Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.
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The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage. (+info)
Identification of a C/G polymorphism in the promoter region of the BRCA1 gene and its use as a marker for rapid detection of promoter deletions.
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Reduced expression of BRCA1 has been implicated in sporadic breast cancer, although the mechanisms underlying this phenomenon remain unclear. To determine whether regulatory mutations could account for the reduced expression, we screened the promoter region by sequencing in 20 patients with sporadic disease. No mutations were detected; however, a new polymorphism consisting of a C-to-G base change within the beta-promoter was identified, with the frequency of the G allele being 0.34. Close to complete linkage disequilibrium was found between this marker and the Pro871 Leu polymorphism, situated in exon 11, which has previously been shown not to be associated with breast or ovarian cancer. This indicates that the C/G polymorphism is also unlikely to play a role in either disease. However, the strength of linkage disequilibrium between these markers permitted their use for rapid screening for genomic deletions within BRCA1. A series of 214 cases with familial breast cancer were analysed using this approach; 88/214 were heterozygous for the promoter polymorphism, thereby excluding a deletion in this region. Among the remaining patients, one hemizygous case reflecting a promoter deletion was successfully identified. Therefore, this study indicates that deletions within the beta-promoter region of BRCA1 are an uncommon event in familial breast cancer. Furthermore, it suggests that mutations within the BRCA1 promoter are unlikely to account for the reported decreased expression of BRCA1 in sporadic disease. (+info)
CDKN2A variants in a population-based sample of Queensland families with melanoma.
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BACKGROUND: Mutations in the CDKN2A gene confer susceptibility to cutaneous malignant melanoma (CMM); however, the population incidence of such mutations is unknown. Polymorphisms in CDKN2A have also been described, but it is not known whether they influence melanoma risk. We investigated the association of CDKN2A mutations and polymorphisms with melanoma risk in a population-based sample of families ascertained through probands with melanoma. METHODS: The 482 Queensland, Australia, families in our sample were characterized previously as having high, intermediate, or low family risk of CMM. Unrelated individuals (n = 200 families/individuals) drawn from the Australian Twin Registry served as control subjects. For individuals in the high-risk group, the entire CDKN2A gene coding region was screened for mutations by use of the polymerase chain reaction, agarose gel electrophoresis, allele-specific oligonucleotide (ASO) hybridization, and single-strand conformation polymorphism analysis. The intermediate- and low-risk families and control subjects were analyzed by ASO hybridization for a total of six recurring mutations as well as for polymorphisms at nucleotides (Nts) 442, 500, and 540. RESULTS: CDKN2A mutations were found only in the high-risk families (nine [10.3%] of 87). The prevalence of the Nt500G (guanosine) polymorphism increased linearly with increasing familial risk (two-sided P = .02) and was highest in the nine (primarily Celtic) families with CDKN2A mutations. After adjustment for ethnic origin, the relationship between risk group and the frequency of the Nt500G allele was weakened (P = .25); however, there was no relationship between ethnic origin and Nt500-polymorphism frequency among the control subjects. CONCLUSIONS: CDKN2A mutations are rare in this population (approximately 0.2% of all melanoma cases in Queensland) and appear to be associated with melanoma in only the most affected families. The Nt500G allele appears to be associated with familial risk, but this association probably reflects Celtic ancestry. (+info)
The relationship between a polymorphism in CYP17 with plasma hormone levels and breast cancer.
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The A2 allele of CYP17 has been associated with polycystic ovarian syndrome, elevated levels of certain steroid hormones in premenopausal women, and increased breast cancer risk. We prospectively assessed the association between the A2 allele of CYP17 and breast cancer risk in a case-control study nested within the Nurses' Health Study cohort. We also evaluated associations between this CYP17 genotype and plasma steroid hormone levels among postmenopausal controls not using hormone replacement to assess the biological significance of this genetic variant. Women with the A2 allele were not at an increased risk of incident breast cancer [OR (odds ratio), 0.85; 95% CI (confidence interval), 0.65-1.12] or advanced breast cancer (OR, 0.84; 95% CI, 0.54-1.32). We did observe evidence that the inverse association of late age at menarche with breast cancer may be modified by the CYP17 A2 allele. The protective effect of later age at menarche was only observed among women without the A2 allele (A1/A1 genotype: for age at menarche > or =13 versus <13; OR, 0.57; 95% CI, 0.36-0.90; A1/A2 and A2/A2 genotypes: OR, 1.05; 95% CI, 0.76-1.45; P for interaction = 0.07). Among controls, we found women with the A2/A2 genotype to have elevated levels of estrone (+14.3%, P = 0.01), estradiol (+13.8%, P = 0.08), testosterone (+8.6%, P = 0.34), androstenedione (+17.1%, P = 0.06), dehydroepiandrosterone (+14.4%, P = 0.02), and dehydroepiandrosterone sulfate (+7.2%, P = 0.26) compared with women with the A1/A1 genotype. These data suggest that the A2 allele of CYP17 modifies endogenous hormone levels, but is not a strong independent risk factor for breast cancer. (+info)
Ethylnitrosourea-induced development of malignant schwannomas in the rat: two distinct loci on chromosome of 10 involved in tumor susceptibility and oncogenesis.
