RESUMO
BRCA1 and BRCA2 are the two major identified causes of inherited breast cancer, with mutations in either gene conferring up to 80-90% lifetime risk of breast cancer in carrier females. Mutations in BRCA1 account for approximately 45% of familial breast cancer and 90% of inherited breast/ovarian cancer, whereas mutations in BRCA2 account for a comparable percentage of inherited breast cancer cases. Over 85 distinct BRCA1 mutations and a growing list of BRCA2 mutations have been identified, with the majority resulting in protein truncation. A specific BRCA1 mutation, 185delAG, has a reported increased carrier frequency of approximately 0.9% in the Ashkenazi Jewish population, but is also found in rare non-Jewish patients with a different haplotype. The 6174delT mutation in BRCA2 was recently identified as a frequent mutation in 8 out of 107 Ashkenazi Jewish women diagnosed with breast cancer by age 50 (ref. 8), as well as in three Ashkenazi male breast cancer patients. We have conducted a large-scale population study to investigate the prevalence of specific BRCA1 and BRCA2 mutations in Ashkenazi Jewish individuals who were unselected for breast cancer. BRCA1 mutation screening on approximately 3,000 Ashkenazi Jewish samples determined a carrier frequency of 1.09% for the 185delAG mutation and 0.13% for the 5382insC mutation. BRCA2 analysis on 3,085 individuals from the same population showed a carrier frequency of 1.52% for the 6174delT mutation. This expanded population-based study confirms that the BRCA1 185delAG mutation and the BRCA2 6174delT mutation constitute the two most frequent mutation alleles predisposing to hereditary breast cancer among the Ashkenazim, and suggests a relatively lower penetrance for the 6174delT mutation in BRCA2.
Assuntos
Genes BRCA1/genética , Testes Genéticos , Judeus/genética , Proteínas de Neoplasias/genética , Deleção de Sequência/genética , Fatores de Transcrição/genética , Alelos , Proteína BRCA2 , Análise Mutacional de DNA , Feminino , Frequência do Gene , Triagem de Portadores Genéticos , Humanos , Israel , Fatores de Risco , Estados UnidosRESUMO
Mutations in BRCA1 (ref. 1) confer an increased risk of female breast cancer. In a genome-wide scan of linkage disequilibrium (LD), a high level of LD was detected among microsatellite markers flanking BRCA1 (ref. 3), raising the prospect that positive natural selection may have acted on this gene. We have used the predictions of evolutionary genetic theory to investigate this further. Using phylogeny-based maximum likelihood analysis of the BRCA1 sequences from primates and other mammals, we found that the ratios of replacement to silent nucleotide substitutions on the human and chimpanzee lineages were not different from one another (P=0.8), were different from those of other primate lineages (P=0.004) and were greater than 1 (P=0.04). This is consistent with the historic occurrence of positive darwinian selection pressure on the BRCA1 protein in the human and chimpanzee lineages. Analysis of genetic variation in a sample of female Australians of Northern European origin showed evidence for Hardy-Weinberg (HW) disequilibrium at polymorphic sites in BRCA1, consistent with the possibility that natural selection is affecting genotype frequencies in modern Europeans. The clustering of between-species variation in the region of the gene encoding the RAD51-interaction domain of BRCA1 suggests the maintenance of genomic integrity as a possible target of selection.
Assuntos
Evolução Molecular , Genes BRCA1/genética , Adaptação Biológica , Animais , Neoplasias da Mama/genética , Feminino , Variação Genética , Genótipo , Humanos , Funções Verossimilhança , Mutação , Pan troglodytes , Filogenia , Polimorfismo GenéticoRESUMO
Mutations in the mouse Brca1 gene cause lethality at different embryonic stages. We have shown that Brca1 mutant embryos, in which the fifth and sixth exons of Brca1 are deleted die before E7.5 and show decreased cellular proliferation. Brca1 mutants also show decreased expression of mdm2, a gene encoding an inhibitor of p53 activity. Thus, we have proposed that the reduction in mdm2 expression in Brca1 (5-6) mutants might lead to increased p53 activity. Consistent with this finding, the expression of p21, which encodes a G1 cell cycle inhibitor and is a target for p53 transcriptional activation was dramatically increased in the Brca1 (5-6) mutants, suggesting that impaired cellular proliferation could be due to a G1 cell-cycle arrest, caused by increased p21 levels. To test this hypothesis, we generated mice double mutant for Brca1 (5-6) and p53, or Brca1 (5-6) and p21. Mutation in either p53 or p21 prolonged the survival of Brca1 (5-6) mutant embryos from E7.5 to E9.5. The development of most Brca1 (5-6): p21 double-mutant embryos was comparable to that of their wild-type littermates, although no mutant survived past E10.5. The fact that mutation of neither p53 nor p21 completely rescued Brca1 (5-6) embryos suggests that their lethality is likely due to a multi-factorial process.
