Within Band Llpl3 (WAGR Locus/Potter Facies/Kidney Development/Somatic Cell Hybrids/In Situ Localization) DAVID J
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 5355-5359, August 1987 Genetics HRASI-selected chromosome transfer generates markers that colocalize aniridia- and genitourinary dysplasia-associated translocation breakpoints and the Wilms tumor gene within band llpl3 (WAGR locus/Potter facies/kidney development/somatic cell hybrids/in situ localization) DAVID J. PORTEOUS*, WENDY BICKMORE*, SHEILA CHRISTIE*, PATRICIA A. BOYD*t, GWEN CRANSTON*, JUDY M. FLETCHER*, JOHN R. GOSDEN*, DEREK ROUT*, ANNE SEAWRIGHT*, KALLE 0. J. SIMOLAt, VERONICA VAN HEYNINGEN*, AND NICHOLAS D. HASTIE* *Medical Research Council Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, United Kingdom; and tDepartment of Medical Genetics, University of Helsinki, Helsinki, Finland 00290 Communicated by M. F. Lyon, April 13, 1987 (receivedfor review February 2, 1987) ABSTRACT We show that chromosome-mediated gene MIC4 and MIC11 (synonymous with MIC1), all map to band transfer can provide an enriched source of DNA markers for llpl3 and are closely associated with WAGR but not invari- predetermined, subchromosomal regions of the human ge- ably deleted (4, 8). Most recently (8), the /3 subunit of nome. Forty-four human DNA recombinants isolated from a follicle-stimulating hormone (FSHB) has been shown to map HRASI-selected chromosome-mediated gene transformant close to the aniridia and Wilms tumor genes. Of the anony- map exclusively to chromosome 11, with several sublocalizing mous DNA markers mapped to the short arm of chromosome to the Wilms tumor region at llpl3. We present a detailed 11, the majority derive from a somatic cell hybrid considered molecular map of the deletion chromosomes 11 from five to retain lip material as the sole human component (11). WAGR (Wilms tumor/aniridia/genitourinary abnormalities/ However, none of these markers has been found to map as mental retardation) syndrome patients, three of which are at close to WAGR as CAT or FSHB (8). We describe here an the limits of cytogenetic resolution but shown here to be alternative, directed approach to the problem of mapping molecularly distinguishable and overlapping. We can define subchromosomal regions (12-14), by isolation of human ten distinct regions of the short arm of chromosome 11, five of DNA recombinants, following HRASJ-selected, chromo- which subdivide band lipl3. We also map two independent some-mediated gene transfer (CMGT) (15, 16). llpl3 translocation breakpoints to within the smallest region of As we dissect the short arm of chromosome 11 and home overlap defined by the WAGR deletions. The first comes from in on the WAGR region, it becomes increasingly important to a patient with familial aniridia, and the second from a patient determine the linear order ofnew markers. Any translocation with Potter facies and genitourinary dysplasia. The close occurring in the llpl3 region is, therefore, valuable as a similarities in map location and affected cell lineage for Wilms mapping tool and ofparticular interest when accompanied by tumor and genitourinary dysplasia suggest that they may be clinical conditions found associated with WAGR syndrome alternative manifestations of mutation at the same locus. (1, 2). We describe two. The first segregates with aniridia in a Finnish family (17). The second was found in a neonate with The association of aniridia, genitourinary abnormalities, and the clinical features of Potter facies (18, 19) and the patho- mental retardation with Wilms tumor (WAGR syndrome; logical features of genitourinary dysplasia, with urethral and genetic locus WAGR) is well established (1, 2). Cytogenetic ureteral atresia and bilateral undescended testes. analysis of independent WAGR syndrome patients has lo- calized the relevant genetic region to band llpl3 (3). We (4) MATERIALS AND METHODS and others (5-8) have utilized available DNA probes and expressed protein markers to start to define the molecular Cell Culture. The mouse myeloma-human lymphoblastoid extent of the various deletions. In most studies, only one somatic cell hybrids NYX3.1, ANX6.14, and SAX3.10 con- patient has been examined (5-7) or the deletions have been tain the deletion chromosomes 11 from three independent large and therefore likely to show loss of markers that map WAGR patients and have been described (4). Three further some distance from the disease-related genes (8). We now hybrids were established by the same procedure. GOX2 attempt to distinguish the smaller deletions, those which take contains the deletion chromosome 11 from patient GOTY (4), us beyond the resolving power of cytogenetic analysis and MAX15 the deletion chromosome 11 from a WAGR towards defining the smallest region of overlap that is within patient, described as case 7 by Shannon et al. (20). SMX13 reach of complete molecular analysis. We make use of five was established from the same patient as for the LHV hybrids independent WAGR deletion hybrids, three of which are at