Detection of hypermethylation BRCA1/2 gene promoter in breast tumours among Moroccan women

Molecular Biology Reports - The promoter region is a key element of gene expression regulation. In mammals, most of the genes present, at the level of their promoter, a large number of islands CpG....     

BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian

OBJECTIVE: Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose women to breast and ovarian cancer. Female carriers of BRCA1 or BRCA2 gene mutations have very high lifetime risks for breast and ovarian cancers. Genetic abnormalities occur in all cancers, so BRCA-related pathways are critical because they serve to safeguard genetic content. Although protecting genetic information is a general function, BRCA-related pathways seem largely specific to preventing breast and ovarian cancer. The objective of this study was to resolve this difference between the theoretical functions of BRCA genes and their specific clinical effects. DATA SOURCES, DATA EXTRACTION, DATA SYNTHESIS: The author collected data published in > 30 epidemiologic studies on the incidence of cancers other than breast or ovarian in mutation carriers and in large populations eligible for mutation testing. Data were extracted and used directly as published whenever possible with a minimum of statistical manipulation. CONCLUSIONS: Although mutations target breast and ovary, a broader spectrum of cancers also occur with statistically significant elevated frequencies. Risks for "all cancers except breast or ovary" are elevated, with some population subgroups differing with regard to how frequently elevated risks were found at individual sites. Additional sites at risk included stomach, pancreas, prostate, and colon. The increased risk ranged from about 20% to 60%, with the greatest increases in risk in stomach and pancreas. The collected data show BRCA-pathway functions are probably required at multiple sites, not just in breast or ovary. Known interactions and relationships among BRCA-related pathways strongly support the idea that their inactivation provides growth or survival advantages for a variety of cancers. The data suggest applying an increased level of clinical alertness to those with defects in BRCA-related pathways. Identifying molecules that confer growth or survival advantages to BRCA-related cancers may provide broadly useful targets for chemotherapy or chemoprevention.     

Downregulation of ARFGEF1 and CAMK2B by promoter hypermethylation in breast cancer cells

To identify novel genes that are regulated by promoter methylation, a combinational approach involving in silico mining followed by molecular assay was performed. From the expression microarray data registered in the European bioinformatics institute (EBI), genes showing downregulation in breast cancer cells were initially screened and then selected by e-Northern analysis using the Unigene database. A series of these in silico methods identified CAMK2B and ARFGEF1 as candidates, and the two genes were revealed to be hypermethylated in breast cancer cell lines and hypomethylated in normal breast cell lines. Additionally, cancer cell lines showed downregulated expression of these genes. Furthermore, treatment of the cancer cell lines with a demethylation agent, 5-Aza-2'-deoxycytidine, recovered expression of CAMK2B and ARFGEF1, implying that hypermethyaltion silenced gene activity in cancer cells. Taken together, promoter methylations of CAMK2B and ARFGEF1 are novel epigenetic markers identified in breast cancer cell lines and can be utilized for the application to clinical cancer tissues.     

BRCA1 and BRCA2 in hereditary breast cancer

The hereditary breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, have established roles in genome integrity maintenance and in the control of homologous recombination. Recent work has produced valuable insights into the mechanisms of action of the gene products. This review summarizes some of these advances, and attempts to place them in the context of known functions of the genes.     

Genotyping of BRCA1, BRCA2, and CHEK2 germline mutations in Russian breast cancer patients using diagnostic biochips

The identification of BRCA1/2 and CHEK2 germline mutations is central to the molecular diagnostics of susceptibility to breast or/and ovarian cancer. A microarray-based rapid genotyping technique has been developed for identifying BRCA1 (185delAG, 300T>G, 4153delA, 5382insC, and 4158 A>G, 5382insC), BRCA2 (695insT and 6174delT), and CHEK2 (1100delC) mutations. It was applied for 412 randomly collected breast-cancer specimens from central Russia. In 25 (6.0%) patients, breast cancer was associated with other tumors of, e.g., ovarian, cervical, or colorectal cancer. BRCA1/2 and CHEK2 mutations were detected in 33 breast-cancer patients (8.0%). The most frequent mutations were BRCA1 5382insC, which was found in 16 patients (3.9%), and CHEK2 1100delC, which was detected in seven patients (1.7%). The suggested diagnostic microarray proved to be an efficient means of identifying BRCA1/2 and CHEK2 founder mutations most frequent in central Russia and can be proposed as a high-throughput diagnostic tool for clinical genetic testing.     

Methylation of the RASSF1A gene in human cancers

Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.     