Assuntos
Ciclinas/genética , Desenvolvimento Embrionário e Fetal , Genes BRCA1/genética , Genes p53/genética , Mutação , Animais , Proteína BRCA1/metabolismo , Southern Blotting , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Sondas de DNA , Embrião de Mamíferos/metabolismo , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Fenótipo , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/metabolismoRESUMO
Certain germline mutations in either BRCA1 or BRCA2 confer a lifetime risk of developing breast cancer that may approach 90%. The BRCA1 185delAG mutation was found in 20% and the BRCA2 6174delT mutation in 8% of Ashkenazi Jewish women with early-onset breast cancer. The 185delAG mutation was observed in 0.9% of 858 Ashkenazi Jews unselected for a personal or family history of cancer. Assuming comparable age-specific penetrances, a carrier frequency of 0.3% was estimated for the 6174delT BRCA2 mutation. To test this hypothesis, we performed a population survey of 1,255 Jewish individuals. In two independent groups, a prevalence of approximately 1% (C.I. 0.6-1.5) was observed for the 6174delT mutation. The relative risk of developing breast cancer by age 42 was estimated to be 9.3 (C.I. 2.5-22.5) for 6174delT, compared to 31 (C.I. 11-77) for 185delAG. Analysis of 107 Ashkenazi Jewish women with breast cancer and a family history of breast or ovarian cancer confirmed a four-fold greater prevalence for the BRCA1 185delAG mutation compared to the BRCA2 6174delT mutation. Our findings suggest a difference in cumulative life-time penetrance for the two mutations. Genetic counseling for the one in 50 Ashkenazi Jewish individuals harbouring specific germline mutations in BRCA1 or BRCA2 must be tailored to reflect the different risks associated with the two mutations.
Assuntos
Triagem de Portadores Genéticos , Judeus/genética , Proteínas de Neoplasias/genética , Deleção de Sequência/genética , Fatores de Transcrição/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2 , Neoplasias da Mama/genética , Feminino , Frequência do Gene , Genes BRCA1/genética , Testes Genéticos , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Fatores de RiscoRESUMO
Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.
Assuntos
Códon sem Sentido/genética , Éxons/genética , Genes BRCA1/genética , Mutação de Sentido Incorreto/genética , Splicing de RNA/genética , Motivos de Aminoácidos , Substituição de Aminoácidos/genética , Proteína BRCA1/química , Proteína BRCA1/genética , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Fases de Leitura Aberta/genética , Fenótipo , Fosfoproteínas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-ArgininaRESUMO
Recently, a 6-kb duplication of exon 13, which creates a frameshift in the coding sequence of the BRCA1 gene, has been described in three unrelated U.S. families of European ancestry and in one Portuguese family. Here, our goal was to estimate the frequency and geographic diversity of carriers of this duplication. To do this, a collaborative screening study was set up that involved 39 institutions from 19 countries and included 3,580 unrelated individuals with a family history of the disease and 934 early-onset breast and/or ovarian cancer cases. A total of 11 additional families carrying this mutation were identified in Australia (1), Belgium (1), Canada (1), Great Britain (6), and the United States (2). Haplotyping showed that they are likely to derive from a common ancestor, possibly of northern British origin. Our results demonstrate that it is strongly advisable, for laboratories carrying out screening either in English-speaking countries or in countries with historical links with Britain, to include within their BRCA1 screening protocols the polymerase chain reaction-based assay described in this report.
