Somatic mutations of the RET proto-oncogene are not required for tumor development in multiple endocrine neoplasia type 2 (MEN 2) gene carriers.

Germ line mutations in one allele of the RET proto-oncogene predispose to the multiple endocrine neoplasia type 2 (MEN 2) syndromes. To investigate whether these inherited mutations alone can cause the development of tumors in vivo (oncogene model) or whether somatic mutations in the homologous RET allele are required for tumorigenesis (tumor suppressor gene model), we analyzed the entire coding region of both alleles of the RET gene in two MEN 2A and two MEN 2B tumors by reverse transcription-PCR and direct sequencing. No tumor-specific mutations could be detected in either allele of the RET gene in these tumors. Unlike the molecular mechanism in other hereditary tumor syndromes, somatic mutations in the homologous allele are apparently not required in MEN 2 tumorigenesis. Thus, RET genes with MEN 2-specific germ line mutations act as dominantly transforming oncogenes in vivo.

Precipitation variability in the Meuse basin in relation to atmospheric circulation.

The distribution of precipitation events in the Meuse basin during the past century has been found to reflect the large-scale atmospheric circulation, as characterised by the Grosswetterlagen system. Statistical analysis of the long observation records (1911-2002) for the basin showed that although the annual (November to October) and winter half-year (November to April) frequencies of wet days > or = 1 mm/day) were nearly stable, the associated precipitation amounts have significantly increased since 1980. From 1980 onwards, the very wet days (> or = 10 mm/day) in the winter half-year have become more frequent. No obvious change was identified for the summer half-year (May to October) very wet days. Both the precipitation amounts of wet and very wet days in the winter half-year and the occurrence of associated atmospheric circulation of the types/sub-types west cyclone, southwest cyclone and northwest cyclone showed a significant increase around 1980.

Reconciling the origins of Africa, India and Madagascar with vertebrate dispersal scenarios.

Africa, India and Madagascar were once part of the supercontinent of Gondwana. This land mass began to fragment approx. 170 million years ago, and by 83 million years, all of the major components we recognize today were separated by tracts of water. Madagascar's fossil record and estimates of the timing of the extant vertebrate radiations in Madagascar are not easily reconciled with this history of fragmentation. Fossil faunas that lived prior to approx. 65 million years had a cosmopolitan flavour, but this was lost after the Cretaceous-Tertiary boundary. Phylogenetic reconstructions of most extant Malagasy vertebrate radiations indicate divergence times that postdate the End-Cretaceous (lemurs, tenrecs, cichlid fish) and even the Early Miocene (chameleons, carnivores, rodents). Most biogeographic explanations of these groups rely, therefore, on Simpson's model of sweepstakes dispersal (see also cover figure), but there are significant problems in applying the model to migrations from Africa to Madagascar, although its application is not so intractable between India and Madagascar. Alternative migration routes for consideration lie: (1) along the suite of fracture zones between Antarctica and Africa/Madagascar (known as the Antarctic-Africa Corridor), which may have been exposed episodically above sea level; (2) along a series of submerged basaltic ridges/plateaus with known or suspected continental crust between Antarctica and Africa/Madagascar/India flanking the Antarctic-Africa Corridor (e.g. the Madagascar Ridge, Mozambique Ridge, Conrad Plateau, Gunnerus Ridge); (3) between Africa and Madagascar along the Davie Ridge (parts of which are known to have been exposed episodically above sea level); (4) along the Deccan hotspot corridor between India and greater Africa.

Reconstructing the evolutionary history of the Lorisidae using morphological, molecular, and geological data.

Major aspects of lorisid phylogeny and systematics remain unresolved, despite several studies (involving morphology, histology, karyology, immunology, and DNA sequencing) aimed at elucidating them. Our study is the first to investigate the evolution of this enigmatic group using molecular and morphological data for all four well-established genera: Arctocebus, Loris, Nycticebus, and Perodicticus. Data sets consisting of 386 bp of 12S rRNA, 535 bp of 16S rRNA, and 36 craniodental characters were analyzed separately and in combination, using maximum parsimony and maximum likelihood. Outgroups, consisting of two galagid taxa (Otolemur and Galagoides) and a lemuroid (Microcebus), were also varied. The morphological data set yielded a paraphyletic lorisid clade with the robust Nycticebus and Perodicticus grouped as sister taxa, and the galagids allied with Arctocebus. All molecular analyses maximum parsimony (MP) or maximum likelihood (ML) which included Microcebus as an outgroup rendered a paraphyletic lorisid clade, with one exception: the 12S + 16S data set analyzed with ML. The position of the galagids in these paraphyletic topologies was inconsistent, however, and bootstrap values were low. Exclusion of Microcebus generated a monophyletic Lorisidae with Asian and African subclades; bootstrap values for all three clades in the total evidence tree were over 90%. We estimated mean genetic distances for lemuroids vs. lorisoids, lorisids vs. galagids, and Asian vs. African lorisids as a guide to relative divergence times. We present information regarding a temporary land bridge that linked the two now widely separated regions inhabited by lorisids that may explain their distribution. Finally, we make taxonomic recommendations based on our results.