the limit of cytogenetic resolution and involve only part of Abbreviations: CMGT, chromosome-mediated gene transfer. Ab- band llpl3. breviations for gene loci on chromosome 11 follow the recommen- The short arm of human chromosome 11 (lip) is one of the dations of McAlpine et al. (ref. 36) and are as follows: WAGR, Wilms tumor/aniridia/genitourinary abnormalities/mental retardation; better mapped regions of the genome (9, 10), but there is still AN2, aniridia; WT, Wilms tumor; HRASI, c-Ha-ras-1; HBB, 13- a relative dearth of genetic markers that map within even the globin; INS, insulin; PTH, parathyroid hormone; CALCI, calcitonin larger WAGR syndrome deletions (4, 8). The genes encoding gene-related peptide 1; LDHA, lactate dehydrogenase A; FSHB, /3 erythrocyte catalase (CAT) and two cell surface markers, subunit of follicle-stimulating hormone; CAT, catalase; PGA, pepsinogen A; APOAI, apolipoprotein A-I; MIC4, MIC8, and MICJJ, cell surface antigens recognized by monoclonal antibodies The publication costs of this article were defrayed in part by page charge F10.44.2, 4F2, and 163.A5, respectively. payment. This article must therefore be hereby marked "advertisement" tPresent address: Biology Division, Department of Bioscience and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Biotechnology, University ofStrathclyde, Glasgow, G4 ONR, U.K. 5355 Downloaded by guest on October 3, 2021 5356 Genetics: Porteous et al. Proc. Natl. Acad. Sci. USA 84 (1987) described by Glaser et al. (8) and carries the 11p13-*11pter chromosomes, the breakpoints are as follows. The deletions region of a reciprocal translocation that segregates with in SATO (hybrid SAX3.10), ANNA (hybrid ANX6.14), and aniridia in a Finnish family (17). The POR hybrids were MARGA (hybrid MAX15) are all near the limit of cytogenetic established by fusion of the fibroblast cell line GM4613 detection; each appears to retain part of band 11p13. The (Human Genetic Mutant Cell Repository, Camden, NJ), deletion in MARGA is confined to a reduction ofband 11p13, established from a neonate with Potter facies and genitourin- in agreement with the original observation of Shannon et al. ary dysplasia, and the mouse cell line RAG (21). POR4 carries (20). The deletion in ANNA extends from the middle of band the derived chromosome 11 (llqter-lpl3::2p11-2pter), 11p13 to just beyond the 11p13/14 interface. It is difficult to and POR11 the derived chromosome 2 (2qter-*2pll::11p- distinguish the centromere-proximal breakpoints in NYMI 13--11pter), from the reciprocal of 2;11 translocation chro- (hybrid NYX3. 1), GOTY (hybrid GOX2), and SATO, but the mosomes present in GM4613. Fusion, hybrid selection, deletion in GOTY is visibly larger than that in SATO, subcloning, and culture conditions were essentially as de- extending into band llpl4, whilst the NYMI deletion is scribed previously (4) and will be detailed elsewhere (J.M.F., certainly the largest of the set, extending into band lipiS. J. A. Fantes, H. Morrison, and V.v.H., unpublished data; We have made extensive use of chromosome 11-encoded A.S., J.M.F., D.J.P., N.D.H., and V.v.H., unpublished cell surface markers in selecting cell hybrids containing the data). 1B-8-1/6 is a mouse-human somatic cell hybrid whose appropriate deletion and translocation chromosomes 11. sole human component is chromosome 11 (22). The HRASJ- Detailed methods (J.M.F., J. A. Fantes, H. Morrison, and selected CMGT cell line E67-1 was derived by morphological V.v.H., unpublished data) and analysis with cloned gene transformation of mouse C127 cells (23), with mitotic chro- markers (A.S., J.M.F., D.J.P., N.D.H., and V.v.H., unpub- mosomes isolated from the human bladder carcinoma cell line lished data) will be described elsewhere. Briefly, all of our EJ-18-8D (24), as described (15, 16). deletion hybrids, except ANX6.14, are negative for the Chromosome Analysis. Extended chromosomes from lym- expression of one or both of the short-arm cell surface phoblastoid cell lines and somatic cell hybrids were examined markers MIC4 and MIC11, and for the CAT gene probe (4). following Giemsa and reverse banding, as described (4). The somatic cell hybrid SMX13 expresses LDHA but not DNA Extraction and Analysis. DNA was extracted from MIC4 or MIC11 and the CAT gene is absent; CAT and both lymphoblastoid cell lines and somatic cell hybrids, as de- cell surface markers must map below the aniridia breakpoint scribed previously (25). Restriction endonucleases (Boeh- in band 11p13. POR4 expresses MIC4 and MICl1 but not ringer Mannheim) were used according to the supplier's LDHA; the converse is true for POR11. The presence of the instructions. Total DNA digests were electrophoresed in expected chromosomes in each hybrid was confirmed 0.8% agarose and transferred to Hybond-N membranes cytogenetically. (Amersham), and the membranes were processed as re~com- Testing the HRASJ-Selected CMGT Cloning Strategy. Two mended by the manufacturer. important conditions must be met by the CMGT process if it Cloning, Probe Isolation and Hybridization.