Assuntos
Neoplasias da Mama/genética , Éxons/genética , Efeito Fundador , Duplicação Gênica , Genes BRCA1/genética , Mutação/genética , Neoplasias Ovarianas/genética , Idade de Início , Neoplasias da Mama/epidemiologia , Neoplasias da Mama Masculina/epidemiologia , Neoplasias da Mama Masculina/genética , Análise Mutacional de DNA , Inglaterra/epidemiologia , Inglaterra/etnologia , Feminino , Mutação da Fase de Leitura/genética , Frequência do Gene/genética , Testes Genéticos , Haplótipos/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Neoplasias Ovarianas/epidemiologiaRESUMO
Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.
Assuntos
Processamento Alternativo , Proteína BRCA1/análise , Núcleo Celular/química , Genes BRCA1/genética , Células 3T3 , Animais , Proteína BRCA1/química , Proteína BRCA1/genética , Células COS , Citoplasma/química , DNA Complementar/genética , Éxons/genética , Humanos , Camundongos , Peso Molecular , RNA Mensageiro/análise , Deleção de SequênciaRESUMO
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is a powerful method to quickly and accurately determine the masses of peptides. Most genetic analyses, however, begin with PCR amplification of a test sequence to generate DNA, which is more difficult than peptides to analyze by MALDI-TOF. We describe a method that produces a PCR product of any continuous region of coding sequence which can then be used to encode an N-terminally tagged test peptide in a coupled in vitro transcription/translation reaction. The test peptide is purified using the tag, and its mass is measured by MALDI-TOF. Truncations and amino acid substitutions in peptides coded for by the breast cancer susceptibility gene BRCA1 were readily identified using this method. The process can be multiplexed and is amenable to automation, providing an efficient, high-throughput means for mutation discovery and genetic profiling.
Assuntos
Análise Mutacional de DNA/métodos , Mutação/genética , Biossíntese Peptídica , Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteína BRCA1/análise , Proteína BRCA1/biossíntese , Proteína BRCA1/química , Proteína BRCA1/genética , Análise Mutacional de DNA/economia , Genes BRCA1/genética , Testes Genéticos/economia , Testes Genéticos/métodos , Humanos , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Deleção de Sequência/genética , Suíça , Transcrição Gênica/genética , Dedos de Zinco/genéticaAssuntos
Neoplasias da Mama/prevenção & controle , Antagonistas de Estrogênios/uso terapêutico , Testes Genéticos , Piperidinas/uso terapêutico , Tamoxifeno/uso terapêutico , Proteína BRCA2 , Neoplasias da Mama/genética , Estrogênios/agonistas , Feminino , Genes BRCA1/genética , Humanos , Proteínas de Neoplasias/genética , Cloridrato de Raloxifeno , Fatores de Transcrição/genéticaRESUMO
Using currently available MS-based methods, accurate mass measurements are essential for the characterization of DNA oligomers. However, there is a lack of specificity in mass peaks when the characterization of individual DNA species in a mass spectrum is dependent solely upon the mass-to-charge ratio (m/z). Here, we utilize nucleotide-specific tagging with stable isotopes to provide internal signatures that quantitatively display the nucleotide content of oligomer peaks in MS spectra. The characteristic mass-split patterns induced by the partially (13)C/(15)N-enriched dNTPs in DNA oligomers indicate the number of labeled precursors and in turn the base substitution in each mass peak, and provide for efficient SNP detection. Signals in mass spectra not only reflect the masses of particular DNA oligomers, but also their specific composition of particular nucleotides. The measurements of mass tags are relative in the mass-split pattern and, hence, the accuracy of the determination of nucleotide substitution is indirectly increased. For high sample throughput, (13)C/(15)N-labeled sequences of interest have been generated, excised in solution and purified for MS analysis in a single-tube format. This method can substantially improve the specificity, accuracy and efficiency of mass spectrometry in the characterization of DNA oligomers and genetic variations.