Analysis of BRCA1 involvement in breast cancer in Indian women

The involvement of the familial breast-ovarian cancer gene (BRCA1) in the molecular pathogenesis of breast cancer among Indian women is unknown. We have used a set of microsatellite polymorphisms to examine the frequency of allele loss at the BRCA1 region on chromosome 17q21, in a panel of 80 human breast tumours. Tumour and blood leukocyte/normal tissue DNA from a series of 80 patients with primary breast cancer was screened by PCR-amplified microsatellite length polymorphisms to detect deletions at three polymorphic BRCA1 loci. PCR-allelotype was valuable in examining allele losses from archival and small tumour samples. Loss of alleles at BRCA1 in the patient set, confirmed a noteworthy role of this gene in the molecular patho-genesis of breast cancer and was in accordance with its well-documented tumour suppressive function.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.Key words: homologous recombination, PARP inhibitor, BRCA1, BRCA2, PTEN, PALB2, EMSY, double strand break repair     

A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes

Mutation screening of the breast and ovarian cancer-predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an "average" deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100 : 1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20 : 1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.     

A paradox and three egnimas about the role of BRCA1 in breast and ovarian cancers

More than 50% of the hereditary forms are associated with germ line mutation in either BRCA1 or BRCA2 genes (BReast CAncer 1/2). The BRCA1 protein is expressed ubiquitously and is likely to play a role in several fundamental processes, including the maintenance of genomic integrity. Paradoxically, BRCA1 appears as a gene essential for proliferation of embryonic cells that simultaneously carries tumor suppressor activity. The nature of the role of BRCA1 in DNA repair and maintenance of genome integrity remains enigmatic. BRCA1 may indeed be a sensor of "abnormal" DNA structures that undergo heterochromatinisation. This model finds some support in the recent report that BRCA1 participates in the maintenance of X-chromosome inactivation, a paradigm for facultative heterochromatinisation. Why are epithelial cells from mammary glands and ovaries the privileged targets for tumorigenesis in women carrying germline mutations in BRCA1? The inheritance of a single defective copy of BRCA1 by women confers a status of susceptibility for developing breast and/or ovarian cancer. The loss of the wild-type allele inherited from the unaffected parent (LOH), commonly observed in the primary breast and ovarian tumors in these susceptible women, represents the event that initiates the tumorigenesis process. This classical two hit model, which assumes that heterozygote cells are "normal" until the LOH occurs stochastically, remains enigmatic.     

Contribution of epigenetic alteration of BRCA1 and BRCA2 genes in breast carcinomas in Tunisian patients

Objective: The aim of this study was to evaluate the contribution of the BRCA1 and BRCA2 promoter methylation in the pathogenesis of sporadic breast cancer in Tunisian patients. Methods: Breast carcinoma tissues (n = 117) and available paired normal breast tissues (n = 65) from Tunisian women who had no family history were investigated for the methylation status of BRCA1 and BRCA2 promoters using methylation-specific PCR. Breast specimens from women without carcinoma (16 fibroadenomas and 5 mastopathies) were used as control. Results: Hypermethylation of BRCA1 and BRCA2 promoters was detected respectively in 60.7% and 69.2% of the carcinoma tissues, and in only 7.7% and 4.6% of the paired normal breast tissues. None of the fibroadenomas and mastopathies showed hypermethylation. Correlations were found between BRCA1 and BRCA2 hypermethylation and decrease in their mRNA expression (p = 0.02 and p = 0.009, respectively). Moreover, BRCA1 methylation correlates with patients age (p = 0.01) and triple negative (ER?, PR?, HER2?) tumors (p = 0.01). Patients with methylated BRCA1 and/or BRCA2 had a significant prolonged survivals compared to those with unmethylated tumors (p = 0.002). Conclusion: Our results suggest an important role of BRCA1 and BRCA2 promoter methylation in breast cancer development in the Tunisian population.     

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.     

Usefulness of polymorphic markers in exclusion of BRCA1/BRCA2 mutations in families with aggregation of breast/ovarian cancers

Founder mutations can account for a large proportion of BRCA1/BRCA2 gene abnormalities in a given population. However there is still a need to study the entire gene in many families, even in countries where founder mutations have been identified. It is possible to decrease the number of cases which are studied by complex and expensive sequencing/Southern blot analyses of BRCA1/BRCA2 genes by exclusion of common BRCA1/BRCA2 alleles in a given family by using polymorphic dinucleotide markers. The goal of o ur study was to assess the effectiveness of this method in exclusion of BRCA1/BRCA2 constitutional mutations. In each family, blood samples for genetic analyses were taken from two affected relatives from the same generation. Six polymorphic microsatellite markers linked to BRCA1/BRCA2 genes were analysed. Results obtained with these markers were verified by applying BRCA1 testing for the most common founder mutations in Poland and using exon by exon" sequencing of coding fragments of the BRCA2 gene. Polymorphic markers useful in BRCA1/BRCA2 analyses included only 3 of 6 examined - D17S855, D13S260 and D13S267. Occurrence of commoalleles of BRCA1 was excluded in 3 families and BRCA2 in 5 out of 30 families. Results obtained by testing for BRCA1 Polish founder mutations and BRCA2 sequencing were in agreement with BRCA1 findings based on polymorphic markers. The only exception was family 994 with BRCA1 exon 5 300T/G mutation, in which BRCA1 mutation carrier was excluded by using D17S855. Among 14 families without BRCA1 Polish founder mutations in this gene were excluded in 2 families and BRCA2 mutation was excluded in one family.     

Analysis of Loss of heterozygosity and immunohistochemistry in BRCA1 gene in sporadic breast cancers

BRCA1 is a tumour suppressor gene (TSG), which predisposes cancer to both breast and ovary. The primary objective of the present study is to ascertain the involvement of BRCA1 gene in the pathogenesis of sporadic breast cancer women in Chennai (South India) by analysing its protein expression by immunohistochemistry (IHC) and loss of heterozygosity (LOH) for confirmation of the involvement of TSG in the study population. We found down regulation of BRCA1 protein (54%) in IHC and it was correlated with the clinicopathological parameters of the patients. We found near significant correlation (P < 0.063) between BRCA1 protein expression and clinicopathological parameters. We found 30% LOH in our study and it was also correlated with the clinicopathological parameters. No correlation was found between LOH and clinicopathological parameters. Though we found no correlation, the results revealed in this study support the involvement of BRCA1 TSG in the pathogenesis of sporadic breast cancer women in Chennai (South India).     

Clinical interpretation and recommendations for patients with a variant of uncertain significance in BRCA1 or BRCA2: a survey of genetic counseling practice

The intent of this study was to document current practices in breast cancer genetic counseling and identify areas of variability for patients with a variant of uncertain significance (VUS) in the BRCA1 or BRCA2 gene. Registered members of the National Society of Genetic Counselors (NSGC) Cancer Special Interest Group (SIG) were sent an invitation via electronic mail to participate in an online questionnaire. The questionnaire was divided into three sections: clinical experience, clinical meaning, and risk perceptions and clinical recommendations for clinical situations involving a VUS. Fifty-seven of the eligible members responded. During the pre-test counseling session for a BRCA risk assessment patient, the vast majority of counselors (80.7%) mention VUS as a possible test result. Nearly half, 49.1%, report having given such a result to their patients at least one to four times. However, only 63.2% felt as though their patients understood the meaning of a VUS result. When asked to conclude the implication of a VUS and make medical management recommendations, the responses were varied. Nevertheless, a good proportion of counselors expressed the importance of testing other family members to help clarify the proband's risk and aid in medical management issues. Although the recent recommendations by the American College of Medical Genetics suggest standards for the interpretation of sequence variations, they do not provide guidelines for making clinical recommendations based on these variations. The results of this study reveal significant diversity in the personal interpretation of a VUS result, leading to various clinical recommendations, and suggest a need for clinical management recommendations as well.     

Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan

The population of Pakistan has been reported to have the highest rate of breast cancer of any Asian population (excluding Jews in Israel) and one of the highest rates of ovarian cancer worldwide. To explore the contribution that genetic factors make to these high rates, we have conducted a case-control study of 341 case subjects with breast cancer, 120 case subjects with ovarian cancer, and 200 female control subjects from two major cities of Pakistan (Karachi and Lahore). The prevalence of BRCA1 or BRCA2 mutations among case subjects with breast cancer was 6.7% (95% confidence interval [CI] 4.1%-9.4%), and that among case subjects with ovarian cancer was 15.8% (95% CI 9.2%-22.4%). Mutations of the BRCA1 gene accounted for 84% of the mutations among case subjects with ovarian cancer and 65% of mutations among case subjects with breast cancer. The majority of detected mutations are unique to Pakistan. Five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delAG, and IVS14-1A-->G) and one BRCA2 mutation (3337C-->T) were found in multiple case subjects and represent candidate founder mutations. The penetrance of deleterious mutations in BRCA1 and BRCA2 is comparable to that of Western populations. The cumulative risk of cancer to age 85 years in female first-degree relatives of BRCA1-mutation-positive case subjects was 48% and was 37% for first-degree relatives of the BRCA2-mutation-positive case subjects. A higher proportion of case subjects with breast cancer than of control subjects were the progeny of first-cousin marriages (odds ratio [OR] 2.1; 95% CI 1.4-3.3; P=.001). The effects of consanguinity were significant for case subjects with early-onset breast cancer (age <40 years) (OR=2.7; 95% CI 1.5-4.9; P=.0008) and case subjects with ovarian cancer (OR=2.4; 95% CI 1.4-4.2; P=.002). These results suggest that recessively inherited genes may contribute to breast and ovarian cancer risk in Pakistan.