Hum Genet (1992) 89 : 187-193 9 Springer-Verlag1992 Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type I (MEN1) locus Catharina Larsson 1, Giinther Weber 1, Eva Kvanta 1, Kathy Lewis 2, Marie Janson 1, Carol Jones 3, Tom Glaser 4, Glen Evans 2, and Magnus Nordenskj61d 1 1Department of Clinical Genetics, KarolinskaHospital, S-10401 Stockholm,Sweden 2Molecular GeneticsLaboratory, SALK Institute, La Jolla, CA 92037, USA 3Eleanor RooseveltInstitute for Cancer Research, Denver, CO 80206, USA 4Howard Hughes Medical Center, Harvard Medical School, Boston, MA 02115, USA Received August 2, 1991 / Revised September 11, 1991 Summary. Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves un- masking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chro- mosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced so- matic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12-13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction frag- ment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene. Introduction Multiple endocrine neoplasia type 1 (MEN1) is an auto- somal, dominantly inherited predisposition to neoplastic lesions of the parathyroids, the neuroendocrine pan- creas, and the anterior pituitary (reviewed in Larsson and NordenskjOld 1990). The genetic defect has previ- ously been mapped to the centromeric part of the long arm of chromosome 11 by genetic linkage to restriction fragment length polymorphism (RFLP) markers in three Offprint requests to: C. Larsson affected families (Larsson et al. 1988). Furthermore, combined family and tumor genotype analysis has shown that pathogenesis of both pancreatic and parathyroid lesions involves unmasking of a recessive mutation at the MEN1 locus (Knudson 1978; Larsson and NordenskjOld 1990). This is accomplished by, e.g., mitotic recombina- tion, deletion or loss of one chromosome 11 complement (Larsson et al. 1988; Thakker et al. 1989; Friedman et al. 1989; Arnold et al. 1989; Bystr6m et al. 1990). Based on these findings, it is possible to localize the gene further by deletion mapping of tumors and by identification of meiotic cross-overs in families segregating the disease. The most recent linkage map around the MEN1 locus includes seven RFLP markers covering approximately 12 cM; llp-(D11S149-D11S288)-PGA-PYGM-(D11S97- DllS146-1NT2)-llqter (Nakamura et al. 1989; Julier et al. 1990). Meiotic recombinations have been identified between MEN1 and markers within this region, but no detailed mapping of the breakpoints was possible be- cause of uninformativity (Nakamura et al. 1989). In ad- dition, deletion mapping of two tumors has suggested that the MEN1 locus is flanked by PYGM and DllS146 (Bystr6m et al. 1990), which are located 5 cM apart from each other (Julier et al. 1990). In order to sublocalize the MEN 1 gene and to make its isolation possible, we under- took to isolate additional markers from the 11q13 region. In this study, unique cosmid clones were isolated and mapped on a panel of radiation-reduced somatic cell hy- brids. New RFLPs were then identified and used to pin- point meiotic cross-overs close to the MEN1 gene. Materials and methods DNA markers The polymorphic probes, D11S16/p32-1, CAT/pINT-800,DLIS149/ pTHH26, D11S288/p3C7, PYGM/pMCMP1, D11S146/pHBI59, DllS35/phi2-22, DllS84/p2-7-1-D6, ETS1/pHE 5.4 and DllS29/