Monica Barile

Common Breast Cancer-Predisposition Alleles Are Associated with Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Oral contraceptive use and breast or ovarian cancer risk in BRCA1/2 carriers: A meta-analysis

BACKGROUND Women with BRCA1 or BRCA2 mutations are at increased risk of breast and ovarian cancer. Oral contraceptives (OC) use has been associated with a reduction in ovarian cancer risk and with a moderately increased breast cancer risk, which tends to level off in the few years after stopping. The association between oral contraceptive and BRCA1 or BRCA2 gene mutations carriers is unclear. METHODS We performed a comprehensive literature search updated to March 2010 of studies on the associations between OC users and breast or ovarian cancer for ascertained BRCA1/2 carriers. We obtained summary risk estimated for ever OC users, for duration of use and time since stopping. RESULTS A total of 2855 breast cancer cases and 1503 ovarian cancer cases, carrying an ascertained BRCA1/2 mutation, were included in our meta-analyses, based on overall 18 studies. Use of OC was associated with a significant reduced risk of ovarian cancer for BRCA1/2 carriers (summary relative risk (SRR)=0.50; 95% confidence interval (CI), 0.33-0.75). We also observed a significant 36% risk reduction for each additional 10 years of OC use (SRR: 0.64; 95% CI, 0.53-0.78; P trend<0.01). We found no evidence of a significant association between OC and breast cancer risk in carriers (SRR: 1.13; 95% CI, 0.88-1.45) and with duration of use. OC formulations used before 1975 were associated with a significant increased risk of breast cancer (SRR: 1.47; 95% 1.06, 2.04), but no evidence of a significant association was found with use of more recent formulations (SRR: 1.17; 95% 0.74, 1.86). CONCLUSIONS OC users carrying an ascertained BRCA1/2 mutation have a reduced risk of ovarian cancer, proportional to the duration of use. There is no evidence that recent OC formulations increase breast cancer risk in carriers.

Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in German and Italian familial breast cancer cases

Recently, the SNPs rs11614913 in hsa‐mir‐196a2 and rs3746444 in hsa‐mir‐499 were reported to be associated with increased breast cancer risk, and the SNP rs2910164 in hsa‐mir‐146a was shown to have an effect on age of breast cancer diagnosis. In order to further investigate the effect of these SNPs, we genotyped a total of 1894 breast cancer cases negative for disease‐causing mutations or unclassified variants in BRCA1 and BRCA2, and 2760 controls from Germany and Italy. We compared the genotype and allele frequencies of rs2910164, rs11614913 and rs3746444 in cases versus controls of the German and Italian series, and of the two series combined; we also investigated the effect of the three SNPs on age at breast cancer diagnosis. None of the performed analyses showed statistically significant results. In conclusion, our data suggested lack of association between SNPs rs2910164, rs11614913 and rs3746444 and breast cancer risk, or age at breast cancer onset.

PALB2 germline mutations in familial breast cancer cases with personal and family history of pancreatic cancer.

These authors contributed equally to this work. *Correspondence to Paolo Radice. Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazion ale dei Tumori, Milan, Italy. E -mail: paolo.radice@istitutotumori.mi.it . Telephone: +39 02.2390.3224. Fax: +39 02.2390.2764

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Identification of fifteen novel germline variants in the BRCA1 3′UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site†

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron–exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3′UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3′UTR for mutations in a large series of BRCA‐mutation negative, population and clinic‐based breast cancer cases, and controls. Fifteen novel BRCA1 3′UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.*528G>C, c.*718A>G, and c.*1271T>C and four found in controls, c.*309T>C, c.*379G>A, c.*823C>T, and c.*264C>T, reduced 3′UTR activity (P < 0.02), whereas two variants found in cases, c.*291C>T and c.*1139G>T, increased 3′UTR activity (P < 0.01). Three case variants, c.*718A>G, c.*800T>C, and c.*1340_1342delTGT, were predicted to create new miRNA binding sites and c.*1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3′UTR reporter activity when coexpressed with the predicted targeting miRNA, miR‐103. This is the most comprehensive identification and analysis of BRCA1 3′UTR variants published to date. Hum Mutat 33:1665–1675, 2012.

Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.

Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.

