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1.
Hum Mutat ; 40(9): 1546-1556, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31294896

RESUMO

Testing for variation in BRCA1 and BRCA2 (commonly referred to as BRCA1/2), has emerged as a standard clinical practice and is helping countless women better understand and manage their heritable risk of breast and ovarian cancer. Yet the increased rate of BRCA1/2 testing has led to an increasing number of Variants of Uncertain Significance (VUS), and the rate of VUS discovery currently outpaces the rate of clinical variant interpretation. Computational prediction is a key component of the variant interpretation pipeline. In the CAGI5 ENIGMA Challenge, six prediction teams submitted predictions on 326 newly-interpreted variants from the ENIGMA Consortium. By evaluating these predictions against the new interpretations, we have gained a number of insights on the state of the art of variant prediction and specific steps to further advance this state of the art.

2.
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.

3.
Hum Mutat ; 40(9): 1593-1611, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31112341

RESUMO

BRCA1 and BRCA2 (BRCA1/2) germline variants disrupting the DNA protective role of these genes increase the risk of hereditary breast and ovarian cancers. Correct identification of these variants then becomes clinically relevant, because it may increase the survival rates of the carriers. Unfortunately, we are still unable to systematically predict the impact of BRCA1/2 variants. In this article, we present a family of in silico predictors that address this problem, using a gene-specific approach. For each protein, we have developed two tools, aimed at predicting the impact of a variant at two different levels: Functional and clinical. Testing their performance in different datasets shows that specific information compensates the small number of predictive features and the reduced training sets employed to develop our models. When applied to the variants of the BRCA1/2 (ENIGMA) challenge in the fifth Critical Assessment of Genome Interpretation (CAGI 5) we find that these methods, particularly those predicting the functional impact of variants, have a good performance, identifying the large compositional bias towards neutral variants in the CAGI sample. This performance is further improved when incorporating to our prediction protocol estimates of the impact on splicing of the target variant.

4.
Hum Mutat ; 40(9): 1557-1578, 2019 09.
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 1,395 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; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with 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 nonpathogenic 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.

5.
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
6.
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.

7.
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.

8.
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.

9.
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.

10.
Apoptosis ; 20(3): 371-82, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25398538

RESUMO

We tested apoptosis levels in in vitro irradiated T-lymphocytes from breast cancer (BC) patients with radiotherapy-induced late effects. Previous results reported in the literature were revised. We also examined the effect of TP53 Arg72Pro polymorphism on irradiation-induced apoptosis (IA). Twenty BC patients, ten with fibrosis and/or telangiectasias and ten matched controls with no late reactions, were selected from those receiving radiotherapy between 1993 and 2007. All patients were followed-up at least 6 years after radiotherapy. Using the combination of both CD3 and CD8 antibodies the in vitro IA was measured in CD3, CD8 and CD4 T-lymphocytes, and CD8 natural killer lymphocytes (CD8 NK) by flow cytometry. The TP53 Arg72Pro genotype was determined by sequencing. Patients with late radiotherapy toxicity showed less IA for all T-lymphocytes except for the CD8 NK. CD8 NK showed the highest spontaneous apoptosis and the lowest IA. IA in patients with toxicity appears to be lower than the control patients only in TP53 Arg/Arg patients (P = 0.077). This difference was not present in patients carrying at least one Pro allele (P = 0.8266). Our data indicate that late side effects induced by radiotherapy of BC are associated to low levels of IA. CD8 NK cells have a different response to in vitro irradiation compared to CD8 T-lymphocytes. It would be advisable to distinguish the CD8 NK lymphocytes from the pool of CD8+ lymphocytes in IA assays using CD8+ cells. Our data suggest that the 72Pro TP53 allele may influence the IA of patients with radiotherapy toxicity.


Assuntos
Neoplasias da Mama/radioterapia , Linfócitos T CD4-Positivos/efeitos da radiação , Linfócitos T CD8-Positivos/efeitos da radiação , Raios gama/efeitos adversos , Células Matadoras Naturais/efeitos da radiação , Subpopulações de Linfócitos/efeitos da radiação , Polimorfismo Genético , Proteína Supressora de Tumor p53/genética , Adulto , Apoptose/efeitos da radiação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibrose , Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Tolerância a Radiação , Telangiectasia/genética , Telangiectasia/metabolismo , Telangiectasia/patologia , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo
11.
Breast Cancer Res Treat ; 147(1): 133-43, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25086635