Assuntos
DNA/química , DNA/genética , Genes BRCA1/genética , Nucleotídeos/análise , Polimorfismo de Nucleotídeo Único/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Composição de Bases , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Heterozigoto , Humanos , Peso Molecular , Nucleotídeos/química , Nucleotídeos/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Especificidade por Substrato , Fatores de TempoRESUMO
BRCA1 expression is repressed by aberrant cytosine methylation in sporadic breast cancer. We hypothesized that aberrant cytosine methylation of the BRCA1 promoter was associated with the transcriptionally repressive effects of histone hypoacetylation and chromatin condensation. To address this question, we developed an in vitro model of study using normal cells and sporadic breast cancer cells with known levels of BRCA1 transcript to produce a 1.4 kb 5-methylcytosine map of the BRCA1 5' CpG island. While all cell types were densely methylated upstream of -728 relative to BRCA1 transcription start, all normal and BRCA1 expressing cells were non-methylated downstream of -728 suggesting that this region contains the functional BRCA1 5' regulatory region. In contrast, the non-BRCA1 expressing UACC3199 cells were completely methylated at all 75 CpGs. Chromatin immunoprecipitations showed that the UACC3199 cells were hypoacetylated at both histones H3 and H4 in the BRCA1 promoter compared to non-methylated BRCA1 expressing cells. The chromatin of the methylated UACC3199 BRCA1 promoter was inaccessible to DNA-protein interactions. These data indicate that the epigenetic effects of aberrant cytosine methylation, histone hypoacetylation and chromatin condensation act together in a discrete region of the BRCA1 5' CpG island to repress BRCA1 transcription in sporadic breast cancer.
Assuntos
Cromatina/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Inativação Gênica , Genes BRCA1/genética , Histonas/metabolismo , Regiões Promotoras Genéticas/genética , 5-Metilcitosina , Acetilação , Mama/citologia , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Linhagem Celular , Cromatina/química , Cromatina/genética , Ilhas de CpG/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/química , Humanos , Testes de Precipitina , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: In response to the isolation of the BRCA1 gene, a breast-ovarian cancer-susceptibility gene, biotechnology companies are already marketing genetic tests to health care providers and to the public. Initial studies indicate interest in BRCA1 testing in the general public and in populations at high risk. However, the optimal strategies for educating and counseling individuals have yet to be determined. PURPOSE: Our goal was to evaluate the impact of alternate strategies for pretest education and counseling on decision-making regarding BRCA1 testing among women at low to moderate risk who have a family history of breast and/or ovarian cancer. METHODS: A randomized trial design was used to evaluate the effects of education only (educational approach) and education plus counseling (counseling approach), as compared with a waiting-list (control) condition (n = 400 for all groups combined). The educational approach reviewed information about personal risk factors, inheritance of cancer susceptibility, the benefits, limitations, and risks of BRCA1 testing, and cancer screening and prevention options. The counseling approach included this information, as well as a personalized discussion of experiences with cancer in the family and the potential psychological and social impact of testing. Data on knowledge of inherited cancer and BRCA1 test characteristics, perceived risk, perceived benefits, limitations and risks of BRCA1 testing, and testing intentions were collected by use of structured telephone interviews at baseline and at 1-month follow-up. Provision of a blood sample for future testing served as a proxy measure of intention to be tested (in the education and counseling arms of the study). The effects of intervention group on study outcomes were evaluated by use of hierarchical linear regression modeling and logistic regression modeling (for the blood sample outcome). All P values are for two-sided tests. RESULTS: The educational and counseling approaches both led to significant increases in knowledge, relative to the control condition (P < .001 for both). The counseling approach, but not the educational approach, was superior to the control condition in producing significant increases in perceived limitations and risks of BRCA1 testing (P < .01) and decreases in perceived benefits (P < .05). However, neither approach produced changes in intentions to have BRCA1 testing. Prior to and following both education only and education plus counseling, approximately one half of the participants stated that they intended to be tested; after the session, 52% provided a blood sample. CONCLUSIONS: Standard educational approaches may be equally effective as expanded counseling approaches in enhancing knowledge. Since knowledge is a key aspect of medical decision-making, standard education may be adequate in situations where genetic testing must be streamlined. On the other hand, it has been argued that optimal decision-making requires not only knowledge, but also a reasoned evaluation of the positive and negative consequences of alternate decisions. Although the counseling approach is more likely to achieve this goal, it may not diminish interest in testing, even among women at low to moderate risk. Future research should focus on the merits of these alternate approaches for subgroups of individuals with different backgrounds who are being counseled in the variety of settings where BRCA1 testing is likely to be offered.
