Background: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5′ ...region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. Methods: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. Results: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. Conclusions: Taken together, our results firmly establish that the BRCA1 5′ region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.
Background
Retinoblastoma (Rb) is a rare intraocular malignant tumor in children with high overall survival. Predisposition to Rb is linked to RB1 germline mutations with high penetrance, but rare ...RB1 low‐penetrance variants are also known. Rb survivors are at risk of second primary malignancies (SPMs), mostly osteosarcoma and soft‐tissue sarcoma. Nevertheless, the risk of primary osteosarcoma developing without prior Rb has not been reported in RB1 germline mutation carriers.
Methods
We report a patient in whom osteosarcoma developed at age 17 as a first primary malignancy within a family context of sarcoma.
Results
Unexpectedly, genetic testing identified a low‐penetrance germline mutation in RB1 NM_000321.2: c.45_76dup; p.(Pro26Leufs*50). In eight additional similar cases from published and unpublished reports of families, first primary osteosarcomas and sarcomas mostly developed in RB1 low‐penetrance mutation carriers without prior Rb.
Conclusion
We propose that first primary sarcoma and osteosarcoma could be a novel clinical presentation of a RB1‐related hereditary predisposition syndrome linked to RB1 low‐penetrance germline mutations. In these families, careful screening of primary non‐Rb cancer and SPMs is required by maintaining enhanced clinical vigilance. Implementing lifelong periodic whole‐body MRI screening might be a complementary strategy for unaffected carrier relatives in these families.
The risk of primary osteosarcoma developing without prior retinoblastoma has not been reported in RB1 germline mutation carriers. We report a patient in whom osteosarcoma developed at age 17 as a first primary malignancy within a family context of sarcoma. Unexpectedly, genetic testing identified a low‐penetrance germline mutation in RB1 NM_000321.2: c.45_76dup, p.(Pro26Leufs*50). We propose that first primary sarcoma and osteosarcoma could be a novel clinical presentation of a RB1‐related hereditary predisposition syndrome linked to RB1 low‐penetrance germline mutations and discuss the screening strategy to implement for unaffected carrier relatives in these families.
PTEN hamartoma tumour syndrome (PHTS) encompasses several clinical syndromes with germline mutations in the PTEN tumour suppressor gene, including Cowden syndrome which is characterised by an ...increased risk of breast and thyroid cancers. Because PHTS is rare, data regarding cancer risks and genotype-phenotype correlations are limited. The objective of this study was to better define cancer risks in this syndrome with respect to the type and location of PTEN mutations.
154 PHTS individuals with a deleterious germline PTEN mutation were recruited from the activity of the Institut Bergonié genetic laboratory. Detailed phenotypic information was obtained for 146 of them. Age and sex adjusted standardised incidence ratio (SIR) calculations, cumulative cancer risk estimations, and genotype-phenotype analyses were performed.
Elevated SIRs were found mainly for female breast cancer (39.1, 95% CI 24.8 to 58.6), thyroid cancer in women (43.2, 95% CI 19.7 to 82.1) and in men (199.5, 95% CI 106.39 to 342.03), melanoma in women (28.3, 95% CI 7.6 to 35.4) and in men (39.4, 95% CI 10.6 to 100.9), and endometrial cancer (48.7, 95% CI 9.8 to 142.3). Cumulative cancer risks at age 70 were 85% (95% CI 70% to 95%) for any cancer, 77% (95% CI 59% to 91%) for female breast cancer, and 38% (95% CI 25% to 56%) for thyroid cancer. The risk of cancer was two times greater in women with PHTS than in men with PHTS (p<0.05).
This study shows a considerably high cumulative risk of cancer for patients with PHTS, mainly in women without clear genotype-phenotype correlation for this cancer risk. New recommendations for the management of PHTS patients are proposed.
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/).
BRCA1 and BRCA2 genes play a crucial role in repairing DNA double-strand breaks through homologous recombination. Their mutations represent a significant proportion of homologous recombination ...deficiency and are a reliable effective predictor of sensitivity of high-grade ovarian cancer (HGOC) to poly(ADP-ribose) polymerase inhibitors. However, their testing by next-generation sequencing is costly and time-consuming and can be affected by various preanalytical factors. In this study, we present a deep learning classifier for BRCA mutational status prediction from hematoxylin-eosin-safran–stained whole slide images (WSI) of HGOC. We constituted the OvarIA cohort composed of 867 patients with HGOC with known BRCA somatic mutational status from 2 different pathology departments. We first developed a tumor segmentation model according to dynamic sampling and then trained a visual representation encoder with momentum contrastive learning on the predicted tumor tiles. We finally trained a BRCA classifier on more than a million tumor tiles in multiple instance learning with an attention-based mechanism. The tumor segmentation model trained on 8 WSI obtained a dice score of 0.915 and an intersection-over-union score of 0.847 on a test set of 50 WSI, while the BRCA classifier achieved the state-of-the-art area under the receiver operating characteristic curve of 0.739 in 5-fold cross-validation and 0.681 on the testing set. An additional multiscale approach indicates that the relevant information for predicting BRCA mutations is located more in the tumor context than in the cell morphology. Our results suggest that BRCA somatic mutations have a discernible phenotypic effect that could be detected by deep learning and could be used as a prescreening tool in the future.
Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction ...of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced
exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (
= 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.