Validity of peak oxygen uptake calculations from heart rate deflection points.

Two graded exercise tests on a cycle ergometer were examined in regard to their predictability of the peak oxygen uptake (VO2peak) based upon the deflection points (Dp) in the heart rate curves. The "constant duration" test (DUR-test) was based upon data from a previous maximal test. The "constant distance" test (DIS-test) was based upon the subjects' body weight and fitness level. In both tests 29 male subjects, 25 to 35 years, pedalled until exhaustion and from these tests the Dp variables (heart rate, DpHR, and the workload, DpW) and the VO2peak were determined. The subjects also underwent the Astrand & Ryhming-test (A&R-test). Deflection points could be detected for 15 subjects (52%) in the DUR-test and for 18 subjects (62%) in the DIS-test. In 13 subjects a Dp in both tests could be determined with a test-retest correlation coefficient of r = 0.66 for the DpHR and r = 0.79 for the DpW. Multiple regression analysis yielded the following equations: VO2peak = 7.7538 + 0.0199 DpW-0.0506 DpHR (n = 15; r = 0.87; SEE = 0.291.min-1) for the DUR-test and VO2peak = 5.8673 + 0.0200 DpW -0.0354 DpHR (n = 18; r = 0.88; SEE = 0.401.min-1) for the DIS-test. Compared to the measured VO2peak of the DUR-test and the DIS-test, the calculated VO2peak from the A&R-test has a correlation coefficient of r = 0.74 and r = 0.77 with a SEE of 0.57 (13.3%) and 0.55 (12.7%) l.min-1, it was concluded that, when a deflection point in the W-HR curve is found, the VO2peak can be calculated more accurately with the developed regression equations in this study than with the A&R-test. However, since the graded exercise cycle tests presented in this paper are only reliable in 33 of the 58 cases, they are useless for evaluating the VO2peak in a practicle sense, especially when no previous information about the appearance of a Dp is available.

Exclusion of the phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene.

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC beta 3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC beta 3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC beta 3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC beta 3 gene were expressed and analyzed. In conclusion, these results exclude the PLC beta 3 gene as a candidate gene for MEN 1.

Exclusion of the nuclear factor-kappa B3 (REL A) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene.

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by neoplasia and hyperplasia in specific endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a region of approximately 900 kb on chromosome 11q13. The nuclear factor-kappa B (NF-kappa B) is a transcription factor with pleiotropic expression, which is involved in the regulation of expression of many cellular genes. The p50/p65 heterodimer is the most abundant form of NF-kappa B. The gene encoding the p65 subunit (NF-kappa B3/REL A) was recently localized in the 900-kb MEN 1 region and was considered a good candidate gene for MEN 1. The structure and nucleotide sequence of the NF-kappa B3 coding region in MEN 1 patients were compared with those of non-MEN 1 subjects, to determine the potential role of this gene in MEN 1 tumorigenesis. Southern blot analysis with constitutional DNA from probands of 14 independent MEN 1 families and DNA from four MEN 1 tumor specimens did not reveal any structural abnormality of the NF-kappa B3 gene. Direct sequencing of cDNAs from two affected subjects from 2 different MEN 1 families, as well as nucleotide sequence analysis of exon/intron boundaries in these patients, did not reveal MEN 1-specific point mutations or other small structural aberrations in the NF-kappa B3 gene. These results make it very unlikely that the NF-kappa B3 gene is the gene responsible for the development of MEN 1.

Criteria for mutation analysis in MEN 1-suspected patients: MEN 1 case-finding.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal, dominantly inherited cancer syndrome, with tumours in various endocrine glands. In 1997 the responsible tumour suppressor gene was identified. MEN1 gene germ-line mutations are detected in the vast majority of MEN 1 patients, however, with regard to case-finding, unfortunately only at a very low frequency in patients with apparently sporadic MEN 1-related tumours. In order to increase the detection rate of disease gene carriers among patients with apparently sporadic MEN 1-related tumours, clinical criteria were needed. DESIGN AND RESULTS: In this study MEN1 gene germ-line mutations were revealed in 16/16 MEN 1 patients/families (100%). Based on our clinical experience with MEN 1 patients/families we formulated clinical criteria to identify disease gene carriers among patients with apparently sporadic MEN 1-related tumours. The criteria for MEN 1-suspected patients are: young age at onset (< 35 years) and/or multiple MEN 1-related lesions in a single organ or two distinct organs affected. Application of these criteria yielded MEN1 gene germ-line mutations in nine of 15 MEN 1-suspected patients (60%), thus identifying novel MEN 1 families. Follow up was also guaranteed for patients not fulfilling these criteria. CONCLUSIONS: The clinical criteria for MEN 1-suspected patients increase the detection rate of germ-line MEN1 gene mutations among patients with apparently sporadic MEN 1-related tumours. These criteria may be used for (presymptomatic) identification of MEN 1 disease gene-carriers, thus enabling early detection of tumour development and timely treatment, as well as genetic counselling.

Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A.

BACKGROUND: Multiple endocrine neoplasia type 2A (MEN-2A) is characterized by medullary thyroid carcinoma in combination with pheochromocytoma and sometimes parathyroid adenoma. Missense mutations in the RET proto-oncogene are associated with MEN-2A. Their detection by DNA analysis allows the identification of carriers of the gene, in whom the risk of medullary thyroid carcinoma is 100 percent. We compared the reliability of biochemical tests with that of DNA analysis in identifying carriers of the MEN2A gene. METHODS: Starting in 1975, we screened 300 subjects in four large families with MEN-2A for expression of the disease, using measurements of plasma calcitonin after stimulation with pentagastrin or calcium and urinary excretion of catecholamines and catecholamine metabolites. We tested for carrier status by DNA analysis, including linkage analysis, and more recently by analysis of mutations in the RET gene. RESULTS: Of 80 MEN2A gene carriers (in 61 of whom carrier status was proved by DNA analysis), 66 had abnormal plasma calcitonin values and medullary thyroid carcinoma. Fourteen young carriers had normal results of plasma calcitonin tests. In 8 of these 14, thyroidectomy revealed small foci of medullary thyroid carcinoma; the remaining 6 have not yet been operated on. Of the other 220 family members, 68 were found by DNA analysis not to carry the MEN2A gene. None of these 68 subjects had medullary thyroid carcinoma or pheochromocytoma; 6 had elevated plasma calcitonin concentrations and underwent thyroidectomy but had only C-cell hyperplasia. CONCLUSIONS: Unlike biochemical tests, DNA analysis permits the unambiguous identification of MEN2A gene carriers.

Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary that represents one of the familial cancer syndromes. The MEN1 locus has been previously localised to chromosome 11q13, and a <300 kb gene-rich region flanked centromerically by PYGM and telomerically by D11S1783 defined by combined meiotic and tumour deletion mapping studies. Two candidate genes, ZFM1 and PPP2R5B, from this region have been previously excluded, and in order to identify additional candidate genes we used a BAC to isolate cDNAs from a bovine parathyroid cDNA library by direct selection. One of the novel genes that we identified, SCG2, proved to be identical to the recently published MEN1 gene, which is likely to be a tumour suppressor gene. The SCG2 transcript was 2.9 kb in all tissues with an additional 4.2 kb transcript also being present in the pancreas and thymus. Mutational analysis of SCG2 in 10 unrelated MEN1 families identified one polymorphism and nine different heterozygous mutations (one missense, four non-sense, one insertional and three deletional frameshifts) that segregated with the disease, hence providing an independent confirmation for the identification of the MEN1 gene.

Construction of a 1.2-Mb sequence-ready contig of chromosome 11q13 encompassing the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by parathyroid, pancreatic, and anterior pituitary tumors. The MEN1 locus has been previously localized to chromosome 11q13, and a 2-Mb gene-rich region flanked by D11S1883 and D11S449 has been defined. We have pursued studies to facilitate identification of the MEN1 gene by narrowing this critical region to a 900-kb interval between the VRF and D11S1783 loci through melotic mapping. This was achieved by investigating 17 cosmids for microsatellite polymorphisms, which defined two novel polymorphisms at the VRF and A0138 loci, and utilizing these to characterize recombinants in MEN1 families. In addition, we have established a 1200-kb sequence-ready contig consisting of 26 cosmids, eight BACs, and eight PACs that encompass this region. The precise locations for 19 genes and three ESTs within this contig have been determined, and three gene clusters consisting of a centromeric group (VRF, FKBP2, PNG, and PLCB3), a middle group (PYGM, ZFM1, SCG1, SCG2 (which proved to be the MEN1 gene), and PPP2R5B), and a telomeric group (H4B, ANG3, ANG2, ANG1, FON, FAU, NOF, NON, and D11S2196E) were observed. These results represent a valuable transcriptional map of chromosome 11q13 that will help in the search for disease genes in this region.

A putative human zinc-finger gene (ZFPL1) on 11q13, highly conserved in the mouse and expressed in exocrine pancreas. The European Consortium on MEN 1.

In the process of identification of the multiple endocrine neoplasia type 1 gene, which was recently published, we isolated a novel gene in the 11q13 region. This gene (named ZFPL1, for zinc-finger protein-like 1) is expressed strongly in the exocrine pancreas as a 1.4-kb polyadenylated RNA encoding a putative protein of 310 amino acids. A mouse EST contig predicts an equally sized murine protein with 91% amino acid sequence identity to the human protein. No significant homology with known proteins could be found through database screening. However, zinc-finger-like domains and leucine-zipper-like motifs in the predicted ZFPL1 protein were identified, suggesting the presence of DNA-binding and dimerization domains possibly involved in transcription regulation. This notion is supported by the presence of a putative bipartite nuclear localization signal. This paper presents the full-length cDNA sequence for this gene, its genomic structure and chromosomal orientation, and expression studies by Northern blot hybridization and RNA in situ hybridization.