RESUMO
BACKGROUND: The mismatch repair gene, MLH1, appears to occur as two main haplotypes at least in white populations. These are referred to as A and G types with reference to the A/G polymorphism at IVS14-19. On the basis of preliminary experimental data, we hypothesised that deviations from the expected frequency of these two haplotypes could exist in carriers of disease associated MLH1 germline mutations. METHODS: We assembled a series (n=119) of germline MLH1 mutation carriers in whom phase between the haplotype and the mutation had been conclusively established. Controls, without cancer, were obtained from each contributing centre. Cases and controls were genotyped for the polymorphism in IVS14. RESULTS: Overall, 66 of 119 MLH1 mutations occurred on a G haplotype (55.5%), compared with 315 G haplotypes on 804 control chromosomes (39.2%, p=0.001). The odds ratio (OR) of a mutation occurring on a G rather than an A haplotype was 1.93 (95% CI 1.29 to 2.91). When we compared the haplotype frequencies in mutation bearing chromosomes carried by people of different nationalities with those seen in pooled controls, all groups showed a ratio of A/G haplotypes that was skewed towards G, except the Dutch group. On further analysis of the type of each mutation, it was notable that, compared with control frequencies, deletion and substitution mutations were preferentially represented on the G haplotype (p=0.003 and 0.005, respectively). CONCLUSION: We have found that disease associated mutations in MLH1 appear to occur more often on one of only two known ancient haplotypes. The underlying reason for this observation is obscure, but it is tempting to suggest a possible role of either distant regulatory sequences or of chromatin structure influencing access to DNA sequence. Alternatively, differential behaviour of otherwise similar haplotypes should be considered as prime areas for further study.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Estudos de Casos e Controles , Cromossomos , Neoplasias Colorretais Hereditárias sem Polipose/etnologia , Europa (Continente) , Frequência do Gene , Triagem de Portadores Genéticos , Mutação em Linhagem Germinativa , Haplótipos , Humanos , Proteína 1 Homóloga a MutL , América do Norte , Proteínas NuclearesRESUMO
Germ-line mutations in the 5' half of the Adenomatous Polyposis Coli (APC) gene are found in about 80% of the patients affected with familial adenomatous polyposis (FAP). The vast majority of these are nonsense or frameshift mutations which result in the loss of the carboxyl terminus of the APC protein. Using an in vivo assay in yeast, we have identified pathogenic germ-line mutations in 26 of 32 (81%) unrelated Swiss families affected with FAP. Nine mutations were novel and eight families were shown to harbor two recurrent mutations. Correlations were attempted between the location of APC germ-line mutations and clinical manifestations of the disease.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Adolescente , Adulto , Criança , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Saúde da Família , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , FenótipoAssuntos
Variação Genética/genética , Haplótipos/genética , Meiose/genética , Proteínas de Neoplasias/genética , Recombinação Genética/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Sequência de Bases , Proteínas de Transporte , Neoplasias Colorretais Hereditárias sem Polipose/genética , Repetições de Dinucleotídeos/genética , Evolução Molecular , Éxons/genética , Frequência do Gene/genética , Mutação em Linhagem Germinativa/genética , Humanos , Cinética , Funções Verossimilhança , Desequilíbrio de Ligação , Repetições de Microssatélites/genética , Proteína 1 Homóloga a MutL , Mutagênese , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares , Polimorfismo Genético/genética , Seleção GenéticaRESUMO
A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.