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1.
Cancer Res ; 80(17): 3593-3605, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32641407

RESUMO

BRCA2 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 BRCA2 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 BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico 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 BRCA2 transcripts. Of 100 BRCA2e3 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 BRCA2 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 BRCA2 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 BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

2.
Oral Oncol ; 108: 104816, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32480311

RESUMO

Fanconi anemia (FA) is a rare genetic disease that is mostly transmitted, according to a recessive model with biallelic germline alterations in one of the 22 genes of the FA pathway, or monoallelic alteration of the 23rd FA gene (RAD51). The FA pathway is implicated in interstrand DNA crosslink repair, induces genome stability, and is a potent driver of tumorigenesis. Patients with FA have a 500 to 1000-fold increased risk of developing head and neck squamous cell carcinoma (HNSCC). Patients with FA developing an HNSCC, usually have severe radiation toxicities. In this context, the modalities of radiation therapy should be adapted. Some patients with FA present a milder phenotype, especially in the case of medullary FA gene spontaneous reversion. Therefore, in an unusual context of HNSCC, such as no risk factors or a young age, it may be very useful to search anemia or development abnormalities, that may unravel a yet undiagnosed FA disease. Besides, in some young patients with HNSCC who did not suffer from FA, a monoallelic germline alteration in an FA gene could be combined with a second risk factor such as HPV infection or APOBEC alteration. Although several in vitro studies showed that normal cells with monoallelic FA gene alteration may have a particular radiosensitivity, these observations have not been confirmed in vivo in FA heterozygotes patients. Finally, some somatic activating alterations have also been found in HSNCC tumor samples and could be associated with radioresistance.

3.
Cancer Res ; 80(7): 1374-1386, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046981

RESUMO

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 BRCA2 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 (n = 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.


Assuntos
Processamento Alternativo , Proteína BRCA2/genética , Predisposição Genética para Doença , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Éxons/genética , Feminino , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética
4.
Clin Chem ; 66(2): 352-362, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32040573

RESUMO

BACKGROUND: Identifying patients with high-grade serous ovarian cancer (HGSOC) who will respond to treatment remains a clinical challenge. We focused on miR-622, a miRNA involved in the homologous recombination repair (HRR) pathway, and we assessed its predictive value in serum prior to first-line chemotherapy and at relapse. METHODS: Serum miR-622 expression was assessed in serum prior to first-line platinum-based chemotherapy in a prospective multicenter study (miRNA Serum Analysis, miRSA, NCT01391351) and a retrospective cohort (Biological Resource Center, BRC), and was also studied at relapse. Progression-free survival (PFS) and overall survival (OS) were used as primary and secondary endpoints prior to first-line chemotherapy and OS as a primary endpoint at relapse. RESULTS: The group with high serum miR-622 expression was associated with a significantly lower PFS (15.4 versus 24.4 months; adjusted HR 2.11, 95% CI 1.2 3.8, P = 0.015) and OS (29.7 versus 40.6 months; adjusted HR 7.68, 95% CI 2.2-26.2, P = 0.0011) in the miRSA cohort. In the BRC cohort, a high expression of miR-622 was also associated with a significantly lower OS (22.8 versus 35.9 months; adjusted HR 1.98, 95% CI 1.1-3.6, P = 0.026). At relapse, high serum miR-622 was associated with a significantly lower OS (7.9 versus 20.6 months; adjusted HR 3.15, 95% CI 1.4-7.2, P = 0.0062). Serum miR-622 expression is a predictive independent biomarker of response to platinum-based chemotherapy for newly diagnosed and recurrent HGSOC. CONCLUSIONS: These results may open new perspectives for HGSOC patient stratification and monitoring of resistance to platinum-based and poly(ADP-ribose)-polymerase-inhibitor-maintenance therapies, facilitating better and personalized treatment decisions.


Assuntos
Ácidos Nucleicos Livres/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Antineoplásicos/uso terapêutico , Intervalo Livre de Doença , Feminino , Humanos , MicroRNAs/sangue , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/diagnóstico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Estudos Retrospectivos
5.
BMC Genomics ; 21(1): 86, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992191

RESUMO

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.


Assuntos
Íntrons , Precursores de RNA , Sítios de Splice de RNA , Processamento de RNA , Processamento Alternativo , Biologia Computacional/métodos , Humanos , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Processamento Pós-Transcricional do RNA , Curva ROC , Reprodutibilidade dos Testes
7.
Bioinformatics ; 36(5): 1634-1636, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617569

RESUMO

SUMMARY: Alternative splicing is an important biological process widely analyzed in molecular diagnostic settings. Indeed, a variant can be pathogenic by splicing alteration and a suspected pathogenic variant (e.g. truncating variant) can be rescued by splicing. In this context, detecting and quantifying alternative splicing is challenging. We developed SpliceLauncher, a fast and easy to use open source tool that aims at detecting, annotating and quantifying alternative splice junctions at high resolution. AVAILABILITY AND IMPLEMENTATION: SpliceLauncher is available at https://github.com/raphaelleman/SpliceLauncher. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Processamento de RNA , Software , Processamento Alternativo
8.
J Med Genet ; 56(7): 453-460, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30890586

RESUMO

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.


