Eighteen new polymorphic markers in the multiple endocrine neoplasia type 1 (MEN1) region

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder in which affected individuals develop tumors primarily in the parathyroids, anterior pituitary, endocrine pancreas, and duodenum. The locus for MEN1 is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has previously placed the MEN1 gene within a 2-Mb interval flanked by markers D11S1883 and D11S449. Loss of heterozygosity (LOH) studies in MEN1 and sporadic tumors have helped narrow the location of the gene to a 600-kb interval between PYGM and D11S449. Eighteen new polymerase chain reaction (PCR)-based polymorphic markers were generated for the MEN1 region, with ten mapping to the PYGM-D11S449 interval. These new markers, along with 14 previously known polymorphic markers, were precisely mapped on a 2.8-Mb (D11S480–D11S913) high-density clone contig-based, physical map generated for the MEN1 region. Received: 21 February 1997 / Accepted: 5 June 1997     

Definition of the MinimalMEN1Candidate Area Based on a 5-Mb Integrated Map of Proximal 11q13: THE EUROPEAN CONSORTIUM ON MEN1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with a high penetrance characterized by tumors of the parathyroid glands, the endocrine pancreas, and the anterior pituitary. TheMEN1gene, a putative tumor suppressor gene, has been mapped to a 3- to 8-cM region in chromosome 11q13 but it remains elusive as yet. We have combined the efforts and resources from four laboratories to form the European Consortium on MEN1 with the aims of establishing the genetic and the physical maps of 11q13 and of further narrowing the MEN1 region. A 5-Mb integrated map of the region was established by fluorescencein situhybridization on both metaphase chromosomes and DNA fibers, by hybridization to DNA from somatic cell hybrids containing various parts of human chromosome 11, by long-range restriction mapping, and by characterization of YACs and cosmids. Polymorphic markers were positioned and ordered by physical mapping and genetic linkage in 86 MEN1 families with 452 affected individuals. Two critical recombinants identified in two affected cases placed theMEN1gene in an ≈2-Mb region aroundPYGM,flanked by D11S1883 and D11S449.     

Improved carrier testing for multiple endocrine neoplasia,type 1, using new microsatellite-type DNA markers

Familial multiple endocrine neoplasia, type 1 (FMEN1), is an autosomal dominant trait generated by hyperfunction of various endocrine glands. The gene for MEN1 has been mapped to chromosome 11q13 by genetic linkage and deletion mapping in tumors. Eight Finnish families, including 46 individuals carrying the risk haplotype, have been typed for four polymorphic microsatellite DNA markers spanning the MEN1 chromosomal region. Three of the loci concerned, D11S913, D11S987, and D11S1337, displayed maximum lod scores (Z _max ) 6.70, 9.88, and 2.54, respectively, with no recombinations with the disease gene, whereas a Z _max of 8.43 was obtained for D11S971 at a recombination fraction of 0.03. Our results indicate that the use of this set of markers considerably improves the diagnostic value of genotyping patients at risk of developing MEN1.     

Mapping eight new polymorphisms in 11q13 in the vicinity of multiple endocrine neoplasia type 1: identification of a new distal recombinant

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that predisposes affected individuals to neoplasms of the parathyroid glands, endocrine pancreas, anterior pituitary, and carcinoids. The MEN1 locus has been localized by family studies to 11q13, flanked by markers PGA and D11S97. Eight new polymorphisms located in three separate radiation-reduced somatic cell hybrid segregation groups were developed. The order of the new markers, within the context of previously described loci, was determined by linkage analysis on the Venezuelan reference pedigree. Four independent MEN1 families, consisting of 57 affected individuals, and 70 individuals at-risk for the disease were genotyped. Sixteen people inherited a chromosome that shows recombination between a linked marker and the disease. The nearest proximal and distal markers that show recombination with the disease are D11S822 and GSTP1, respectively, thereby narrowing the candidate region for MEN1 by 50% on the distal side. Using these loci in haplotype analysis, an accurate presymptomatic molecular diagnostic test has been developed. These new markers in 11q13 linked to MEN1 also facilitate the genetic and physical characterization of this very gene-rich region.     

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11g13, and exclusion of μ-calpain as the multiple endocrine neoplasia type 1 gene

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11g13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MENI-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM-SEA-D11S913-D115970-D11S97-D11S146-INT2-D11S971-D11S533-11gter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding μ-calpain (CAPN1) located CAPN1 to llg13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11g13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.     

Improved predictive test for MEN2, using flanking dinucleotide repeats and RFLPs.

Gene(s) for the autosomal dominant endocrine cancer syndromes, multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B (MEN2B), and familial medullary thyroid carcinoma (MTC1) all map to the pericentromeric region of chromosome 10. Predictive testing for the inheritance of mutant alleles in individuals at risk for these disorders has been limited by the availability of highly informative and closely linked flanking markers. We describe the development of eight new markers, including two PCR-based dinucleotide repeat polymorphisms and six RFLPs that flank the disease loci. One of the dinucleotide repeat markers (sJRH-1) derives from the RBP3 locus on 10q11.2 and has a PIC of .88. The other dinucleotide repeat (sTCL-1) defines a new locus, D10S176, that maps by in situ hybridization to 10p11.2 and has a PIC of .68. We have constructed a new genetic linkage map of the pericentromeric region of chromosome 10, on the basis of 13 polymorphisms at six loci, which places the MEN2A locus between the dinucleotide repeat markers, with odds of 5,750:1 over the next most likely position. Using this set of markers, predictive genetic testing of 130 at-risk individuals from six families segregating MEN2A revealed that 95% were jointly informative with flanking markers, representing a significant improvement in genetic testing capabilities.     

Genetic analysis of 24 French families with multiple endocrine neoplasia type 2A.

The gene for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to the pericentromeric region of chromosome 10 by linkage analysis. Thirty-four families with multiple cases of medullary carcinoma of the thyroid (MTC), including 24 families with origins in France, have been typed with nine polymorphic markers spanning the centromere of chromosome 10. No recombination was observed between the MEN2A locus and either of the four loci D10Z1 (lod score 12.79), D10S102 (lod score 6.38), D10S94 (lod score 7.76), and D10S34 (lod score 5.94). There was no evidence for genetic linkage heterogeneity in the panel of 34 families. Haplotypes were constructed for a total of 11 polymorphisms in the MEN2A region, for mutation-bearing chromosomes in 24 French families and for 100 spouse controls. One haplotype was present in four MEN2A families but was not observed in any control (P less than .01). Two additional families share a core segment of this haplotype near the MEN2A gene. It is likely that these six families have a common affected ancestor. Because the incidence of pheochromocytoma among carriers varies from 0% to 74% within these six families, it is probable that additional factors modify the expression of the MEN2A gene.     

A linkage group of five DNA markers on human chromosome 10

Five chromosome 10 DNA markers (D10S1, D10S3, D10S4, D10S5, and RBP3) were typed in five large pedigrees with multiple endocrine neoplasia type 2A (MEN-2A) and in five non-MEN-2A pedigrees. Linkage analyses showed that these loci and the locus for MEN-2A (MEN2A) are in one linkage group spanning at least 70 cM. The order of the marker loci is RBP3-D10S5-D10S3-D10S1-D10S4, with interlocus recombination frequencies of 7, 13-19, 19, and 19%, respectively, all on the same side of MEN2A. Analyses of sex-specific recombination frequencies indicated no significant differences between males and females for any of the map intervals studied. Previous localization of D10S5 and RBP3 to the proximal region of the long arm and the pericentric region, respectively, comparison of results with other studies, and our preliminary results with other chromosome 10 markers suggest that the D10S4 end of the map extends into the long arm. Our linkage map has been constructed using only two- and three-locus analyses. It will be possible to combine our results with those of other groups to construct a more detailed and accurate genetic map of chromosome 10.     

Multiple endocrine neoplasia type 1 (MEN1) in two Asian families

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disease characterized by neoplasia of the parathyroid glands, anterior pituitary and endocrine pancreas, is rarely reported in Asian populations. The MEN1 gene, mapped to chromosome 11q13 but yet to be cloned, has been found to be homogeneous in Caucasian populations through linkage analysis. Here, two previously unreported Asian kindreds with MEN1 are described; link-age analysis using microsatellite polymorphic markers in the MEN1 region was carried out. The first kindred, of Mongolian-Chinese origin, is a multigeneration family with over 150 living members, eight of whom are affected toB. T. Teh and S. I. Hii are to be considered as joint first authors     

Exclusion of ZFM1 as a candidate gene for multiple endocrine neoplasia type 1 (MEN1)

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and linkage studies and a 3.8-cM region flanked by PYGM and D11S97 has been defined. The zinc finger in the MEN1 locus (ZFM1) gene, which has also been mapped to this region, represents a candidate gene for MEN1. The ZFM1 gene, which consists of 14 exons, encodes a 623-amino acid protein and exons 2, 8 and 12 encode the putative nuclear localisation signal, a zinc finger motif, and a proline-rich region, respectively. We have investigated these potentially functional regions for germ-line mutations by single-stranded conformational polymorphism (SSCP) analysis in 64 unrelated MEN1 patients. In addition, we performed DNA sequence analysis of all the 14 exons and 13 of the 26 exon-intron boundaries in four unrelated MEN1 patients. A 6-bp deletion that resulted in the loss of two proline residues at codons 479 and 480 in exon 12 was found in one MEN1 patient. However, this did not co-segregate with MEN1 in the family and represented a rare polymorphism. Analysis by SSCP, DNA sequencing, northern blotting, Southern blotting and pulsed field gel electrophoresis revealed no additional genetic abnormalities of ZFM1 in the other MEN1 patients. Thus, our results indicate that ZFM1 is excluded as a candidate gene for MEN1. Received: 29 October 1996 / Revised: 16 December 1996     

