region.     

WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin

Denys-Drash syndrome (DDS) is characterized by early onset nephropathy, pseudohermaphroditism in males and a high risk for developing Wilms' tumour (WT). The exact cause of DDS is unknown but germline mutations in the Wilms' tumour suppressor gene (WT1) have recently been described in the majority of DDS patients studied. These mutations occur de novo and are clustered around the zinc finger (ZF) coding exons of the WT1 gene. Analysis of exons 2–10 of the WT1 gene in constitutional DNA from five patients with DDS was carried out using the polymerase chain reaction (PCR) and direct DNA sequencing. In four out of the five patients, heterozygous germline mutations were found: a novel point mutation in exon 8 (ZF₂) at codon 377 altering the wild-type histidine to arginine, and three previously described point mutations in exon 9 (ZF₃) in the codons corresponding to amino acids ³⁹⁴Arg and ³⁹⁶Asp. In one patient, no mutations could be demonstrated. In three patients where parental DNA was available, the mutations were shown to have occurred de novo. Furthermore, since tumour DNA in two of these cases had lost the wild-type allele, polymorphic markers from the short arm of chromosome 11 were used to determine the parental origin of the mutant chromosome. In both cases, the mutant chromosome was shown to be of paternal origin. Since the majority of published WT1 mutations in DDS patients alter a RsrII restriction site in exon 9, we were able to perform PCR-based diagnosis in a female patient with early renal insufficiency and normal external genitalia.     

Detection of a cryptic paracentric inversion within band 11p13 in familial aniridia by fluorescence in situ hybridization

We report the first familial case of dominantly inherited aniridia with a cryptic inversion within band 11p13. High-resolution chromosome analysis gave a suspicion of a tiny constitutional aberration around band 11p13 and fluorescence in situ hybridization using 11p cosmids successfully confirmed that the aniridia patients of this family have an inversion within band 11p13. The distal breakpoint of the inversion is telomeric to a candidate aniridia gene (AN2) and suggests that more genes might be involved in the etiology of aniridia. In situ hybridization is a powerful tool to detect cryptic rearrangements in sporadic or familial patients with aniridia. This family indicated the importance of careful observation of the 11p13 region of aniridia patients, even if the aniridia was autosomal dominantly inherited.     

Sheep gene mapping: assignment of ALDOB, CYP19, WT and SOX2 by somatic cell hybrid analysis

Twenty-four hamster-sheep hybrid cell lines representing eleven ovine synteny groups were used to make syntenic assignments for seven loci ALDOB (aldolase B, fructose biphosphate); AMH (anti-Müllerian hormone); CYP19 [cytochrome P₄₅₀ aromatase, subfamily XIX (aromatization of androgens)]; WT (Wilms' tumour gene); SOX2 (SRY-related HMG-box gene 2); FSHB (follicle-stimulating hormone, beta polypeptide); and SRY (sex region of Y chromosome). These loci were assigned to synteny groups U11(chr2) ( ALDOB ); U19 ( AMH ); U3(chr7) ( CYP19 ); and to chromosomes 15 ( WT ) and 1 ( SOX2 ). SRY defines the hybrids containing the Y chromosome.     

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.     

A panel of restriction fragment length polymorphisms for chromosomal band 11p13

Summary A panel of seven chromosome 11p13 restriction fragment length polymorphisms (RFLPs) detected by five DNA probes is described. Two alleles were identified for each polymorphism, and Mendelian segregation of alleles was observed. Allele frequencies range from 0.13/0.87 to 0.44/0.56. This panel of 11p13 RFLPs will be useful for linkage studies investigating the role of 11p13 genes in Wilms' tumor, aniridia, and genitourinary anomalies. Additionally, these RFLPs will be important tools for studying tumor-specific 11p13 alterations in Wilms' tumor and other cancers.     

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.     

Hitch-hiking from HRAS1 to the WAGR locus with CMGT markers.

The clinical association of Wilms' tumour with aniridia, genitourinary abnormalities and mental retardation (WAGR syndrome) is characterised cytogenetically by variable length, constitutional deletion of the short arm of chromosome 11, which always includes at least part of band 11p13. HRAS1-selected chromosome mediated gene transfer (CMGT) generated a transformant, E65-6, in which the only human genes retained map either to band 11p13 or, with HRAS1, in the region 11p15.4-pter. Human recombinants isolated from E65-6 were mapped to a panel of five WAGR deletion hybrids and two clinically related translocations. We show that E65-6 is enriched congruent to 400-fold for 11p15.4-pter markers and congruent to 200-fold for 11p13 markers. 'Hitch-hiking' from HRAS1 with CMGT markers has allowed us to define seven discrete intervals which subtend band 11p13. Both associated translocations co-locate within the smallest region of overlap for the WAGR locus, which has been redefined by identifying a new interval closer than FSHB.