Assuntos
Neoplasias da Mama/genética , Aconselhamento , Tomada de Decisões , Suscetibilidade a Doenças/diagnóstico , Genes BRCA1/genética , Predisposição Genética para Doença , Consentimento Livre e Esclarecido , Neoplasias Ovarianas/genética , Educação de Pacientes como Assunto , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Análise de Regressão , RiscoRESUMO
BACKGROUND: The natural history of ovarian cancer is not well understood and, to date, there is conflicting evidence as to whether or not there is a demonstrable precursor lesion. Some women at high risk of developing ovarian cancer because of their family history elect to have a prophylactic oophorectomy. To determine whether or not a recognizable premalignant lesion could be defined in familial ovarian carcinogenesis, we reviewed ovarian tissue specimens from women whose ovaries were removed prophylactically before gene testing became available and who were tested subsequently for BRCA1 or BRCA2 gene mutations. METHODS: We analyzed ovarian tissue specimens from 37 women. The specimens were examined for the presence of the following four features: inclusion cysts, clefts and fissures, ovarian epithelial metaplasia, and the presence of papillae on the ovarian surface epithelium. The specimens were also examined closely for the presence of dysplasia and occult neoplasia. Furthermore, the occurrence of endometriosis and benign ovarian tumors was documented in these women. The protein truncation test, nonradioactive single-stranded conformation polymorphism analysis, and heteroduplex analysis, followed by DNA sequencing, were used to identify BRCA1 or BRCA2 mutations in either blood samples or ovarian tissue specimens. RESULTS: Eleven women had inherited a mutated BRCA1 or BRCA2 gene; 26 women had not. There was no difference between these groups for any of the features studied. CONCLUSIONS: Our data suggest that many of the histologic "abnormalities" described in "normal" ovaries are, in fact, variations of the normal and are not associated with the development of cancer.
Assuntos
Genes BRCA1/genética , Genes Supressores de Tumor/genética , Heterozigoto , Mutação/genética , Ovariectomia , Ovário/patologia , Adulto , Biomarcadores Tumorais/análise , Feminino , Humanos , Pessoa de Meia-Idade , Risco , Fatores de Risco , Reino UnidoRESUMO
BACKGROUND: Breast carcinomas occurring in carriers of BRCA1 gene mutations may have a distinctly different pathway of molecular pathogenesis from those occurring in noncarriers. Data from murine models implicate loss of p53 (also known as TP53) gene function as a critical early event in the malignant transformation of cells with a BRCA1 mutation. Therefore, breast tumors from BRCA1 mutation carriers might be expected to exhibit a high frequency of p53 mutations. This study examined the frequency of p53 mutations in the breast tumors of Ashkenazi Jewish carriers and noncarriers of BRCA1 mutations. METHODS: Tumor DNA from carriers and noncarriers of BRCA1 mutations was screened for mutations in exons 4 through 10 of the p53 gene by use of the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis of the amplified DNA. Direct sequencing was performed on gene fragments that showed altered mobility in SSCP analysis. RESULTS: Mutations in the p53 gene were detected in 10 of 13 tumors from BRCA1 mutation carriers versus 10 of 33 tumors from non-carriers (two-sided P = .007). The p53 mutations were distributed throughout exons 4 through 10 and included both protein-truncating and missense mutations in both groups. CONCLUSIONS: A statistically significantly higher frequency of p53 mutations was found in breast tumors from carriers of BRCA1 mutations than from noncarriers, which adds to the accumulating evidence that loss of p53 function is an important step in the molecular pathogenesis of BRCA1 mutation-associated breast tumors. This finding may have implications for understanding phenotypic differences and potential prognostic differences between BRCA1 mutation-associated hereditary breast cancers and sporadic cancers.
