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1.
Hum Mutat ; 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31343793

RESUMO

BRCA1 and BRCA2 (BRCA1/2) genetic variants that disrupt messenger RNA splicing are commonly associated with increased risks of developing breast/ovarian cancer. The majority of splicing studies published to date rely on qualitative methodologies (i.e., Sanger sequencing), but it is necessary to incorporate semi-quantitative or quantitative approaches to accurately interpret the clinical significance of spliceogenic variants. Here, we characterize the splicing impact of 31 BRCA1/2 variants using semi-quantitative capillary electrophoresis of fluorescent amplicons (CE), Sanger sequencing and allele-specific assays. A total of 14 variants were found to disrupt splicing. Allelic-specific assays could be performed for BRCA1 c.302-1G>A and BRCA2 c.516+2T>A, c.1909+1G>A, c.8332-13T>G, c.8332-2A>G, c.8954-2A>T variants, showing a monoallelic contribution to full-length transcript expression that was concordant with semi-quantitative data. The splicing fraction of alternative and aberrant transcripts was also measured by CE, facilitating variant interpretation. Following Evidence-based Network for the Interpretation of Germline Mutant Alleles criteria, we successfully classified eight variants as pathogenic (Class 5), five variants as likely pathogenic (Class 4), and 14 variants as benign (Class 1). We also provide splicing data for four variants classified as uncertain (Class 3), which produced a "leaky" splicing effect or introduced a missense change in the protein sequence, that will require further assessment to determine their clinical significance.

2.
Cancer Res ; 79(16): 4258-4270, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31213465

RESUMO

Taxanes are the mainstay of treatment in triple-negative breast cancer (TNBC), with de novo and acquired resistance limiting patient's survival. To investigate the genetic basis of docetaxel resistance in TNBC, exome sequencing was performed on matched TNBC patient-derived xenografts (PDX) sensitive to docetaxel and their counterparts that developed resistance in vivo upon continuous drug exposure. Most mutations, small insertions/deletions, and copy number alterations detected in the initial TNBC human metastatic samples were maintained after serial passages in mice and emergence of resistance. We identified a chromosomal amplification of chr12p in a human BRCA1-mutated metastatic sample and the derived chemoresistant PDX, but not in the matched docetaxel-sensitive PDX tumor. Chr12p amplification was validated in a second pair of docetaxel-sensitive/resistant BRCA1-mutated PDXs and after short-term docetaxel treatment in several TNBC/BRCA1-mutated PDXs and cell lines, as well as during metastatic recurrence in a patient with BRCA1-mutated breast cancer who had progressed on docetaxel treatment. Analysis of clinical data indicates an association between chr12p amplification and patients with TNBC/basal-like breast cancer, a BRCA1 mutational signature, and poor survival after chemotherapy. Detection of chr12p amplification in a cohort of TNBC PDX models was associated with an improved response to carboplatin. Our findings reveal tumor clonal dynamics during chemotherapy treatments and suggest that a preexisting population harboring chr12p amplification is associated with the emergence of docetaxel resistance and carboplatin responsiveness in TNBC/BRCA1-mutated tumors. SIGNIFICANCE: Chr12p copy number gains indicate rapid emergence of resistance to docetaxel and increased sensitivity to carboplatin, therefore sequential docetaxel/carboplatin treatment could improve survival in TNBC/BRCA1 patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4258/F1.large.jpg.

3.
Hum Mutat ; 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31131967

RESUMO

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.

4.
Hum Mutat ; 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31112363

RESUMO

BRCA1 and BRCA2 (BRCA1/2) pathogenic sequence variants (PSVs) confer elevated risks of multiple cancers. However, most BRCA1/2 PSVs reports focus on European ancestry individuals. Knowledge of the PSV distribution in African descent individuals is poorly understood. We undertook a systematic review of the published literature and publicly available databases reporting BRCA1/2 PSVs also accessed the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) database to identify African or African descent individuals. Using these data, we inferred which of the BRCA PSVs were likely to be of African continental origin. Of the 43,817 BRCA1/2 PSV carriers in the CIMBA database, 469 (1%) were of African descent. Additional African descent individuals were identified in public databases (n = 291) and the literature (n = 601). We identified 164 unique BRCA1 and 173 unique BRCA2 PSVs in individuals of African ancestry. Of these, 83 BRCA1 and 91 BRCA2 PSVs are of likely or possible African origin. We observed numerous differences in the distribution of PSV type and function in African origin versus non-African origin PSVs. Research in populations of African ancestry with BRCA1/2 PSVs is needed to provide the information needed for clinical management and decision-making in African descent individuals worldwide.

