Mélanie Léoné

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5′ and 3′ sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation. Hum Mutat 33:1228–1238, 2012.

Prevalence of BRCA1 and BRCA2 germline mutations in young breast cancer patients: A population-based study

Our aim was to estimate the prevalence of mutations in the BRCA1 and BRCA2 genes among unselected incident cases of breast cancer in young women. We identified 158 incident breast cancer cases diagnosed before age 46 years in predefined geographic areas in Girona and Tarragona, Spain, during 1995–1997. Of these, 136 (86%) provided information on family history of cancer and were screened for BRCA1 and BRCA2 mutations. Nine of the 136 (6.6%) were found to carry BRCA deleterious mutations (MUT) (1 BRCA1 and 8 BRCA2), and 20 were detected with rare BRCA variants of unknown significance (UV). Both MUT and US BRCA alterations were more frequent in younger patients: 7 (11.6%) MUT and 12 (19.3%) UV carriers were found in the group of 62 patients younger than 40 years, whereas 2 (2.7%) MUT and 9 (12%) US carriers were identified in the group of 74 patients aged 40–45. Family history of breast and ovarian cancers suggestive of hereditary condition (at least 2 first‐ or second‐degree relatives affected with breast cancer or at least 1 relative affected with ovarian cancer or early‐onset breast cancer) was absent for 5 of 9 MUT carriers. This suggests that BRCA screening policies based on family history of cancer would miss a considerable proportion of BRCA mutations. Mutations in the BRCA1 and BRCA2 genes explain at least 10% of breast cancer cases diagnosed before age 40 years. The contribution of these genes to early‐onset breast cancer is likely to be even higher given that certain UV cases might be disease‐associated.

Contribution of BRCA1 and BRCA2 germ-line mutations to the incidence of breast cancer in young women: Results from a prospective population-based study in France

The prevalence of BRCA1/2 germ‐line mutations was assessed in a prospective population‐based series of early‐onset breast cancer (BC) patients in France, and the usefulness of a clinical assessment of hereditary BC risk, based on multiple criteria including pedigree structure, was evaluated. Through the Rhône region BC registry, 232 women diagnosed with BC before 46 years of age were included. They were tested for BRCA1/2 mutations an average of 10 months after diagnosis. All the women were classified according to their family history of cancer: high risk of hereditary breast cancer (HBC), low risk of HBC, isolated BC, and unknown HBC risk. Deleterious mutations were observed in 21 women (9.1%): 15 (6.5%) BRCA1 and 6 (2.6%) BRCA2. Mutations were more prevalent in women who developed BC before age 41 than in women who developed BC between ages 41 and 45 (12.8% versus 5.2%, respectively, P = 0.04). A high prevalence of BRCA1/2 mutations was found among women in the high‐risk category with particular family features (i.e., small family size, predominantly male pedigree, specific cancers; 23.5%) and among women with isolated BC before age 41 and with five or fewer close adult female relatives (16.6%). According to the 10% probability level recommended by the American Society of Clinical Oncology guidelines for genetic testing of cancer, BRCA1/2 mutation screening should be considered for all women diagnosed before age 41, except for those with isolated BC in a large pedigree including multiple unaffected female relatives. The clinical assessment of HBC risk that we have developed should help in the decision to perform genetic testing.

The TP53 Arg72Pro and MDM2 309G > T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

Background:The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance.Methods:To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework.Results:No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93–1.10, Ptrend=0.77; MDM2: HR=0.96, 95%CI: 0.84–1.09, Ptrend=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87–1.12, Ptrend=0.83; MDM2: HR=0.98, 95%CI: 0.80–1.21, Ptrend=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association.Conclusion:There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort

Abstract Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

Full in-frame exon 3 skipping of BRCA2 confers high risk of breast and/or ovarian cancer

Germline pathogenic variants in the BRCA2 gene are associated with a cumulative high risk of breast/ovarian cancer. Several BRCA2 variants result in complete loss of the exon-3 at the transcript level. The pathogenicity of these variants and the functional impact of loss of exon 3 have yet to be established. As a collaboration of the COVAR clinical trial group (France), and the ENIGMA consortium for investigating breast cancer gene variants, this study evaluated 8 BRCA2 variants resulting in complete deletion of exon 3. Clinical information for 39 families was gathered from Portugal, France, Denmark and Sweden. Multifactorial likelihood analyses were conducted using information from 293 patients, for 7 out of the 8 variants (including 6 intronic). For all variants combined the likelihood ratio in favor of causality was 4.39*1025. These results provide convincing evidence for the pathogenicity of all examined variants that lead to a total exon 3 skipping, and suggest that other variants that result in complete loss of exon 3 at the molecular level could be associated with a high risk of cancer comparable to that associated with classical pathogenic variants in BRCA1 or BRCA2 gene. In addition, our functional study shows, for the first time, that deletion of exon 3 impairs the ability of cells to survive upon Mitomycin-C treatment, supporting lack of function for the altered BRCA2 protein in these cells. Finally, this study demonstrates that any variant leading to expression of only BRCA2 delta-exon 3 will be associated with an increased risk of breast and ovarian cancer.