Assuntos
Neoplasias da Mama/etnologia , Neoplasias da Mama/genética , Genes BRCA1/genética , Genes p53/genética , Heterozigoto , Judeus/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , DNA de Neoplasias/genética , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
BACKGROUND: Most human female cells contain two X chromosomes, only one of which is active. The process of X-chromosome inactivation, which occurs early in development, is usually random, producing tissues with equal mixtures of cells having active X chromosomes of either maternal or paternal origin. However, nonrandom inactivation may occur in a subset of females. If a tumor suppressor gene were located on the X chromosome and if females with a germline mutation in one copy of that suppressor gene experienced nonrandom X-chromosome inactivation, then some or all of the tissues of such women might lack the wild-type suppressor gene function. This scenario could represent a previously unrecognized mechanism for development of hereditary cancers. We investigated whether such a mechanism might contribute to the development of hereditary ovarian cancers. METHODS: Patterns of X-chromosome inactivation were determined by means of polymerase chain reaction amplification of the CAG-nucleotide repeat of the androgen receptor (AR) gene after methylation-sensitive restriction endonuclease digestion of blood mononuclear cell DNA from patients with invasive (n = 213) or borderline (n = 44) ovarian cancer and control subjects without a personal or family history of cancer (n = 50). BRCA1 gene status was determined by means of single-strand conformational polymorphism analysis and DNA sequencing. All statistical tests were two-sided. RESULTS AND CONCLUSIONS: Among individuals informative for the AR locus, nonrandom X-chromosome inactivation was found in the DNA of 53% of those with invasive cancer versus 28% of those with borderline cancer (P = .005) and 33% of healthy control subjects (P = .016). Nonrandom X-chromosome inactivation can be a heritable trait. Nine of 11 AR-informative carriers of germline BRCA1 mutations demonstrated nonrandom X-chromosome inactivation (.0002 < P < .008, for simultaneous occurrence of both). IMPLICATIONS: Nonrandom X-chromosome inactivation may be a predisposing factor for the development of invasive, but not borderline, ovarian cancer.
Assuntos
DNA de Neoplasias/genética , Mecanismo Genético de Compensação de Dose , Evolução Molecular , Genes BRCA1/genética , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/genética , Estudos de Casos e Controles , DNA de Neoplasias/metabolismo , Feminino , Humanos , Perda de Heterozigosidade , Metilação , Neoplasias Ovarianas/metabolismoRESUMO
BACKGROUND: BRCA1 and BRCA2 are the two major susceptibility genes involved in hereditary breast cancer. This study was undertaken to provide reliable population-based estimates of genetic influence and to characterize the nature and prevalence of BRCA1 and BRCA2 germline mutations in early-onset breast cancer. METHODS: In a series comprising all women diagnosed with breast cancer under the age of 41 years in southern Sweden during 1990 through 1995 (n = 262), family history of cancer was evaluated in 95% (n = 250) of the case subjects and germline mutations in BRCA1 and BRCA2 were analyzed in 89% (n = 234). All statistical tests were two-sided. RESULTS: A total of 97 case subjects had at least one first- or second-degree relative with breast or ovarian cancer; 34 (14%; 95% confidence interval [CI] = 9.6% to 18%) cases had at least two first- or second-degree relatives, 22 (8.8%; 95%CI = 5.3% to 12%) had one first-degree relative, and 41 (16%; 95% CI = 12% to 21%) had one second-degree relative with either cancer. If two females affected with breast or ovarian cancer who were related through an unaffected male were also defined as first-degree relatives, then a higher number of case subjects, 120 (48%; 95% CI = 42% to 54%), had at least one first-degree or second-degree relative with breast or ovarian cancer. Sixteen (6.8%; 95% CI = 4.0% to 11%) BRCA1 mutation carriers and five (2.1%; 95% CI = 0.70% to 4.9%) BRCA2 mutation carriers were identified. Among case subjects with one first- or more than one first- or second-degree relative with breast or ovarian cancer, BRCA mutations were more frequent (P<.001) than among the case subjects without this degree of family history. BRCA mutations were also statistically significantly more common among women with bilateral breast cancer than among women with unilateral breast cancer (P =.002). BRCA mutations were more common among younger case subjects than among older ones (P =.0027). CONCLUSIONS: Almost half (48%) of women in southern Sweden with early-onset breast cancer have some family history of breast or ovarian cancer, and 9.0% of early-onset breast cancer cases are associated with a germline mutation in BRCA1 or BRCA2. Mutation carriers were more prevalent among young women, women with at least one first- or second-degree relative with breast or ovarian cancer, and women with bilateral breast cancer.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1/genética , Genes Supressores de Tumor/genética , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/genética , Vigilância da População , Adulto , Idade de Início , Neoplasias da Mama/epidemiologia , Feminino , Testes Genéticos , Heterozigoto , Humanos , Neoplasias Ovarianas/epidemiologia , Prevalência , Suécia/epidemiologiaRESUMO