RESUMO

BRCA1 and BRCA2 are the most well-known breast and ovarian cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. Recently, RAD51C, a new Fanconi Anemia gene, essential for homologous recombination repair, has been reported to be a rare hereditary breast and ovarian cancer susceptibility gene. Indeed, several pathogenic mutations have been identified in BRCA1/BRCA2-negative hereditary breast and ovarian cancer families. Here, we present the results of the screening of RAD51C mutations in a large series of 516 BRCA1/BRCA2-negative Spanish patients from breast and/or ovarian cancer families, and the evaluation of these results in the context of all RAD51C carriers. RAD51C mutation screening was performed by DNA analysis for all index cases. All the genetic variants identified were analyzed in silico for splicing and protein predictions. cDNA analysis was performed for three selected variants. All previous RAD51C mutation studies on breast and/or ovarian cancer were reviewed. We identified three inactivating RAD51C mutations. Two mutations were found in breast and ovarian cancer families and one mutation in a site-specific breast cancer family. Based on the mean age of ovarian cancer diagnosis in RAD51C carriers, we would recommend prophylactic bilateral salpingo-ophorectomy in premenopausal RAD51C mutation carriers. Our results support that RAD51C is a rare breast and ovarian cancer susceptibility gene and may contribute to a small fraction of families including breast and ovarian cancer cases and families with only breast cancer. Thus, RAD51C testing should be offered to hereditary breast and/or ovarian cancer families without selecting for specific cancer origin.


Assuntos
Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Neoplasias Ovarianas/genética , Adulto , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Família , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/epidemiologia , Prognóstico , Espanha/epidemiologia
12.
Hum Mol Genet ; 23(14): 3666-80, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24569164

RESUMO

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.


Assuntos
Processamento Alternativo , Proteína BRCA1/sangue , Proteína BRCA1/genética , Mama/metabolismo , Feminino , Humanos , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
13.
Int J Cancer ; 134(9): 2088-97, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24130102

RESUMO

RAD51D mutations have been recently identified in breast (BC) and ovarian cancer (OC) families. Although an etiological role in OC appears to be present, the association of RAD51D mutations and BC risk is more unclear. We aimed to determine the prevalence of germline RAD51D mutations in Spanish BC/OC families negative for BRCA1/BRCA2 mutations. We analyzed 842 index patients: 491 from BC/OC families, 171 BC families, 51 OC families and 129 patients without family history but with early-onset BC or OC or metachronous BC and OC. Mutation detection was performed with high-resolution melting, denaturing high-performance liquid chromatography or Sanger sequencing. Three mutations were found in four families with BC and OC cases (0.82%). Two were novel: c.1A>T (p.Met1?) and c.667+2_667+23del, leading to the exon 7 skipping and one previously described: c.674C>T (p.Arg232*). All were present in BC/OC families with only one OC. The c.667+2_667+23del cosegregated in the family with one early-onset BC and two bilateral BC cases. We also identified the c.629C>T (p.Ala210Val) variant, which was predicted in silico to be potentially pathogenic. About 1% of the BC and OC Spanish families negative for BRCA1/BRCA2 are carriers of RAD51D mutations. The presence of several BC mutation carriers, albeit in the context of familial OC, suggests an increased risk for BC, which should be taken into account in the follow-up and early detection measures. RAD51D testing should be considered in clinical setting for families with BC and OC, irrespective of the number of OC cases in the family.


Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença/genética , Neoplasias Ovarianas/genética , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Feminino , Genes BRCA1 , Genes BRCA2 , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Espanha
14.
Gynecol Oncol ; 131(2): 460-3, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23911796

RESUMO

OBJECTIVE: About 5%-10% of breast cancer is due to inherited disease predisposition. Currently, mutations in the BRCA1 and BRCA2 genes explain less than 25% of the familial clustering of breast cancer, and additional susceptibility genes are suspected. The BCCIP gene plays an important role in the regulation of gene transcription and cell proliferation and could be involved in the maintenance of genomic integrity. The BCCIP protein binds in mammalian cells to the longest conserved region of the BRCA2 protein and is required for BRCA2 stability and function, making a critical contribution to the function of BRCA2 in mediating homologous recombination. Variants in the BCCIP gene could affect the BRCA2 functionality and be associated to the familial breast/ovarian carcinogenesis. Therefore, BCCIP gene is a potential candidate for being involved in heritable cancer susceptibility. METHODS: We have screened the entire coding region and splice junctions of BCCIP in affected index cases from 215 Spanish breast/ovarian cancer families for germ line defects, using direct sequencing. RESULTS: Mutation analysis revealed 3 different intronic sequence changes. CONCLUSIONS: Based on the in silico and in vitro RNA analyses of these sequence alterations, none of them were predicted to be pathogenic or associated with cancer susceptibility. Our results indicate that BCCIP germ line mutations are unlikely to be a major contributor to familial breast/ovarian cancer risk in our population.


Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama Masculina/genética , Análise Mutacional de DNA , Éxons , Saúde da Família , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Íntrons , Masculino , Polimorfismo de Nucleotídeo Único
15.
J Cancer Res Clin Oncol ; 139(3): 529-32, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23371468

RESUMO

PURPOSE: About 5-10 % of breast cancer is due to inherited disease predisposition. Currently known susceptibility genes such as BRCA1 and BRCA2 explain less than 25 % of familial aggregation of breast cancer, which suggests the involvement of additional genetic susceptibility. The SHFM1 [split hand/foot malformation (ectrodactyly) type 1] gene plays an important role in the regulation of gene transcription and cell proliferation and may be involved in the maintenance of genomic integrity. It is a potential candidate for being involved in heritable cancer susceptibility due to its biological function. The SHFM1 protein binds in mammalian cells to the longest conserved region of the BRCA2 protein and is required for BRCA2 stability and function, making a critical contribution to the BRCA2 function in mediating homologous recombination. Therefore, variants in the SHFM1 gene could affect the BRCA2 functionality and be associated with the familial breast/ovarian carcinogenesis. METHODS: We have screened the entire coding region and splice junctions of SHFM1 in affected index cases from 369 Spanish breast/ovarian cancer families for germ line defects, using direct sequencing. RESULTS: Mutation analysis revealed seven different sequence changes. Based on the in silico analyses of these sequence alterations, as well as their occurrence in cases and controls, none of them, however, were predicted to be pathogenic or associated with cancer susceptibility. CONCLUSIONS: To our knowledge, this is the most comprehensive study reporting the mutation screening of the SHFM1 gene in familial breast/ovarian cancer cases. No evidence for the association with breast/ovarian cancer was observed.


Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Neoplasias Primárias Múltiplas/genética , Neoplasias Ovarianas/genética , Complexo de Endopeptidases do Proteassoma/genética , Neoplasias da Mama/complicações , Neoplasias da Mama/epidemiologia , Neoplasias da Mama Masculina/epidemiologia , Neoplasias da Mama Masculina/genética , Carcinoma/complicações , Carcinoma/epidemiologia , Análise Mutacional de DNA , Família , Feminino , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Masculino , Neoplasias Primárias Múltiplas/epidemiologia , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/epidemiologia , Polimorfismo de Nucleotídeo Único
16.
PLoS One ; 7(10): e47892, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23112866

RESUMO

Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.


Assuntos
Proteínas Argonauta/genética , Metilação de DNA , Infertilidade Masculina/genética , Espermatogênese , Espermatozoides/metabolismo , Adulto , Proteínas de Transporte/genética , DNA/genética , Regulação para Baixo , Humanos , Infertilidade Masculina/patologia , Masculino , Pessoa de Meia-Idade , RNA/genética , Síndrome de Células de Sertoli/genética , Síndrome de Células de Sertoli/patologia , Espermatozoides/patologia
17.
Breast Cancer Res Treat ; 133(1): 273-83, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22434521

RESUMO

Large genomic rearrangements (LGRs) at the BRCA2 locus explain a non-negligible proportion of hereditary breast and ovarian cancer (HBOC) syndromes. The multiplex ligation and probe amplification (MLPA) assay has permitted in recent years to identify several families carrying LGRs at this locus, but very few such alterations have been fully characterized at the molecular level. Yet, molecular characterization is essential to identify recurrent alterations, to analyze the genetic mechanisms underlying such alterations, or to investigate potential genotype/phenotype relationships. We have used MLPA to identify BRCA2 LGRs in 7 out of 813 Spanish HBOC families previously tested negative for BRCA1 and BRCA2 small genomic alterations (substitutions and indels) and BRCA1 LGRs. We used a combination of long-range PCR, restriction mapping, and cDNA analysis to characterize the alterations at the molecular level. We found that Del Exon1-Exon2, Del Exon12-Exon16 and Del Exon22-Exon24 explain one family each, while Del Exon2 appears to be a Spanish founder mutation explaining four independent families. Finally, we have combined our data with a comprehensive review of the literature to reevaluate the genetic mechanisms underlying LGRs at the BRCA2 locus. Our study substantially increases the spectrum of BRCA2 LGRs fully characterized at the molecular level. Further on, we provide data to suggest that non-allelic homologous recombination has been overestimated as a mechanism underlying these alterations, while the opposite might be true for microhomology-mediated events.