MC1R variation and melanoma risk in relation to host/clinical and environmental factors in CDKN2A positive and negative melanoma patients

Host, environmental and genetic factors differently modulate cutaneous melanoma (CM) risk across populations. Currently, the main genetic risk determinants are germline mutations in the major known high‐risk susceptibility genes, CDKN2A and CDK4, and variants of the low‐risk gene MC1R, which is key in the pigmentation process. This case–control study aimed at investigating the influence of the main host and environmental risk factors and of MC1R variation on CM risk in 390 CDKN2A‐negative and 49 CDKN2A‐positive Italian individuals. Multivariate analysis showed that MC1R variation, number of nevi and childhood sunburns doubled CM risk in CDKN2A‐negative individuals. In CDKN2A‐positive individuals, family history of CM and presence of atypical nevi, rather than MC1R status, modified risk (20.75‐ and 2.83‐fold, respectively). Occupational sun exposure increased CM risk (three to sixfold) in both CDKN2A‐negative and CDKN2A‐positive individuals, reflecting the occupational habits of the Ligurian population and the geographical position of Liguria.

The SNP rs895819 in miR-27a is not associated with familial breast cancer risk in Italians

MicroRNAs (miRNAs) are a class of small non-coding RNA implicated in gene expression; deregulation of miRNAs, accounted by somatic variations, may lead to initiation and progression of several types of cancers [1]. It has been postulated that also common germline variants located within miRNA genes may be associated with cancer risk. In particular, the single nucleotide polymorphisms (SNPs) rs2910164, rs11614913, rs3746444, and rs6505162 located within miR-146a, miR-196a2, miR-499, and miR423, respectively, resulted all associated with breast cancer risk [2, 3]. However, a subsequent large study on SNPs in miR-146a, miR-192a2, and miR-499 did not replicate these findings [4]. Two following meta-analyses confirmed the association for the SNP in miR-196a2 and the lack of association for the SNP in miR-146a [5, 6]. The rare allele of the rs12975333 in miRNA-125a was found strongly associated with breast cancer risk in Antwerp, Belgium [7], but a multicenter study reported no allele carriers failing to confirm this association [8]. Recently, the SNP rs895819 located in the miRNA-27a gene was investigated in a series of 1,217 German familial breast cancer cases and 1,422 unrelated German controls and the rare [G] allele was shown to have a protective effect with odds ratio (OR) = 0.88 [95 % confidence interval (CI) 0.78–0.99, P = 0.0287]. Additional analyses indicated that the protective effect was limited to cases with age at diagnosis \50 years (OR = 0.83, 95 % CI 0.70–0.98, P = 0.0314), whereas a stronger effect was detected in bilateral breast cancer cases (OR = 0.70, 95 % CI 0.52–0.95, P = 0.0238) [9]. Aimed at re-testing these findings, we investigated the effect of rs895819 on breast cancer risk by genotyping a

Methylation of O 6-methylguanine-DNA methyltransferase (MGMT) promoter gene in triple-negative breast cancer patients

Triple-negative breast cancers are characterized by the triple-negative (ER/PgR/Her2 negative) phenotype, are frequently associated with BRCA gene mutation, and are not candidate to currently available endocrine and HER2-targeted treatments. MGMT is involved in direct DNA repair exerted by cleavage of mutagenic alkyl adducts within DNA, and its epigenetic silencing confers susceptibility to DNA-damaging alkylating agents in glioblastomas and melanomas. MGMT methylation status has not been extensively investigated in breast cancer patients. The goal of our study was to evaluate the MGMT methylation status in TNBC patients, for most of which BRCA1 and BRCA2 mutational status was known. We evaluated MGMT methylation status by methylation-specific PCR (MSP) in formalin-fixed and paraffin-embedded tumor specimens from 92 TNBC patients. By using the GelDoc system (Biorad) software, the cases were further classified as follows: 0 (absence of methylated signal), 1 (prevalence of unmethylated signal, U/M ratio >1), 2 (prevalence of methylated signal, U/M ratio <1), and 3 (absence of unmethylated signal). MSP products were obtained in 89 (96.7%) of the cases. Overall, 15 (16.9%) cases were classified as 0, 33 (37.1%) cases as 1, 39 (43.8%) cases as 2, and 2 (2.2%) cases as 3. The 48 cases classified as 0 and 1 were considered as MGMT unmethylated, and the 41 cases classified as 2 and 3 as MGMT methylated. The prevalence of MGMT methylation in patients with BRCA1 mutated, wild-type, and unknown was 30.2% (13/43), 63.6% (14/22), and 58.3% (14/24), respectively. MGMT methylation was unrelated to the main clinical pathological characteristics, with the exception of a weak association with advanced age. In conclusion, our data suggest that in TNBC with wild-type BRCA1, the direct DNA repair system may be frequently (63.6%) silenced by MGMT methylation. The evaluation of the MGMT status could offer a new adjunct in predicting tumor response to alkylating drugs in TNBC patients.