Linkage of the multiple endocrine neoplasia type 2B gene (MEN2B) to chromosome 10 markers linked to MEN2A

The syndrome of multiple endocrine neoplasia type 2B (MEN 2B) resembles that of MEN 2A in that both include medullary carcinoma of the thyroid, pheochromocytoma, and autosomal dominant inheritance, but is distinct in that MEN 2B patients have neuromas of the mucous membranes. MEN2A has been linked to RBP3, D10S5, FNRB, D10S15, and D10Z1 near the centromere of chromosome 10. We examined linkage between MEN2B and RFLPs on chromosome 10 in all available members in two or three generations of 14 kindreds. The centromere marker D10Z1 was linked to MEN2B with a peak lod score of 5.42 at theta = 0.02. One possible recombinant was observed between D10Z1 and MEN2B. Multipoint analysis of RFLPs at FNRB, D10Z1, RBP3, and D10S15 gave a peak lod score of 7.12 at the midpoint between D10Z1 and RBP3 on the long arm (band q11). The most likely gene order FNRB-D10Z1-MEN2B was 27 times more likely than MEN2B-FNRB-D10Z1 and 31/2 times more likely than FNRB-MEN2B-D10Z1. Additional data will be required to establish the order of these loci with confidence.     

Linked markers flanking the gene for multiple endocrine neoplasia type 2A

The inherited cancer syndrome multiple endocrine neoplasia type 2A (MEN2A) has recently been mapped to chromosome 10. We have typed 29 families with this disorder with DNA markers from the pericentromeric region of chromosome 10. Two markers, RBP3 and MCK2, were tightly linked to the MEN2A gene at recombination fractions of less than 3%. Multipoint analysis of the linkage data suggests that the gene is located within a 3-cM interval defined by the markers RBP3/MCK2 on one side and TB14.34 on the other. No evidence for locus heterogeneity was detected in any of the 27 families from 14 countries who were informative for the markers tested. The data confirm and refine the original assignment and provide the basis for presymptomatic screening for this disorder.     

Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to the same region of chromosome 10 as multiple endocrine neoplasia type 2A.

Medullary thyroid carcinoma (MTC) occurs as a component of three well-described autosomal dominant familial cancer syndromes. Multiple endocrine neoplasia type 2A (MEN 2A) is characterized by MTC, pheochromocytomas, and parathyroid hyperplasia. Patients with the rarer multiple endocrine neoplasia type 2B (MEN 2B) syndrome develop MTC and pheochromocytomas, as well as mucosal neuromas, ganglioneuromatosis of the gastrointestinal tract, and a characteristic "marfanoid" habitus. Finally, MTC is transmitted in an autosomal dominant pattern in some families without associated pheochromocytomas or parathyroid hyperplasia (familial medullary thyroid carcinoma, MTC1(2). Sixty-one members of two well-characterized kindreds segregating MTC1 and 34 [corrected] members of six families segregating MEN2B were genotyped using a panel of RFLP probes from the pericentromeric region of chromosome 10 near a locus for MEN 2A. Statistically significant linkage was observed between the chromosome 10 centromere-specific marker D10Z1 and MTC1 (maximum pairwise lod score 5.88 with 0% recombination) and D10Z1 and MEN2B (maximum pairwise lod score 3.58 with 0% recombination). A maximum multipoint lod score of 4.08 was obtained for MEN2B at the position of D10Z1. In addition, 92 members of a previously unreported large MEN2A kindred were genotyped, and linkage to the pericentromeric region of chromosome 10 is reported (maximum pairwise lod score of 11.33 with 0% recombination between MEN2A and RBP3). These results demonstrate that both a locus for familial MTC and a locus for MEN 2B map to the pericentromeric region of chromosome 10, in the same region as a locus for MEN 2A. The finding that each of these three clinically distinct familial cancer syndromes maps to the same chromosomal region suggests that all are allelic mutations at the same locus or represent a cluster of genes involved in the regulation of neuroendocrine tissue development.     

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)     

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.     

A new polymorphic marker (D10S97) tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus

Familial multiple endocrine neoplasia type 2A (MEN 2A) is a cancer syndrome that is inherited as an autosomal dominant with high penetrance. Its clinical features are medullary carcinoma of the thyroid, pheochromocytomas, and hyperparathyroidism. A new polymorphic locus D10S97 (probe: KW6SacI) detects a codominant EcoRI polymorphism that is tightly linked to the MEN2A locus. The peak lod score for linkage between D10S97 with MEN2A is 13.03 at =0.00. The polymorphic locus D10S97 maps, by linkage analysis, into the previously defined interval between FNRB and RBP3 to which MEN2A has been assigned. We present physical mapping data showing that the probe pKW6 originates from 10p13 and that the polymorphic locus D10S97 in 10q11.2 is detected by cross-hybridization.     

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     

A new DNA marker (D10S94) very tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus.

Combined somatic cell hybrid and linkage studies between D10S94 and five pericentromeric loci (FNRB, D10Z1, MEN2A, RBP3, and D10S15) have localized the new DNA sequence pcl1/A1S-6-c23 at D10S94 to 10q11.2. No recombinants were observed between D10S94 and D10Z1 or MEN2A. D10S94 maps in proximal 10q11.2 very near to MEN2A. There are three possible orders for the six loci that we investigated from the centromeric region of chromosome 10. At present the genetic data do not allow us to order MEN2A with respect to D10Z1 and D10S94. The three possible orders are FNRB-D10Z1-D10S94-MEN2A-RBP3-D10S15, FNRB-D10Z1-MEN2A-D10S94-RBP3-D10S15, and FNRB-MEN2A-D10Z1-D10S94-RBP3-D10S15. In view of the fact that no recombinants between D10S94 and MEN2A or between D10S94 and D10Z1 were observed, the combined haplotypes formed from RFLPs and D10Z1 and D10S94 will increase the informativeness and accuracy of genotype prediction for at-risk members of the families having the MEN 2A syndrome, particularly when the affected parent is female. The localization of D10S94 with respect to MEN2A will prove valuable in experiments directed toward cloning the MEN2A locus.     