5.
J Clin Oncol ; 37(25): 2257-2269, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31050911

RESUMO

The DNA damage response (DDR) pathway coordinates the identification, signaling, and repair of DNA damage caused by endogenous or exogenous factors and regulates cell-cycle progression with DNA repair to minimize DNA damage being permanently passed through cell division. Severe DNA damage that cannot be repaired may trigger apoptosis; as such, the DDR pathway is of crucial importance as a cancer target. Poly (ADP-ribose) polymerase (PARP) is the best-known element of the DDR, and several PARP inhibitors have been licensed. However, there are approximately 450 proteins involved in DDR, and a number of these other targets are being investigated in the laboratory and clinic. We review the most recent evidence for the clinical effect of PARP inhibition in breast and ovarian cancer and explore expansion into the first-line setting and into other tumor types. We critique the evidence for patient selection techniques and summarize what is known about mechanisms of PARP inhibitor resistance. We then discuss what is known about the preclinical rationale for targeting other members of the DDR pathway and the associated tumor cell genetics that may confer sensitivity to these agents. Examples include DNA damage sensors (MLH1), damage signaling molecules (ataxia-telangiectasia mutated; ataxia-telangiectasia mutated-related and Rad3-related; CHK1/2; DNA-dependent protein kinase, catalytic subunit; WEE1; CDC7), or effector proteins for repair (POLQ [also referred to as POLθ], RAD51, poly [ADP-ribose] glycohydrolase). Early-phase clinical trials targeting some of these molecules, either as a single agent or in combination, are discussed. Finally, we outline the challenges that must be addressed to maximize the therapeutic opportunity that targeting DDR provides.

6.
Nat Commun ; 10(1): 1741, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988301

RESUMO

Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas
7.
Int J Cancer ; 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30927264

RESUMO

Multigene panels provide a powerful tool for analyzing several genes simultaneously. We evaluated the frequency of pathogenic variants (PV) in customized predefined panels according to clinical suspicion by phenotype and compared it to the yield obtained in the analysis of our clinical research gene panel. We also investigated mutational yield of opportunistic testing of BRCA1/2 and mismatch repair (MMR) genes in all patients. A total of 1,205 unrelated probands with clinical suspicion of hereditary cancer were screened for germline mutations using panel testing. Overall, 1,048 females and 157 males were analyzed, mean age at cancer diagnosis was 48; 883 had hereditary breast/ovarian cancer-suspicion, 205 hereditary nonpolyposis colorectal cancer (HNPCC)-suspicion, 73 adenomatous-polyposis-suspicion and 44 with other/multiple clinical criteria. At least one PV was found in 150 probands (12%) analyzed by our customized phenotype-driven panel. Tumoral MMR deficiency predicted for the presence of germline MMR gene mutations in patients with HNPCC-suspicion (46/136 vs. 0/56 in patients with and without MMR deficiency, respectively). Opportunistic testing additionally identified five MSH6, one BRCA1 and one BRCA2 carriers (0.6%). The analysis of the extended 24-gene panel provided 25 additional PVs (2%), including in 4 out of 51 individuals harboring MMR-proficient colorectal tumors (2 CHEK2 and 2 ATM). Phenotype-based panels provide a notable rate of PVs with clinical actionability. Opportunistic testing of MMR and BRCA genes leads to a significant straightforward identification of MSH6, BRCA1 and BRCA2 mutation carriers, and endorses the model of opportunistic testing of genes with clinical utility within a standard genetic counseling framework.

8.
Breast Cancer Res Treat ; 174(2): 543-550, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30552643

RESUMO

PURPOSE: Disruption of splicing motifs by genetic variants can affect the correct generation of mature mRNA molecules leading to aberrant transcripts. In some cases, variants may alter the physiological transcription profile composed of several transcripts, and an accurate in vitro evaluation is crucial to establish their pathogenicity. In this study, we have characterized a novel PALB2 variant c.3201+5G>T identified in a breast cancer family. METHODS: Peripheral blood RNA was analyzed in two carriers and ten controls by RT-PCR and Sanger sequencing. The splicing profile was also characterized by semi-quantitative capillary electrophoresis and quantitative PCR. RAD51 foci formation and PALB2 LOH status were evaluated in primary breast tumor samples from the carriers. RESULTS: PALB2 c.3201+5G>T disrupts intron 11 donor splice site and modifies the abundance of several alternative transcripts (∆11, ∆12, and ∆11,12), also present in control samples. All transcripts are predicted to encode for non-functional proteins. Semi-quantitative and quantitative analysis of PALB2 full-length transcript indicated haploinsufficiency in carriers. One tumor exhibited PALB2 LOH and RAD51 assay indicated homologous recombination deficiency in both tumors. CONCLUSIONS: Our results support a pathogenic classification for PALB2 c.3201+5G>T, highlighting the impact of variants causing an imbalanced expression of natural RNA isoforms in cancer susceptibility.


Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Mutação em Linhagem Germinativa , Polimorfismo de Nucleotídeo Único , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de RNA
9.
Clin Cancer Res ; 25(9): 2717-2724, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30563931

RESUMO

PURPOSE: To assess talazoparib activity in germline BRCA1/2 mutation carriers with advanced breast cancer. PATIENTS AND METHODS: ABRAZO (NCT02034916) was a two-cohort, two-stage, phase II study of talazoparib (1 mg/day) in germline BRCA mutation carriers with a response to prior platinum with no progression on or within 8 weeks of the last platinum dose (cohort 1) or ≥3 platinum-free cytotoxic regimens (cohort 2) for advanced breast cancer. Primary endpoint was confirmed objective response rate (ORR) by independent radiological assessment. RESULTS: We enrolled 84 patients (cohort 1, n = 49; cohort 2, n = 35) from May 2014 to February 2016. Median age was 50 (range, 31-75) years. Triple-negative breast cancer (TNBC) incidence was 59% (cohort 1) and 17% (cohort 2). Median number of prior cytotoxic regimens for advanced breast cancer was two and four, respectively. Confirmed ORR was 21% [95% confidence interval (CI), 10-35; cohort 1] and 37% [95% CI, 22-55; cohort 2]. Median duration of response was 5.8 and 3.8 months, respectively. Confirmed ORR was 23% (BRCA1), 33% (BRCA2), 26% (TNBC), and 29% (hormone receptor-positive). The most common all-grade adverse events (AE) included anemia (52%), fatigue (45%), and nausea (42%). Talazoparib-related AEs led to drug discontinuation in 3 (4%) patients. In an exploratory analysis, longer platinum-free interval was associated with higher response rate in cohort 1 (0% ORR with interval <8 weeks; 47% ORR with interval >6 months). CONCLUSIONS: Talazoparib exhibited promising antitumor activity in patients with advanced breast cancer and germline BRCA mutation.

10.
EMBO Mol Med ; 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30377213

RESUMO

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2-related cancers. A test to identify additional HRR-deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2-related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi-sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2-related cancers.

11.
Eur J Cancer ; 106: 45-53, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30471647

RESUMO

European Breast Cancer Council manifesto and supporting article on genetic risk prediction testing in breast cancer, presented at the 11th European Breast Cancer Conference in Barcelona, Spain.

12.
Eur J Cancer ; 106: 54-60, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30471648

RESUMO

An international panel of experts representing 17 European countries and Israel convened to discuss current needs and future developments in BRCA testing and counselling and to issue consensus recommendations. The experts agreed that, with the increasing availability of high-throughput testing platforms and the registration of poly-ADP-ribose-polymerase inhibitors, the need for genetic counselling and testing will rapidly increase in the near future. Consequently, the already existing shortage of genetic counsellors is expected to worsen and to compromise the quality of care particularly in individuals and families with suspected or proven hereditary breast or ovarian cancer. Increasing educational efforts within the breast cancer caregiver community may alleviate this limitation by enabling all involved specialities to perform genetic counselling. In the therapeutic setting, for patients with a clinical suspicion of genetic susceptibility and if the results may have an immediate impact on the therapeutic strategy, the majority voted that BRCA1/2 testing should be performed after histological diagnosis of breast cancer, regardless of oestrogen receptor and human epidermal growth factor receptor 2 (HER2) status. Experts also agreed that, in the predictive and therapeutic setting, genetic testing should be limited to individuals with a personal or family history suggestive of a BRCA1/2 pathogenic variant and should also include high-risk actionable genes beyond BRCA1/2. Of high-risk actionable genes, all pathological variants (i.e. class IV and V) should be reported; class III variants of unknown significance, should be reported provided that the current lack of clinical utility of the variant is expressly stated. Genetic counselling should always address the possibility that already tested individuals might be re-contacted in case new information on a particular variant results in a re-classification.