Assuntos
Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Recombinação Genética , Proteína BRCA1/genética , Sequência de Bases , Pontos de Quebra do Cromossomo , Análise Mutacional de DNA , Éxons , Feminino , Haplótipos , Humanos , Linhagem , Alinhamento de Sequência , Deleção de Sequência , Espanha
18.
Hum Reprod ; 27(6): 1556-67, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22447625

RESUMO

BACKGROUND: Assessment of male fertility is traditionally based on microscopic evaluation of semen. However, the classical semen parameters do not adequately reflect sperm function, and their clinical value in predicting fertility is limited. We hypothesize that the sperm expression profile could reflect the fertilizing quality of spermatozoa and could be more informative for predicting the in vivo reproductive fitness of men with normal semen parameters. METHODS: Sperm gene expression patterns of 68 normozoospermic donors (43 Phase I and 25 Phase II), used for therapeutic IUI, were analysed via TaqMan Arrays. RESULTS: Significant differences in the expression of individual genes were observed between groups of donors with the lowest and highest pregnancy rates (PRs) after IUI. Additionally, we have developed a molecular means to classify the fertility status of semen donors for IUI based on the expression signature of four genes. In the Phase I study, this model had 90% sensitivity and 97% specificity for discriminating donors resulting in low PRs (cut-off value: <13.6%), far better than that obtained from the combination of sperm parameters. The translation of the model was validated in Phase II donors resulting in a sensitivity of 71.5% and a specificity of 78%. CONCLUSIONS: Our findings contribute to the search for the most valuable genetic markers which are potentially useful as tools for predicting pregnancy. Our expression model could complement classical semen analysis in order to identify sperm donors with a less favourable IUI reproductive outcome despite having normal semen parameters. It may also be useful for the study of sperm function in couples with unexplained infertility.


Assuntos
Perfilação da Expressão Gênica , Infertilidade Masculina/genética , Inseminação Artificial Heteróloga , Espermatozoides/metabolismo , Doadores de Tecidos , Adulto , Feminino , Fertilidade/genética , Marcadores Genéticos , Humanos , Inseminação Artificial , Masculino , Gravidez , Taxa de Gravidez , Análise do Sêmen , Sensibilidade e Especificidade , Espermatozoides/química
19.
J Androl ; 31(4): 346-57, 2010 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20075417

RESUMO

DNA mismatch repair (MMR) genes have been described to participate in crossover events during meiotic recombination, which is, in turn, a key step of spermatogenesis. This evidence suggests that MMR family gene expression may be altered in infertile men with defective sperm production. In order to determine the expression profile of MMR genes in impaired human spermatogenesis, we performed transcript levels analysis of MMR genes (MLH1, MLH3, PMS2, MSH4, and MSH5), and other meiosis-involved genes (ATR, HSPA2, and SYCP3) as controls, by real-time reverse transcription-polymerase chain reaction in testis from 13 patients with spermatogenic failure, 5 patients with primary germ cell tumors, and 10 controls with conserved spermatogenesis. Correlation of the expression values with the histological findings was also performed. The MMR gene expression values, with the exception of PMS2, are significantly decreased in men with spermatogenic failure. The pattern of MMR reduction correlates with the severity of damage, being maximum in maturation arrest. Specifically, expression of the testicular MSH4 gene could be useful as a surrogate marker for the presence of intratesticular elongated spermatid in patients with nonobstructive azoospermia, contributing to predict the viability of assisted reproduction. Interestingly, a reduction in the MSH4 and MSH5 transcript concentration per spermatocyte was also observed. The decreased expression level of other meiosis-specific genes, such as HSPA2 and SYCP3, suggests that the spermatocyte capacity to express meiosis-related genes is markedly reduced in spermatogenic failure, contributing to meiosis impairment and spermatogenic blockade.


Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Infertilidade Masculina/genética , Meiose , Espermatogênese/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adulto , Processamento Alternativo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteínas MutL , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Testículo/metabolismo , Testículo/patologia
20.
Fertil Steril ; 88(4): 988-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17434513

RESUMO

No functional SYCP3 exonic SNP was found in infertile Spanish patients with meiosis arrest, suggesting that SYCP3 mutations very likely are not relevant in Spain in genetic susceptibility to meiosis arrest, just as in other studied European populations.


Assuntos
Infertilidade Masculina/genética , Meiose , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Humanos , Masculino , Espanha , Complexo Sinaptonêmico/genética
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