Exclusion of the 13-kDa rapamycin binding protein gene (FKBP2) as a candidate gene for multiple endocrine neoplasia type 1

The MEN1 gene is considered to be a tumour suppressor gene and has been localised to a 1-Mb region of 11q13.1. In this study, we report the physical localisation of the 13-kDa FK506 and rapamycin binding protein gene (FKBP2) to the cosmid marker D11S750, which is located inside the MEN1 region of non-recombination. The product of this gene is involved in signal transduction and is thus a candidate cell growth regulator or tumour suppressor gene. Northern studies have revealed that FKBP2 is expressed in those tissues predisposed to hyperplasia in MEN1; however, single-strand conformation polymorphism analysis and direct sequencing of DNAs from affected members of MEN1 kindreds and sporadic tumour DNAs have been performed and no mutations have been found. These studies exclude FKBP2 as a candidate gene for MEN1.     

Fine-scale mapping of the gene responsible for multiple endocrine neoplasia type 1 (MEN 1).

We have constructed a high-resolution genetic linkage map in the vicinity of the gene responsible for multiple endocrine neoplasia type 1 (MEN1). The mutation causing this disease, inherited as an autosomal dominant, predisposes carriers to development of neoplastic tumors in the parathyroid, the endocrine pancreas, and the anterior lobe of the pituitary. The 12 markers on the genetic linkage map reported here span nearly 20 cM, and linkage analysis of MEN1 pedigrees has placed the MEN1 locus within the 8-cM region between D11S480 and D11S546. The markers on this map will be useful for prenatal or presymptomatic diagnosis of individuals in families that segregate a mutant allele of the MEN1 gene.     

The clinical implications of a positive calcitonin test for C-cell hyperplasia in genetically unaffected members of an MEN2A kindred.

C-cell hyperplasia precedes the development of medullary thyroid carcinoma in multiple endocrine neoplasia type 2A (MEN2A). Identification of abnormal calcitonin levels after a provocative stimulus is a technique that has been widely used to diagnose this preneoplastic condition in an early stage during the development of medullary thyroid carcinoma, when total thyroidectomy is likely to be curative. In a MEN2A kindred, we identified seven individuals with abnormal calcitonin test results, whose carrier state was questionable. Five of these people were thyroidectomized, and C-cell hyperplasia was diagnosed. Four of these individuals were the offspring of a mother who is at risk for the development of MEN2A but who has had normal calcitonin test results throughout the years and of a father who is not at risk but who has had abnormal test results over a period of 10 years, without evidence of progressive elevation. None of these people developed other manifestations of MEN2A. DNA analysis using markers linked to the MEN2A gene demonstrated, with > 99% likelihood, that none of the individuals who could be genotyped was a gene carrier. C-cell hyperplasia due to some mechanism other than the presence of the MEN2A gene may also occur in MEN2A kindreds. DNA analysis offers an important additional tool for proper diagnosis in the clinical management of MEN2A families.     

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.     

Genomic and Yeast Artificial Chromosome Long-Range Regular Article Linking Six Loci in 10q11.2 and Spanning the Multiple Endocrine Neoplasia Type 2A (MEN2A) Region

Multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited cancers that have in common the clinical feature of medullary thyroid carcinoma (MTC). We have performed both genomic long-range restriction mapping and yeast artificial chromosome (YAC) contig assembly and restriction mapping to establish physical linkage, order, and distances between six loci in 10q11.2 near the genes responsible for these hereditary cancers. RET, D10S94, D10S182, and D10S102 have been mapped in genomic DNA. RET, D10S94, D10S182, D10F3853, and the 10q11.2 sequences detected by DNA marker DM124 are encompassed by a 1-Mb YAC contig. Six physically linked loci are within 1.4 Mb and have an order and orientation of 10cen, D10F38S3, DM124, RET, D10S94, D10S182, D10S102, 10qter. Mutations in the RET proto-oncogene have recently been demonstrated to be associated with MEN 2A and FMTC. RET is located within a genetically defined MEN2A candidate interval between D10S141 and D10S94; MEN2B has been mapped to a larger, overlapping region between D10S141 and a more distal locus, RBP3. Both our genomic physical map and our YAC contig span the entire MEN2A candidate region and overlap with that of MEN2B . These maps will facilitate the identification of genes that can be considered candidates for MEN2B and the identification of tumor-specific alterations important in sporadic MTC.     

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.     

Extensive sequence polymorphisms associated with chromosome 10 alpha satellite DNA and its close linkage to markers from the pericentromeric region

Summary Extensive sequence polymorphisms exist in the chromosome 10 alpha satellite DNA (the D10Z1 locus). Polymorphic morphs revealed by the enzymes PstI, EcoRV, and HincII, can be unambiguously scored and make this centromeric region an excellent genetic marker for the study of multiple endocrine neoplasia, type 2A (MEN2A), as well as for chromosome 10 linkage studies in general. Strong positive lod scores and linkage distance relationships between D10Z1 and DNA markers from the chromosome 10 pericentromeric region, especially FNRB and RBP3, known to be on either side of the centromere, provide independent support for mapping of all these loci.     

Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene.

We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species. Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA. Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.     

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.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset ≥60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds.     

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.