An efficient strategy for gene mapping using multipoint linkage analysis: exclusion of the multiple endocrine neoplasia 2A (MEN2A) locus from chromosome 13.

Members of four families in which multiple endocrine neoplasia type 2A (MEN-2A) is segregating were typed for seven DNA markers and one red cell enzyme marker on chromosome 13. Close linkage was excluded between the MEN2A locus and each marker locus tested. By means of multipoint analysis and the genetic map of chromosome 13 developed by Leppert et al., MEN2A was excluded from any position between the most proximal marker locus (D13S6) and the most distal marker locus (D13S3) and from within 12 cMorgans outside these two loci, respectively. However, the support of exclusion within an interval was diminished under the assumption of a substantially larger genetic map in females. The strategy of multipoint analysis, which excluded between 1.5 and 2.0 times more chromosome 13 than did two-point analysis, demonstrates the utility of linkage maps in mapping disease genes.     

Linkage analysis of multiple endocrine neoplasia type 1 with INT2 and other markers on chromosome 11

We evaluated linkage between the locus for multiple endocrine neoplasia type 1 (MEN1) and several polymorphic DNA markers on chromosome 11 in a single large pedigree. On the basis of the finding of a basic fibroblast growth factor (bFGF)-like substance circulating in plasma of MEN1 patients, we chose a bFGF-related gene known to be localized to 11q13 as one of the markers. This gene locus, INT2, was found to be closely linked to the MEN1 gene. Pairwise and multipoint analyses with INT2 confirm the recent finding by C. Larsson et al. (1988, Nature (London) 332: 85-87) of MEN1 linkage to another marker, skeletal muscle glycogen phosphorylase, at 11q13.     

Isolation and high-resolution mapping of new DNA markers from the pericentromeric region of chromosome 10.

The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including D10Z1, D10S94, D10S97, and D10S102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small subchromosomal fragments that include both D10S94 and D10Z1. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a lambda library made with pp11A DNA. We have completed regional mapping of 22 of those clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between D10S34 and RBP3. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A.     

A refined linkage map for DNA markers around the pericentromeric region of chromosome 10

A refined genetic linkage map for the pericentromeric region of human chromosome 10 has been constructed from data on 12 distinct polymorphic DNA loci as well as the locus for multiple endocrine neoplasia type 2A (MEN 2A), a dominantly inherited cancer syndrome. The map extends from D10S24 (at 10p13-p12.2) to D10S3 (at 10q21-q23) and is about 70 cM long. Overall, higher female than male recombination frequencies were observed for this region, with the most remarkable female excess in the immediate vicinity of the centromere, as previously reported. Most of the DNA markers in this map are highly informative for linkage and the majority of the interlocus intervals are no more than 6 cM apart. Thus this map should provide a fine framework for future efforts in more detailed mapping studies around the centromeric area. A set of ordered cross-overs identified in this work is a valuable resource for rapidly and accurately localizing new DNA clones isolated from the pericentromeric region.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic and physical mapping on chromosome 4 narrows the localization of the gene for facioscapulohumeral muscular dystrophy (FSHD).

We have used a combination of classical RFLPs and PCR-based polymorphisms including CA repeats and single-strand conformation polymorphisms to generate a fine-structure genetic map of the distal long arm of chromosome 4q. This map is now genetically linked to the pre-existing anchor map of 4pter-4q31 and generates, for the first time, a complete linkage map of this chromosome. The map consists of 32 anchor loci placed with odds of greater than 1,000:1. The high-resolution map in the cytogenetic region surrounding 4q35 provides the order 4cen-D4S171-F11-D4S187-D4S163-D4S139-4q ter. When we used somatic cell hybrids from a t(X;4)(p21;q35) translocation, these five markers fell into three groups consistent with the genetic map-D4S171 and F11 in 4pter-4q35, D4S163 and D4S139 in 4q35-4qter, and D4S187 as a junction fragment between these two regions. These markers are in tight linkage to the gene for facioscapulo-humeral muscular dystrophy (FSHD) mapped to this region by several collaborating investigators and provide a framework for further detailed analysis of this region.     

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.