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Inbred rodent strains with differing sensitivity to experimental tumor induction provide model systems for the detection of genes that either are responsible for cancer predisposition or modify the process of carcinogenesis. Rats of the inbred BD strains differ in their susceptibility to the induction of neural tumors by N-ethyl-N-nitrosourea (EtNU). Newborn BDIX rats that are exposed to EtNU (80 microg/g body weight; injected s.c.) develop malignant schwannomas predominantly of the trigeminal nerves with an incidence >85%, whereas BDIV rats are entirely resistant. A T:A-->A:T transversion mutation at nucleotide 2012 of the neu (erbB-2) gene on chromosome 10, presumably the initial event in EtNU-induced schwannoma development, is later followed by loss of the wild-type neu allele. Genetic crosses between BDIX and BDIV rats served: (a) to investigate the inheritance of susceptibility; (b) to obtain animals informative for the mapping of losses of heterozygosity (LOH) in tumors with polymorphic simple sequence length polymorphisms (SSLPs); and (c) to localize genes associated with schwannoma susceptibility by linkage analysis with SSLPs. Schwannoma development was strongly suppressed in F1 animals (20% incidence). All of the F1 schwannomas displayed LOH on chromosome 10, with a consensus region on the telomeric tip encompassing D10Rat3, D10Mgh16 and D10Rat2 but excluding neu. A strong bias toward losing the BDIV alleles suggests the involvement of a BDIV-specific tumor suppressor gene(s). Targeted linkage analysis with chromosome 10 SSLPs in F2 intercross and backcross animals localized schwannoma susceptibility to a region around D10Wox23, 30 cM centromeric to the tip. Ninety-four % of F1 tumors exhibited additional LOH at this region. Two distinct loci on chromosome 10 may thus be connected with susceptibility to the induction and development of schwannomas in rats exposed to EtNU. (+info)
A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson's Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson's Disease.
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Autosomal recessive juvenile parkinsonism (AR-JP, PARK2; OMIM 602544), one of the monogenic forms of Parkinson's disease (PD), was initially described in Japan. It is characterized by early onset (before age 40), marked response to levodopa treatment and levodopa-induced dyskinesias. The gene responsible for AR-JP was recently identified and designated parkin. We have analysed the 12 coding exons of the parkin gene in 35 mostly European families with early onset autosomal recessive parkinsonism. In one family, a homozygous deletion of exon 4 could be demonstrated. By direct sequencing of the exons in the index patients of the remaining 34 families, eight previously undescribed point mutations (homozygous or heterozygous) were detected in eight families that included 20 patients. The mutations segregated with the disease in the families and were not detected on 110-166 control chromosomes. Four mutations caused truncation of the parkin protein. Three were frameshifts (202-203delAG, 255delA and 321-322insGT) and one a nonsense mutation (Trp453Stop). The other four were missense mutations (Lys161Asn, Arg256Cys, Arg275Trp and Thr415Asn) that probably affect amino acids that are important for the function of the parkin protein, since they result in the same phenotype as truncating mutations or homozygous exon deletions. Mean age at onset was 38 +/- 12 years, but onset up to age 58 was observed. Mutations in the parkin gene are therefore not invariably associated with early onset parkinsonism. In many patients, the phenotype is indistinguishable from that of idiopathic PD. This study has shown that a wide variety of different mutations in the parkin gene are a common cause of autosomal recessive parkinsonism in Europe and that different types of point mutations seem to be more frequently responsible for the disease phenotype than are deletions. (+info)
Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder.
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A genetic contribution to the pathogenesis of panic disorder has been demonstrated by clinical genetic studies. Molecular genetic studies have focused on candidate genes suggested by the molecular mechanisms implied in the action of drugs utilized for therapy or in challenge tests. One class of drugs effective in the treatment of panic disorder is represented by monoamine oxidase A inhibitors. Therefore, the monoamine oxidase A gene on chromosome X is a prime candidate gene. In the present study we investigated a novel repeat polymorphism in the promoter of the monoamine oxidase A gene for association with panic disorder in two independent samples (German sample, n = 80; Italian sample, n = 129). Two alleles (3 and 4 repeats) were most common and constituted >97% of the observed alleles. Functional characterization in a luciferase assay demonstrated that the longer alleles (3a, 4 and 5) were more active than allele 3. Among females of both the German and the Italian samples of panic disorder patients (combined, n = 209) the longer alleles (3a, 4 and 5) were significantly more frequent than among females of the corresponding control samples (combined, n = 190, chi2 = 10.27, df = 1, P = 0.001). Together with the observation that inhibition of monoamine oxidase A is clinically effective in the treatment of panic disorder these findings suggest that increased monoamine oxidase A activity is a risk factor for panic disorder in female patients. (+info)
Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families.
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Systemic lupus erythematosus (SLE) is the prototype of human autoimmune diseases. Its genetic component has been suggested by familial aggregation (lambdas = 20) and twin studies. We have screened the human genome to localize genetic intervals that may contain lupus susceptibility loci in a sample of 188 lupus patients belonging to 80 lupus families with two or more affected relatives per family using the ABI Prism linkage mapping set which includes 350 polymorphic markers with an average spacing of 12 cM. Non-parametric multipoint linkage analysis suggests evidence for predisposing loci on chromosomes 1 and 18. However, no single locus with overwhelming evidence for linkage was found, suggesting that there are no 'major' susceptibility genes segregating in families with SLE, and that the genetic etiology is more likely to result from the action of several genes of moderate effect. Furthermore, the support for a gene in the 1q44 region as well as in the 1p36 region is clearly found only in the Mexican American families with SLE but not in families of Caucasian ethnicity, suggesting that consideration of each ethnic group separately is crucial. (+info)