Abstract
Background. Assessment of the age-dependent cancer risk conferred by germline predicted pathogenic variants (PPV) in cancer susceptibility genes is often hampered by the way the data are ...collected. Cohort-based data frequently contain an overrepresentation of patients carrying a gene variant of interest and an underrepresentation of cancer-free gene variant carriers. In order to overcome this problem, penetrance estimates can be grounded on family-based study designs, through the evaluation of index patients (IP) and their family relatives. The purpose of the TUMOSPEC study is to estimate the penetrance of PPV in genes whose literature data are currently inaccurate or limited and to determine their associated tumour spectrum. This will lead to an appropriate assessment of the clinical utility of testing these genes. Methods. IP are enrolled consecutively among patients who are being offered a germline genetic test in a hereditary breast and ovary cancer (HBOC) context in a participating cancer genetics clinics. A panel of 24 genes (ATM, BAP1, BARD1, BRIP1, CHEK2, FANCM, FAM175A, MRE11A, NBN, STK11, RINT1, XRCC2, PALB2, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, RAD50, RAD51B, RAD51C, RAD51D, TP53) is tested in parallel of BRCA1 and BRCA2. If a PPV is identified, the IP is asked to give her/his first- and second-degree relatives and cousins address, regardless of their health status. Each relative is then contacted by the study coordinating centre (CC) to be invited. Each participant completes an epidemiological questionnaire addressing personal medical history and exposure to various risk factors, and provides a saliva sample to determine if he/she carries the familial PPV. The CC collects questionnaires, family history, clinical and genetic data. Results. Enrolment for the feasibility study takes place between Sep. 2017 and Dec. 2019 at each of the 46 participating french clinics. In June 2019, the study included 3,298 IP. So far, the CC received 1,241 TUMOSPEC panel results (37.6%) with a mean delay of 5 months 0.7 -17.5. Among them, 169 carried a PPV in at least one of the genes (totalizing 183 PPV). Additionally, 44 IP with PPV identified beforehand were enrolled (table 1). Relatives’ invitation of these 213 IP began in June 2018. Among the relatives of the first 29 index cases contacted so far, 4.6 relatives per family consented to participate. Conclusions. The feasibility study showed that inclusion process is well adapted and that the communication between the various partners (clinicians, biologists, investigators and study participants) is quite smooth. Rates of inclusion for invited relatives (50%), for IP questionnaire completion (45%) and relatives biological sample collection (50%) are also very satisfactory and yet underestimated due to the recent start of relatives inclusions. Overall, this study shows that it is feasible to conduct a large-scale study to gather sufficient number of positive families for each gene included in the panel in a reasonable interval of time. This on-going national effort will allow to appropriately assess cancer risks cancer in families with a PPV in a gene often included in HBOC multi-gene panels. This is an essential step to optimize clinical management guidelines specific to each gene, and will represent a valuable resource for future research on the genetics of breast and ovary cancers.
Number of PPV identified in IPGeneATMCHEK2PALB2RAD51CBRIP1RAD51DMSH6RAD50PMS2MRE11ANBNMSH2MLH1CDH1BARD1TP53STK11XRCC2RINT1BAP1RAD51BFAM175AFANCMPTENNumber of PPV512019161111111010999865544322110
Citation Format: Olivier Caron, Séverine Eon-Marchais, Sarah Bonnet-Boissinot, Juana Beauvallet, Marie-Gabrielle Dondon, Chrystelle Colas, Florence Coulet, Capucine Delnatte, Claude Houdayer, Christine Lasset, Jérôme Lemonnier, Michel Longy, Catherine Nogues, Dominique Stoppa-Lyonnet, Dominique Vaur, Fabienne Lesueur, Nadine Andrieu, TUMOSPEC Investigators Group, UNICANCER Groupe Génétique et Cancer. Feasibility of a nation-wide family-based study to assess cancer risks in families with a predicted pathogenic variant identified through hereditary breast and ovary multi-gene panel testing: The TUMOSPEC study abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-12.
is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance ...(VUS). Among all
VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored
exon 3 (
e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for
haploinsufficiency.
predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL
transcripts. Of 100
e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL
transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL
with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL
exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of
variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that
tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of
leaky splicing variants, some of which may not increase cancer risk.
Three partially overlapping breast cancer polygenic risk scores (PRS) comprising 77, 179 and 313 SNPs have been proposed for European-ancestry women by the Breast Cancer Association Consortium (BCAC) ...for improving risk prediction in the general population. However, the effect of these SNPs may vary from one country to another and within a country because of other factors.
To assess their associated risk and predictive performance in French women from (1) the CECILE population-based case-control study, (2) BRCA1 or BRCA2 (BRCA1/2) pathogenic variant (PV) carriers from the GEMO study, and (3) familial breast cancer cases with no BRCA1/2 PV and unrelated controls from the GENESIS study.
All three PRS were associated with breast cancer in all studies, with odds ratios per standard deviation varying from 1.7 to 2.0 in CECILE and GENESIS, and hazard ratios varying from 1.1 to 1.4 in GEMO. The predictive performance of PRS313 in CECILE was similar to that reported in BCAC but lower than that in GENESIS (area under the receiver operating characteristic curve (AUC) = 0.67 and 0.75, respectively). PRS were less performant in BRCA2 and BRCA1 PV carriers (AUC = 0.58 and 0.54 respectively).
Our results are in line with previous validation studies in the general population and in BRCA1/2 PV carriers. Additionally, we showed that PRS may be of clinical utility for women with a strong family history of breast cancer and no BRCA1/2 PV, and for those carrying a predicted PV in a moderate-risk gene like ATM, CHEK2 or PALB2.
•Three polygenic risk scores for breast cancer assessed in 3 groups of French women.•All of them associated with breast cancer but effects varied depending on group.•Predictive ability is higher in women with no BRCA1 or BRCA2 pathogenic variant.•Opportunity to refine risk stratification in carriers of a moderate-risk variant.•Effects of SNPs in polygenic risk scores vary depending on country and population.
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