13.
J Med Genet ; 2018 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-30472649

RESUMO

BACKGROUND: Genetic analysis of BRCA1 and BRCA2 for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility. METHODS: We analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 during conventional analysis. Rare variants (MAF <0.005) predicted to create/activate splice sites were selected for further characterisation in patient RNA. The splicing outcome was analysed by RT-PCR and Sanger sequencing, and allelic imbalance was also determined when heterozygous exonic loci were present. RESULTS: A novel transcript was detected in BRCA1 c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population. CONCLUSION: Screening of BRCA1/2 intronic regions has identified the first BRCA1 deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.

14.
Eur J Cancer ; 104: 91-103, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30342310

RESUMO

Patients with hereditary gastrointestinal (GI) cancers represent a substantial fraction of the overall affected population. Although awareness for hereditary GI cancer syndromes is on the rise, identification of patients and measures of surveillance are often unclear in everyday clinical routine. Therefore, the European Society of Digestive Oncology expert discussion 2018 at the World Congress on Gastrointestinal Cancer focussed on screening and surveillance of hereditary colorectal, gastric and pancreatic cancers. An international panel of experts and opinion leaders developed the here presented recommendations based on published evidence and on profound clinical expertise to facilitate clinical routine in identification and caretaking of patients with familial GI cancers.

15.
J Cancer Res Clin Oncol ; 144(12): 2495-2513, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30306255

RESUMO

PURPOSE: Few and small studies have been reported about multigene testing usage by massively parallel sequencing in European cancer families. There is an open debate about what genes should be tested, and the actionability of some included genes is under research. METHODS: We investigated a panel of 34 known high/moderate-risk cancer genes, including 16 related to breast or ovarian cancer (BC/OC) genes, and 63 candidate genes to BC/OC in 192 clinically suspicious of hereditary breast/ovarian cancer (HBOC) Spanish families without pathogenic variants in BRCA1 or BRCA2 (BRCA1/2). RESULTS: We identified 16 patients who carried a high- or moderate-risk pathogenic variant in eight genes: 4 PALB2, 3 ATM, 2 RAD51D, 2 TP53, 2 APC, 1 BRIP1, 1 PTEN and 1 PMS2. These findings led to increased surveillance or prevention options in 12 patients and predictive testing in their family members. We detected 383 unique variants of uncertain significance in known cancer genes, of which 35 were prioritized in silico. Eighteen loss-of-function variants were detected in candidate BC/OC genes in 17 patients (1 BARD1, 1 ERCC3, 1 ERCC5, 2 FANCE, 1 FANCI, 2 FANCL, 1 FANCM, 1 MCPH1, 1 PPM1D, 2 RBBP8, 3 RECQL4 and 1 with SLX4 and XRCC2), three of which also carry pathogenic variants in known cancer genes. CONCLUSIONS: Eight percent of the BRCA1/2 negative patients carry pathogenic variants in other actionable genes. The multigene panel usage improves the diagnostic yield in HBOC testing and it is an effective tool to identify potentially new candidate genes.

16.
J Clin Oncol ; : JCO2018786558, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30240327

RESUMO

PURPOSE: This multicenter phase II trial evaluated lurbinectedin (PM01183), a selective inhibitor of active transcription of protein-coding genes, in patients with metastatic breast cancer. A unicenter translational substudy assessed potential mechanisms of lurbinectedin resistance. PATIENTS AND METHODS: Two arms were evaluated according to germline BRCA1/2 status: BRCA1/2 mutated (arm A; n = 54) and unselected ( BRCA1/2 wild-type or unknown status; arm B; n = 35). Lurbinectedin starting dose was a 7-mg flat dose and later, 3.5 mg/m2 in arm A. The primary end point was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors (RECIST). The translational substudy of resistance mechanisms included exome sequencing (n = 13) and in vivo experiments with patient-derived xenografts (n = 11) from BRCA1/2-mutated tumors. RESULTS: ORR was 41% (95% CI, 28% to 55%) in arm A and 9% (95% CI, 2% to 24%) in arm B. In arm A, median progression-free survival was 4.6 months (95% CI, 3.0 to 6.0 months), and median overall survival was 20.0 months (95% CI, 11.8 to 26.6 months). Patients with BRCA2 mutations showed an ORR of 61%, median progression-free survival of 5.9 months, and median overall survival of 26.6 months. The safety profile improved with lurbinectedin dose adjustment to body surface area. The most common nonhematologic adverse events seen at 3.5 mg/m2 were nausea (74%; grade 3, 5%) and fatigue (74%; grade 3, 21%). Neutropenia was the most common severe hematologic adverse event (grade 3, 47%; grade 4, 10%). Exome sequencing showed mutations in genes related to the nucleotide excision repair pathway in four of seven tumors at primary or acquired resistance and in one patient with short-term stable disease. In vivo, sensitivity to cisplatin and lurbinectedin was evidenced in lurbinectedin-resistant (one of two) and cisplatin-resistant (two of three) patient-derived xenografts. CONCLUSION: Lurbinectedin showed noteworthy activity in patients with BRCA1/2 mutations. Response and survival was notable in those with BRCA2 mutations. Additional clinical development in this subset of patients with metastatic breast cancer is warranted.