Assuntos
Processamento Alternativo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Alelos , Perfilação da Expressão Gênica , Estudos de Associação Genética/métodos , Mutação em Linhagem Germinativa , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido , Sítios de Splice de RNA
9.
Viruses ; 11(3)2019 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909570

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. We used a yeast two-hybrid approach to identify cell partners of MOPV and LASV Z matrix protein in which two autophagy adaptors were identified, NDP52 and TAX1BP1. Autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. Here, we demonstrate that autophagy is transiently induced by MOPV, but not LASV, in infected cells two days after infection. Impairment of the early steps of autophagy significantly decreased the production of MOPV and LASV infectious particles, whereas a blockade of the degradative steps impaired only MOPV infectious particle production. Our study provides insights into the role played by autophagy during MOPV and LASV infection and suggests that this process could partially explain their different pathogenicity.


Assuntos
Arenavirus/fisiologia , Autofagia , Vírus Lassa/fisiologia , Animais , Arenavirus/patogenicidade , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus Lassa/patogenicidade , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Técnicas do Sistema de Duplo-Híbrido , Células Vero
10.
Viruses ; 12(1)2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31906112

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.


Assuntos
Arenaviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , Vírus Lassa/fisiologia , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Arenaviridae/genética , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Vírus Lassa/genética , Proteínas Repressoras/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Células Vero , Proteínas Virais/genética
12.
Genet Med ; 20(12): 1677-1686, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29988077

RESUMO

PURPOSE: Integration of gene panels in the diagnosis of hereditary breast and ovarian cancer (HBOC) requires a careful evaluation of the risk associated with pathogenic or likely pathogenic variants (PVs) detected in each gene. Here we analyzed 34 genes in 5131 suspected HBOC index cases by next-generation sequencing. METHODS: Using the Exome Aggregation Consortium data sets plus 571 individuals from the French Exome Project, we simulated the probability that an individual from the Exome Aggregation Consortium carries a PV and compared it to the estimated frequency within the HBOC population. RESULTS: Odds ratio conferred by PVs within BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATM, BRIP1, CHEK2, and MSH6 were estimated at 13.22 [10.01-17.22], 8.61 [6.78-10.82], 8.22 [4.91-13.05], 4.54 [2.55-7.48], 5.23 [1.46-13.17], 3.20 [2.14-4.53], 2.49 [1.42-3.97], 1.67 [1.18-2.27], and 2.50 [1.12-4.67], respectively. PVs within RAD51C, RAD51D, and BRIP1 were associated with ovarian cancer family history (OR = 11.36 [5.78-19.59], 12.44 [2.94-33.30] and 3.82 [1.66-7.11]). PALB2 PVs were associated with bilateral breast cancer (OR = 16.17 [5.48-34.10]) and BARD1 PVs with triple-negative breast cancer (OR = 11.27 [3.37-25.01]). Burden tests performed in both patients and the French Exome Project population confirmed the association of PVs of BRCA1, BRCA2, PALB2, and RAD51C with HBOC. CONCLUSION: Our results validate the integration of PALB2, RAD51C, and RAD51D in the diagnosis of HBOC and suggest that the other genes are involved in an oligogenic determinism.


Assuntos
Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Proteína BRCA1/genética , Proteína BRCA2/genética , França/epidemiologia , Predisposição Genética para Doença , Testes Genéticos , Variação Genética/genética , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/epidemiologia , Síndrome Hereditária de Câncer de Mama e Ovário/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Fatores de Risco , Sequenciamento Completo do Exoma
13.
Oncotarget ; 9(25): 17334-17348, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29707112

RESUMO

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.

14.
Nucleic Acids Res ; 46(15): 7913-7923, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29750258

RESUMO

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/).


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Variação Genética , Sítios de Splice de RNA/genética , Processamento de RNA , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Humanos , Cooperação Internacional , Internet , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
Artigo em Inglês | MEDLINE | ID: mdl-31517176

RESUMO

Purpose: To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. Methods: Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Results: Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Conclusion: Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

16.
Eur J Hum Genet ; 25(12): 1345-1353, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29255180

RESUMO

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have recently been involved in breast and ovarian cancer predisposition: RAD51B, RAD51C, and RAD51D in ovarian cancer, RAD51B and XRCC2 in breast cancer. The aim of this study was to estimate the contribution of deleterious variants in the five RAD51 paralogs to breast and ovarian cancers. The five RAD51 paralog genes were analyzed by next-generation sequencing technologies in germline DNA from 2649 consecutive patients diagnosed with breast and/or ovarian cancer. Twenty-one different deleterious variants were identified in the RAD51 paralogs in 30 patients: RAD51B (n = 4), RAD51C (n = 12), RAD51D (n = 7), XRCC2 (n = 2), and XRCC3 (n = 5). The overall deleterious variant rate was 1.13% (95% confidence interval (CI): 0.72-1.55%) (30/2649), including 15 variants in breast cancer only cases (15/2063; 0.73% (95% CI: 0.34-1.11%)) and 15 variants in cases with at least one ovarian cancer (15/570; 2.63% (95% CI: 1.24-4.02%)). This study is the first evaluation of the five RAD51 paralogs in breast and ovarian cancer predisposition and it demonstrates that deleterious variants can be present in breast cancer only cases. Moreover, this is the first time that XRCC3 deleterious variants have been identified in breast and ovarian cancer cases.


