RESUMO
BACKGROUND: The genetics of sporadic and non-syndromic familial colorectal cancer (CRC) is not well defined. However, genetic factors that promote the development of precursor lesions, i.e. adenomas, might also predispose to CRC. Recently, an association of colorectal adenoma with two variants (c.507C>T;p.L169L and c.511G>T;p.A171S) of the ileal sodium dependent bile acid transporter gene (SLC10A2) has been reported. Here, we reconstructed haplotypes of the SLC10A2 gene locus and tested for association with non-syndromic familial and sporadic CRC compared to 'hyper-normal' controls who displayed no colorectal polyps on screening colonoscopy. METHODS: We included 150 patients with sporadic CRC, 93 patients with familial CRC but exclusion of familial adenomatous polyposis and Lynch's syndrome, and 204 'hyper-normal' controls. Haplotype-tagging SLC10A2 gene variants were identified in the Hapmap database and genotyped using PCR-based 5' exonuclease assays with fluorescent dye-labelled probes. Haplotypes were reconstructed using the PHASE algorithm. Association testing was performed with both SNPs and reconstructed haplotypes. RESULTS: Minor allele frequencies of all SLC10A2 polymorphisms are within previously reported ranges, and no deviations from Hardy-Weinberg equilibrium are observed. However, we found no association with any of the SLC10A2 haplotypes with sporadic or familial CRC in our samples (all P values > 0.05). CONCLUSION: Common variants of the SLC10A2 gene are not associated with sporadic or familial CRC. Hence, albeit this gene might be associated with early stages of colorectal neoplasia, it appears not to represent a major risk factor for progression to CRC.
Assuntos
Neoplasias Colorretais/genética , Predisposição Genética para Doença , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Adulto , Idoso , Estudos de Casos e Controles , Neoplasias Colorretais Hereditárias sem Polipose/genética , Feminino , Frequência do Gene , Genótipo , Alemanha , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos ProspectivosRESUMO
A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.
Assuntos
Proteínas de Ligação a DNA , Deleção de Genes , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Pareamento Incorreto de Bases/genética , Proteínas de Transporte , Neoplasias Colorretais/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Humanos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas NuclearesRESUMO
Familial adenomatous polyposis (FAP) exhibits a variable phenotype even in carriers of the same adenomatous polyposis coli germline mutation. Xenobiotic-metabolizing enzymes such as N-acetyltransferases (NATs) and glutathione S-transferases (GSTs) were reported to modify the individual risk for colorectal cancer. We examined whether the polymorphisms of the NAT2, GSTM1, and GSTT1 enzymes affect age at diagnosis of first colorectal adenomas or extracolonic manifestations in 411 FAP patients. Neither age at diagnosis of colorectal adenomas nor occurrence of extra-intestinal tumors differed significantly between genotypes at the NAT2 and GSTM1 loci, whereas GSTT1 polymorphism showed an uncertain association with extra-intestinal manifestations. Combinations of supposed at risk genotypes of the three enzymes showed no significant differences either. Thus, NAT2, GSTM1, or GSTT1 are unlikely to modify the disease phenotype in FAP patients.
Assuntos
Polipose Adenomatosa do Colo/genética , Arilamina N-Acetiltransferase/genética , Glutationa Transferase/genética , Polimorfismo Genético/genética , Polipose Adenomatosa do Colo/enzimologia , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Genótipo , Mutação em Linhagem Germinativa , HumanosRESUMO
The evaluation of radioactivity accidentally released into the atmosphere involves determining the radioactivity levels of rainwater samples. Rainwater scavenges atmospheric airborne radioactivity in such a way that surface contamination can be deduced from rainfall rate and rainwater radioactivity content. For this purpose, rainwater is usually collected in large surface collectors and then measured by gamma-spectrometry after such treatments as evaporation or iron hydroxide precipitation. We found that collectors can be adapted to accept large surface (diameter 47mm) cartridges containing a strongly acidic resin (Dowex AG 88) which is able to quantitatively extract radioactivity from rainwater, even during heavy rainfall. The resin can then be measured by gamma-spectrometry. The detection limit is 0.1Bq per sample of resin (80g) for (137)Cs. Natural (7)Be and (210)Pb can also be measured and the activity ratio of both radionuclides is comparable with those obtained through iron hydroxide precipitation and air filter measurements. Occasionally (22)Na has also been measured above the detection limit. A comparison between the evaporation method and the resin method demonstrated that 2/3 of (7)Be can be lost during the evaporation process. The resin method is simple and highly efficient at extracting radioactivity. Because of these great advantages, we anticipate it could replace former rainwater determination methods. Moreover, it does not necessitate the transportation of large rainwater volumes to the laboratory.