17.
Front Genet ; 9: 366, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233647

RESUMO

In silico tools for splicing defect prediction have a key role to assess the impact of variants of uncertain significance. Our aim was to evaluate the performance of a set of commonly used splicing in silico tools comparing the predictions against RNA in vitro results. This was done for natural splice sites of clinically relevant genes in hereditary breast/ovarian cancer (HBOC) and Lynch syndrome. A study divided into two stages was used to evaluate SSF-like, MaxEntScan, NNSplice, HSF, SPANR, and dbscSNV tools. A discovery dataset of 99 variants with unequivocal results of RNA in vitro studies, located in the 10 exonic and 20 intronic nucleotides adjacent to exon-intron boundaries of BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, ATM, BRIP1, CDH1, PALB2, PTEN, RAD51D, STK11, and TP53, was collected from four Spanish cancer genetic laboratories. The best stand-alone predictors or combinations were validated with a set of 346 variants in the same genes with clear splicing outcomes reported in the literature. Sensitivity, specificity, accuracy, negative predictive value (NPV) and Mathews Coefficient Correlation (MCC) scores were used to measure the performance. The discovery stage showed that HSF and SSF-like were the most accurate for variants at the donor and acceptor region, respectively. The further combination analysis revealed that HSF, HSF+SSF-like or HSF+SSF-like+MES achieved a high performance for predicting the disruption of donor sites, and SSF-like or a sequential combination of MES and SSF-like for predicting disruption of acceptor sites. The performance confirmation of these last results with the validation dataset, indicated that the highest sensitivity, accuracy, and NPV (99.44%, 99.44%, and 96.88, respectively) were attained with HSF+SSF-like or HSF+SSF-like+MES for donor sites and SSF-like (92.63%, 92.65%, and 84.44, respectively) for acceptor sites. We provide recommendations for combining algorithms to conduct in silico splicing analysis that achieved a high performance. The high NPV obtained allows to select the variants in which the study by in vitro RNA analysis is mandatory against those with a negligible probability of being spliceogenic. Our study also shows that the performance of each specific predictor varies depending on whether the natural splicing sites are donors or acceptors.

18.
Genet Med ; 2018 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-30008476

RESUMO

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

19.
J Natl Cancer Inst ; 110(8): 914-917, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29529211

RESUMO

Heat shock proteins (HSPs) are molecular chaperones that maintain proteins in their correct conformation to ensure stability and protect carcinoma cells from apoptosis. HSP90 inhibitors (HSP90i) block multiple targets simultaneously, and despite responses in a selected population, no HSP90i have yet been approved. We present a patient with a lung tumor with an exceptional response to cisplatin/gemcitabine in combination with HSP90i, which nowadays continues with HSP90i maintenance after three years. Whole-exome sequencing of the lung tumor unveiled a BRCA1/2 deficiency mutational signature, and mutation analysis confirmed a germline BRCA1 mutation. The striking efficacy of HSP90i plus chemotherapy vs chemotherapy alone was reproduced in a patient-derived xenograft (PDX) model from a breast cancer patient with a BRCA1 mutation (mean tumor volume [SD], No. of tumors: vehicle 8.38 [7.07] mm3, n = 3; HSP90i 4.18 [1.93] mm3, n = 5; cisplatin plus gemcitabine 3.31 [1.95] mm3, n = 5; cisplatin plus gemcitabine plus HSP90i 0.065 [0.076] mm3, n = 6). This case and the PDX demonstrate the efficacy for therapeutic inhibition of HSP90 in a BRCA-mutated patient, opening a new potential avenue for better identifying patients who might benefit most from HSP90i.

20.
Hum Mutat ; 39(5): 593-620, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29446198

RESUMO

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.

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