Assuntos
Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Rad51 Recombinase/genética , Adulto , Idoso , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Feminino , Mutação em Linhagem Germinativa , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia
17.
Eur J Hum Genet ; 25(10): 1147-1154, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28905878

RESUMO

Interpretation of variants of unknown significance (VUS) is a major challenge for laboratories performing molecular diagnosis of hereditary breast and ovarian cancer (HBOC), especially considering that many genes are now known to be involved in this syndrome. One important way these VUS can have a functional impact is through their effects on RNA splicing. Here we present a custom RNA-Seq assay plus bioinformatics and biostatistics pipeline to analyse specifically alternative and abnormal splicing junctions in 11 targeted HBOC genes. Our pipeline identified 14 new alternative splices in BRCA1 and BRCA2 in addition to detecting the majority of known alternative spliced transcripts therein. We provide here the first global splicing pattern analysis for the other nine genes, which will enable a comprehensive interpretation of splicing defects caused by VUS in HBOC. Previously known splicing alterations were consistently detected, occasionally with a more complex splicing pattern than expected. We also found that splicing in the 11 genes is similar in blood and breast tissue, supporting the utility and simplicity of blood splicing assays. Our pipeline is ready to be integrated into standard molecular diagnosis for HBOC, but it could equally be adapted for an integrative analysis of any multigene disorder.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Testes Genéticos/métodos , Neoplasias Ovarianas/genética , Análise de Sequência de RNA/métodos , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Feminino , Genoma Humano , Humanos , Neoplasias Ovarianas/diagnóstico
18.
Fam Cancer ; 16(2): 167-171, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27783335

RESUMO

Germline allele specific expression (ASE), resulting in a lowered expression of one of the BRCA1 alleles, has been described as a possible predisposition marker in Hereditary Breast or Ovarian Cancer (HBOC), usable for molecular diagnosis in HBOC. The main objective of this prospective case-control study was to compare the proportion of ASE between controls without familial history of breast or ovarian cancer, and HBOC cases without BRCA1 or BRCA2 deleterious mutation. BRCA1 ASE evaluated on three SNPs among controls and HBOC patients without deleterious mutation were assessed by pyrosequencing. The allelic ratios and the proportion of ASE were compared between controls and cases using a Student's t test and a Fisher exact test, respectively. The linearity and reproducibility of the ASE dosage was demonstrated with R2 > 0.99 and a coefficient of variation below 10 %, and ASE was detected in two positive controls harbouring BRCA1 truncated mutations. In the heterozygote population, composed of 99/264 controls (37.5 %) and 96/227 patients (42.3 %), we detected a 5 % ASE without truncated mutations, in each population. We failed to detect any significant difference of ASE between controls and patients. So far, BRCA1 Allelic specific expression is not usable in routine diagnosis as a possible predisposition marker in HBOC patients except for the detection of truncated mutations.


Assuntos
Desequilíbrio Alélico/genética , Proteína BRCA1/genética , Genes BRCA1 , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Heterozigoto , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Adulto Jovem
19.
Oncotarget ; 7(48): 79485-79493, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27825131

RESUMO

Highlighting tumoral mutations is a key step in oncology for personalizing care. Considering the genetic heterogeneity in a tumor, software used for detecting mutations should clearly distinguish real tumor events of interest that could be predictive markers for personalized medicine from false positives. OutLyzer is a new variant-caller designed for the specific and sensitive detection of mutations for research and diagnostic purposes. It is based on statistic and local evaluation of sequencing background noise to highlight potential true positive variants. 130 previously genotyped patients were sequenced after enrichment by capturing the exons of 22 genes. Sequencing data were analyzed by HaplotypeCaller, LofreqStar, Varscan2 and OutLyzer. OutLyzer had the best sensitivity and specificity with a fixed limit of detection for all tools of 1% for SNVs and 2% for Indels. OutLyzer is a useful tool for detecting mutations of interest in tumors including low allele-frequency mutations, and could be adopted in standard practice for delivering targeted therapies in cancer treatment.


Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , Análise de Sequência de DNA/métodos , Éxons , Frequência do Gene , Genótipo , Humanos , Medicina de Precisão , Software
20.
Mol Oncol ; 10(7): 981-92, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27083764

RESUMO

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross-studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin(®) kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter-sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT-qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two-fold lower inter-sample variability than the widely used endogenous miR-16-5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up-to 0.97 between the microarrays and individual RT-qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.


Assuntos
Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , MicroRNAs/sangue , MicroRNAs/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/sangue , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência
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