BACKGROUND: The cytochrome P450c17alpha enzyme functions in the steroid biosynthesis pathway, and altered endogenous steroid hormone levels have been reported to be associated with a T to C polymorphism in the 5' promoter region of the CYP17 gene. Because steroid hormone exposure is known to influence breast cancer risk, we conducted a population-based, case-control-family study to assess the relationship between the CYP17 promoter polymorphism and early-onset breast cancer. METHODS: Case subjects under 40 years of age at diagnosis of a first primary breast cancer, population-sampled control subjects, and the relatives of both case and control subjects were interviewed to record family history of breast cancer and other risk factors. CYP17 genotype was determined in 369 case subjects, 284 control subjects, and 91 relatives of case subjects. Genotype distributions were compared by logistic regression, and cumulative risk was estimated by a modified segregation analysis. All statistical tests were two-tailed. RESULTS: Compared with the TT genotype (i.e., individuals homozygous for the T allele), the TC genotype was not associated with increased breast cancer risk (P: =.7). Compared with the TT and TC genotypes combined, the CC genotype was associated with a relative risk of 1. 81 (95% confidence interval [CI] = 1.15-2.86; P: =.01) before adjustment for measured risk factors and 1.63 (95% CI = 1.00-2.64; P: =.05) after adjustment. There was an excess of CC genotypes in case subjects who had at least one affected first- or second-degree relative, compared with control subjects unstratified by family history of breast cancer (23% versus 11%; P: =.006), and these case subjects had a threefold to fourfold higher risk than women of other groups defined by genotype and family history of breast cancer. Analysis of breast cancer in first- and second-degree relatives of case subjects with the CC genotype, excluding two known carriers of a deleterious mutation in BRCA1 or BRCA2, gave a relative hazard in women with the CC genotype of 3.48 (95% CI = 1.13-10.74; P: =.04), which is equivalent to a cumulative risk of 16% to age 70 years. CONCLUSIONS: The CC genotype may modify the effect of other familial risk factors for early-onset breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Polimorfismo Genético , Esteroide 17-alfa-Hidroxilase/genética , Adulto , Fatores Etários , Idade de Início , Alelos , Austrália , Estudos de Casos e Controles , Primers do DNA , DNA de Neoplasias/análise , Feminino , Genes BRCA1/genética , Genes Supressores de Tumor/genética , Genótipo , Humanos , Modelos Logísticos , Mutação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RiscoRESUMO
BACKGROUND: The two-hit hypothesis for the genesis of cancer predicts that cancer can develop when the wild-type allele of a tumor suppressor gene is lost in an individual with a germline mutation in that gene. Neither loss of heterozygosity (LOH) for BRCA1 nor mutations of the TP53 (also known as p53) gene have been documented prior to invasion in ovarian cancers arising in women with germline BRCA1 mutations. Such documentation is difficult because lesions are rarely identified in ovarian epithelium. We, therefore, looked for LOH at microsatellite polymorphisms linked to the BRCA1 and TP53 tumor suppressor loci in an incidental carcinoma in situ of the ovary removed prophylactically from a woman with a germline BRCA1 mutation. METHODS: By use of laser-capture microdissection, we obtained pure populations of atypical ovarian epithelial cells and normal stromal cells. DNA was extracted, amplified with primers flanking polymorphic microsatellites linked to BRCA1 (D17S855 and D17S579) and TP53 (TP53 and D17S786), and analyzed for LOH at these microsatellites. We also tested for p53 expression in the abnormal epithelium by immunohistochemistry. RESULTS: Both of the markers linked to TP53 showed LOH, as did an intragenic BRCA1-linked marker (D17S855). The other microsatellite marker for BRCA1 was uninformative. Immunohistochemical staining with an antibody to p53 showed strong immunoreactivity confined to the atypical epithelium. CONCLUSIONS: BRCA1, as well as TP53, can undergo LOH prior to stromal invasion in BRCA1-associated ovarian cancer. Strong immunoreactivity for p53 suggests the presence of mutated p53 in these cells as well. These findings suggest that loss of function of these two tumor suppressor genes occurs early in ovarian carcinogenesis in BRCA1 mutation carriers.
Assuntos
Carcinoma in Situ/genética , Genes BRCA1/genética , Mutação em Linhagem Germinativa , Perda de Heterozigosidade , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Anticorpos Antineoplásicos/análise , Carcinoma in Situ/patologia , Primers do DNA , DNA de Neoplasias/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Ligação Genética , Humanos , Imuno-Histoquímica , Repetições de Microssatélites , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Polimorfismo Genético , Proteína Supressora de Tumor p53/imunologiaRESUMO
BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.