GEMO, a national resource to study genetic modifiers of breast and ovarian cancer risk in BRCA1 and BRCA2 pathogenic variant carriers

Women carrying a pathogenic variant (PV) in the BRCA1 or BRCA2 (BRCA1/2) genes are at high lifetime risk of developing breast cancer (BC) and ovarian cancer (OC), but estimation of the cumulative risk of cancer to age 70 years varies substantially between studies and populations. Initial estimations were obtained from selected high-risk families with multiple cases, such as those ascertained through the Breast Cancer Linkage Consortium used to identify disease loci (1). In the first retrospective studies conducted on such families, estimates for BC ranged from 40 to 87% for BRCA1 PV carriers and from 27 to 84% for BRCA2 PV carriers and estimates for OC ranged from 16 to 68% for BRCA1 PV carriers and from 11 to 27% for BRCA2 PV carriers (1–4). Recently, the largest prospective cohort conducted to date reported cumulative risks of BC to age 80 years of 72% for BRCA1 PV carriers and 69% for BRCA2 PV carriers (5). In the same study, cumulative risks of OC to age 80 years were 44% for BRCA1 PV carriers and 17% for BRCA2 PV carriers. Variation in cancer risks within or between BRCA1/2 families, with respect to age at diagnosis or type of cancer, can be explained by other genetic factors and/or lifestyle and reproductive factors (6–10). Genome-wide association studies (GWAS) conducted by the Breast Cancer Association Consortium (BCAC) have identified 172 common single-nucleotide polymorphisms (SNPs) associated with small increases in breast and/or ovarian cancer risk in the general population (11). A subset of these SNPs modifies the risk of breast and ovarian cancer risk for BRCA1/2 PV carriers (12–14) but most of the variability has not been explained yet (15). Breast and ovarian cancer risks in BRCA1/2 PV carriers might also vary according to the location of the variant and/or its origin (14, 16–19). Genetic testing for BRCA1 and BRCA2 has been part of genetic counseling in European Union countries and North America since their discovery in the 90s, and has greatly improved recommendations about clinical management options and the most appropriate treatments. Nonetheless, both retrospective and prospective studies on large datasets of BRCA1/2 PV carrier families are still very much needed to refine individual cancer risk estimates by considering other genetic and lifestyle/environmental factors, and they will also contribute to a better understanding of the correlation between mutant BRCA1/2 alleles and phenotype. In particular, accurate age-specific risk estimates for the different types of cancer would be useful when choosing risk reduction strategies such as prophylactic bilateral mastectomy or salphingo-oophorectomy. The Genetic Modifiers of BRCA1 and BRCA2 (GEMO) Group is the French multidisciplinary, collaborative framework for the investigation of genetic factors modifying cancer risk in Hereditary Breast and Ovarian cancer (HBOC) families segregating BRCA1/2 PVs. Its primary aims are to contribute to large-scale national and international projects to identify genetic modifiers and to facilitate the translation of research results to the clinical setting. This is achieved by establishing a resource of blood DNA samples from individuals carrying a PV together with family and clinical data through the nation-wide network of cancer genetic clinics. Here we report on the progress of the GEMO study, the characteristics of the 5,303 actual participants and the prevalence and spectrum of BRCA1/2 cancer-associated variants identified so far.

Occurrence of a non deleterious gene conversion event in the BRCA1 gene

The duplication in the primate lineage of a portion of the breast and ovarian cancer susceptibility gene BRCA1 has created a BRCA1 pseudogene 45 kb away. Non‐allelic homologous recombination (NAHR) between BRCA1 and BRCA1P1 has generated recurrent deleterious germ‐line 37‐kb deletions encompassing the first two exons of BRCA1, accounting for several breast and ovarian cancer families in various populations. In principle, NAHR intermediates resolution could also lead through a non‐crossover configuration to interlocus gene conversion (IGC), but none had been described as yet. Here, we report for the first time an IGC event identified in a breast and ovarian cancer family involving exactly the same segment as that involved in the 37‐kb deletions. Close examination of the consequences of this IGC event showed that it does not impact BRCA1 expression. Detailed analysis of the regions of homology between BRCA1 and its pseudogene revealed the specificity of the segment where recombination systematically occurs.