Assuntos
Técnicas de Química Analítica/métodos , Monitoramento Ambiental/métodos , Radioisótopos/análise , Chuva/química , Poluentes Radioativos da Água/análise , Resinas de Troca Iônica/química , Espectrometria gama/métodosRESUMO
INTRODUCTION: About 20% of colorectal cancer (CRC) patients show some kind of familiarity, which might be caused by yet unknown combinations of low penetrance susceptibility genes. We aimed to identify genetic factors for familial CRC (fCRC) in a unique study design that includes phenotypic extremes as represented by fCRC cases and 'hyper-normal' controls without CRC history and no adenomatous polyps on colonoscopy. MATERIALS AND METHODS: Candidate gene variants were determined by allele-specific amplification (SLC10A2 c.169C>T and c.171G>T) and restriction fragment length polymorphism assays (CCND1 c.870A>G; CDH1 -160C>A; TP53 R72P; VDR T2M). In total, 98 patients with fCRC, 96 patients with sporadic CRC, and 220 hyper-normal controls were included. RESULTS: The minor allele of the CDH1 -160C>A polymorphism occurred significantly more often in controls compared to fCRC cases (OR = 0.664; p = 0.042). Homozygosity of the minor allele was significantly associated with affiliation to the control group (OR = 0.577; p = 0.029), indicating that both heterozygous and homozygous carriers of the common allele are at-risk for CRC. With respect to the CCND1 c.870A>G mutation, comparison of fCRC and sporadic CRC cases showed that A/A homozygosity was more common than G/G homozygosity among fCRC patients compared to controls (OR = 2.119; p = 0.045). However, no differences in allele or genotype frequencies were detected between sporadic CRC cases and controls, and no associations were observed for SLC10A2, TP53, and VDR polymorphisms. CONCLUSIONS: We report a potential association of variants in the CCND1 and CDH1 genes with fCRC using a unique study design with phenotypic extremes.
Assuntos
Caderinas/genética , Neoplasias Colorretais/genética , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Polimorfismo Genético , Adulto , Idoso , Antígenos CD , Estudos de Casos e Controles , Colonoscopia , Neoplasias Colorretais/patologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Linhagem , Fenótipo , Regiões Promotoras Genéticas , Estudos Prospectivos , Medição de RiscoRESUMO
UNLABELLED: Mutations of the base excision repair gene MUTYH have been reported as underlying genetic defects in autosomal-recessive familial adenomatous polyposis (FAP). Our aim was to determine the frequency of the most common mutations (p.Tyr165Cys and p.Gly382Asp) in patients with strong evidence for familial colorectal cancer (fCRC). METHODS: We recruited 93 patients with fCRC but no indication for monogenic CRC syndromes (FAP, hereditary non-polyposis colorectal cancer). Tumors showed regular expression of MLH1 and MSH2, and microsatellite instability was excluded. Sporadic CRC patients (n=93) and 'hyper-normal' controls without any adenomas in screening colonoscopies (n=93) were studied for comparison. RESULTS: In the fCRC group, two patients carried biallelic mutations (p.Tyr165Cys/p.Tyr165Cys, p.Tyr165Cys/p.Gly382Asp), while four patients displayed a heterozygous genotype (3 x p.Tyr165Cys/wt, 1 x p.Gly382Asp/wt). In contrast, only two p.Gly382Asp/wt patients were detected in the sporadic CRC group and one p.Gly382Asp carrier was observed in 'hyper-normal' controls, and the p.Tyr165Cys risk allele was absent in both control groups. Association tests demonstrated an increased odds ratio (OR) for CRC in carriers of the p.Tyr165Cys risk allele among fCRC patients, as compared to sporadic CRC patients and controls (OR 2.38; p=0.03). CONCLUSIONS: In our cohort the prevalence of pathogenic MUTYH mutations was increased among fCRC patients compared to sporadic CRC and controls. The association of the p.Tyr165Cys mutation with fCRC indicates that this variant represents a susceptibility factor in a defined subgroup of CRC patients with a positive family history.
Assuntos
Neoplasias Colorretais/genética , DNA Glicosilases/genética , Predisposição Genética para Doença , Idoso , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da PolimeraseRESUMO
Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease-causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu-repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols.