Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

Evaluation of germline BRCA1 and BRCA2 mutations in a multi-ethnic Asian cohort of ovarian cancer patients.

OBJECTIVE: Despite the discovery of breast and ovarian cancer predisposition genes BRCA1 and BRCA2 more than two decades ago, almost all the available data relate to women of European ancestry, with only a handful of studies in Asian populations. In this study, we determined the frequency of germline alterations in BRCA1 and BRCA2 in ovarian cancer patients from a multi-ethnic cross-sectional cohort of Asian ovarian cancer patients from Malaysia. METHODS: From October 2008 to February 2015, we established a hospital-based cohort of ovarian cancer patients and the germline status of all 218 women with invasive epithelial ovarian cancer was tested using targeted amplification and sequencing of the intron-exon junctions and exonic sequences of BRCA1, BRCA2, PALB2 and TP53. RESULTS: BRCA1 and BRCA2 mutations were found in 8% (17 cases) and 3% (7 cases) of the ovarian cancer patients, respectively. Mutation carriers were diagnosed at a similar age to non-carriers, but were more likely to be Indian, have serous ovarian cancer, and have more relatives with breast or ovarian cancer. Nonetheless, 42% (10/24) of mutation carriers did not have any family history of breast or ovarian cancer and offering genetic counselling and genetic testing only to women with family history would mean that 35% (6/17) of BRCA1 mutation carriers and 57% (4/7) of BRCA2 mutation carriers would not be offered genetic testing. CONCLUSIONS: Our data suggest that, similar to Caucasians, a significant proportion of Asian ovarian cancer was attributed to germline mutations in BRCA1 and to a lesser extent in BRCA2.

Recurrent mutation testing of BRCA1 and BRCA2 in Asian breast cancer patients identify carriers in those with presumed low risk by family history.

Although the breast cancer predisposition genes BRCA1 and BRCA2 were discovered more than 20 years ago, there remains a gap in the availability of genetic counselling and genetic testing in Asian countries because of cost, access and inaccurate reporting of family history of cancer. In order to improve access to testing, we developed a rapid test for recurrent mutations in our Asian populations. In this study, we designed a genotyping assay with 55 BRCA1 and 44 BRCA2 mutations previously identified in Asian studies, and validated this assay in 267 individuals who had previously been tested by full sequencing. We tested the prevalence of these mutations in additional breast cancer cases. Using this genotyping approach, we analysed recurrent mutations in 533 Malaysian breast cancer cases with <10 % a priori risk, and found 1 BRCA1 (0.2 %) and 5 BRCA2 (0.9 %) carriers. Testing in a hospital-based unselected cohort of 532 Singaporean breast cancer cases revealed 6 BRCA1 (1.1 %) and 3 BRCA2 (0.6 %) carriers. Overall, 2 recurrent BRCA1 and 1 BRCA2 mutations in Malays, 3 BRCA1 and 2 BRCA2 mutations in Chinese and 1 BRCA1 mutation in Indians account for 60, 24 and 20 % of carrier families, respectively. By contrast, haplotype analyses suggest that a recurrent BRCA2 mutation (c.262_263delCT) found in 5 unrelated Malay families has at least 3 distinct haplotypes. Taken together, our data suggests that panel testing may help to identify carriers, particularly Asian BRCA2 carriers, who do not present with a priori strong family history characteristics.

A novel splice site mutation in the noncoding region of BRCA2: implications for Fanconi anemia and familial breast cancer diagnostics.

Fanconi anemia (FA) is a rare recessive disorder with chromosomal instability, congenital abnormalities, and a high cancer risk. The breast cancer susceptibility gene BRCA2 (FANCD1) is one of the 16 genes involved in this recessive disease. We have identified a novel mutation of the splice donor site of intron 1 in the noncoding region of BRCA2 in a Japanese FA family. This mutation may account for the FA phenotype in a patient originally reported to have biallelic mutations in BRCA2. Subsequent functional studies revealed that one of the mutations, K2729N, was a neutral change. As reported here, a more careful analysis resulted in the identification of a novel splice site mutation. Functional analysis using a mouse embryonic stem cell-based assay revealed that it causes aberrant splicing, reduced transcript levels and hypersensitivity to DNA damaging agents, suggesting that it is likely to be pathogenic. Although similar pathogenic variants in the noncoding region of BRCA1 and 2 were not identified in a cohort of 752 familial breast cancer cases, we still think this finding is relevant for mutation analysis in Hereditary Breast and Ovarian Cancer Syndrome families in a diagnostic setting.

Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations.

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ~2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ~5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750-1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ~650 years ago, and into the Iraqi-Jewish community ~450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.

1'S-1'-acetoxyeugenol acetate: a new chemotherapeutic natural compound against MCF-7 human breast cancer cells.

Medicinal plants containing active natural compounds have been used as an alternative treatment for cancer patients in many parts of the world especially in Asia (Itharat et al. 2004). In this report, we describe the cytotoxic and apoptotic properties of 1'S-1'-acetoxyeugenol acetate (AEA), an analogue of 1'S-1'-acetoxychavicol acetate (ACA), isolated from the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) on human breast cancer cells. Data from MTT cell viability assays indicated that AEA induced both time- and dose-dependent cytotoxicity with an IC(50) value of 14.0 µM within 36 h of treatment on MCF-7 cells, but not in HMEC normal control cells. Both annexin V-FITC/PI flow cytometric analysis and DNA fragmentation assays confirmed that AEA induced cell death via apoptosis. AEA was also found to induce cell cycle arrest in MCF-7 cells at the G(0)/G(1) phase with no adverse cell cycle arrest effects on HMEC normal control cells. It was concluded that AEA isolated from the Malaysian tropical ginger represents a potential chemotherapeutic agent against human breast cancer cells with higher cytotoxicity potency than its analogue, ACA.

CHEK2*1100delC does not contribute to risk to breast cancer among Malay, Chinese and Indians in Malaysia.

A truncating mutation (1100delC) in the cell cycle checkpoint kinase-2 gene, CHEK2, has been identified as a risk factor for familial and sporadic breast cancer in some Northern and Western European populations. However, the prevalence of CHEK2*1100delC in breast cancer appears to be population dependent. We analysed the prevalence of CHEK2*1100delC in 668 breast cancer cases, of which 542 were invasive breast cancers, from a hospital-based cohort of breast cancer patients from Kuala Lumpur, Malaysia. The variant was not found in any patients in this cohort, suggesting that CHEK2*1100delC is rare in our population, and unlikely to contribute significantly to risk to breast cancer among the Malay, Chinese and Indian ethnic groups in Malaysia. This suggests that screening for this allele should not be routinely conducted in Malaysia.

Synthesis, characterization, DNA-binding study and anticancer properties of ternary metal(II) complexes of edda and an intercalating ligand.

A series of ternary metal(ii) complexes {M(phen)(edda); 1a (Cu), 1b (Co), 1c (Zn), 1d (Ni); H(2)edda = N,N(')-ethylenediaminediacetic acid} of N,N'-ethylene-bridged diglycine and 1,10-phenanthroline were synthesized and characterized by elemental analysis, FTIR, UV-visible spectroscopy and magnetic susceptibility measurement. The interaction of these complexes with DNA was investigated using CD and EPR spectroscopy. MTT assay results of 1a-1c , screened on MCF-7 cancer cell lines, show that synergy between the metal and ligands results in significant enhancement of their antiproliferative properties. Preliminary results from apoptosis and cell cycle analyses with flow cytometry are reported. seems to be able to induce cell cycle arrest at G(0)/G(1). The crystal structure of 1a is also included.

Spindle assembly checkpoint and centrosome abnormalities in oral cancer.

Like many solid tumours, oral squamous cell carcinomas (OSCC) invariably exhibit chromosomal instability (CIN) leading to aneuploidy. The mechanisms responsible for CIN in OSCC, however, are largely unknown. This study examined the fidelity of the spindle checkpoint, together with the number, structure and function of centrosomes in a series of well-characterised aneuploid immortal OSCC-derived cell lines that harbour p53 and p16(INK4A) defects. The spindle checkpoints were fully functional in 2 of 7 cell lines and attenuated in the remaining 5 cell lines. Overexpression of the spindle checkpoint protein, Cdc20, was observed in 2 of the cell lines with attenuated checkpoints. Defects in centrosome number, size and localisation were detected in 5 of the cell lines. Clonal cell populations contained cells with both normal and abnormal numbers of centrosomes, suggesting that the control of centrosome number may be inherently unstable in OSCC-derived cell lines. Centrosomal abnormalities were then examined in tissue samples of oral epithelial dysplasias and carcinomas. Abnormal centrosomes were detected in all the tissues examined albeit in a low percentage of cells (<1% to >5%). The percentage of cells containing centrosome abnormalities was significantly higher in the carcinomas than in the dysplasias (p<0.02) and in the poorly differentiated SCCs relative to their moderately differentiated (p<0.04) and well-differentiated (p<0.01) counterparts. We suggest that the genetic alterations associated with the development of the immortal phenotype, together with spindle checkpoint and centrosome defects, are responsible, albeit in part, for the complex karyotypes observed in OSCC. The presence of centrosome abnormalities in oral dysplasias raises the possibility that such defects might contribute to malignant progression.