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1.
Hum Mutat ; 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31433103

RESUMO

Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.

2.
Breast Cancer Res Treat ; 177(3): 723-733, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302855

RESUMO

BACKGROUND: In healthy BRCA1/2 mutation carriers, bilateral risk-reducing mastectomy (BRRM) strongly reduces the risk of developing breast cancer (BC); however, no clear survival benefit of BRRM over BC surveillance has been reported yet. METHODS: In this Dutch multicenter cohort study, we used multivariable Cox models with BRRM as a time-dependent covariable to estimate the associations between BRRM and the overall and BC-specific mortality rates, separately for BRCA1 and BRCA2 mutation carriers. RESULTS: During a mean follow-up of 10.3 years, 722 out of 1712 BRCA1 (42%) and 406 out of 1145 BRCA2 (35%) mutation carriers underwent BRRM. For BRCA1 mutation carriers, we observed 52 deaths (20 from BC) in the surveillance group, and 10 deaths (one from BC) after BRRM. The hazard ratios were 0.40 (95% CI 0.20-0.90) for overall mortality and 0.06 (95% CI 0.01-0.46) for BC-specific mortality. BC-specific survival at age 65 was 93% for surveillance and 99.7% for BRRM. For BRCA2 mutation carriers, we observed 29 deaths (7 from BC) in the surveillance group, and 4 deaths (no BC) after BRRM. The hazard ratio for overall mortality was 0.45 (95% CI 0.15-1.36). BC-specific survival at age 65 was 98% for surveillance and 100% for BRRM. CONCLUSION: BRRM was associated with lower mortality than surveillance for BRCA1 mutation carriers, but for BRCA2 mutation carriers, BRRM may lead to similar BC-specific survival as surveillance. Our findings support a more individualized counseling based on BRCA mutation type.

3.
Int J Cancer ; 145(2): 401-414, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30623411

RESUMO

A subset of genetic variants found through screening of patients with hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome impact RNA splicing. Through target enrichment of the transcriptome, it is possible to perform deep-sequencing and to identify the different and even rare mRNA isoforms. A targeted RNA-seq approach was used to analyse the naturally-occurring splicing events for a panel of 8 breast and/or ovarian cancer susceptibility genes (BRCA1, BRCA2, RAD51C, RAD51D, PTEN, STK11, CDH1, TP53), 3 Lynch syndrome genes (MLH1, MSH2, MSH6) and the fanconi anaemia SLX4 gene, in which monoallelic mutations were found in non-BRCA families. For BRCA1, BRCA2, RAD51C and RAD51D the results were validated by capillary electrophoresis and were compared to a non-targeted RNA-seq approach. We also compared splicing events from lymphoblastoid cell-lines with those from breast and ovarian fimbriae tissues. The potential of targeted RNA-seq to detect pathogenic changes in RNA-splicing was validated by the inclusion of samples with previously well characterized BRCA1/2 genetic variants. In our study, we update the catalogue of normal splicing events for BRCA1/2, provide an extensive catalogue of normal RAD51C and RAD51D alternative splicing, and list splicing events found for eight other genes. Additionally, we show that our approach allowed the identification of aberrant splicing events due to the presence of BRCA1/2 genetic variants and distinguished between complete and partial splicing events. In conclusion, targeted-RNA-seq can be very useful to classify variants based on their putative pathogenic impact on splicing.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Processamento de RNA , Análise de Sequência de RNA/métodos , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Eletroforese Capilar , Feminino , Predisposição Genética para Doença , Humanos , Mutação
4.
Hum Mutat ; 39(12): 2025-2039, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30204945

RESUMO

The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5' noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

5.
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.

6.
J Med Genet ; 55(1): 15-20, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28490613

RESUMO

BACKGROUND: We previously showed that the BRCA1 variant c.5096G>A p.Arg1699Gln (R1699Q) was associated with an intermediate risk of breast cancer (BC) and ovarian cancer (OC). This study aimed to assess these cancer risks for R1699Q carriers in a larger cohort, including follow-up of previously studied families, to further define cancer risks and to propose adjusted clinical management of female BRCA1*R1699Q carriers. METHODS: Data were collected from 129 BRCA1*R1699Q families ascertained internationally by ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium members. A modified segregation analysis was used to calculate BC and OC risks. Relative risks were calculated under both monogenic model and major gene plus polygenic model assumptions. RESULTS: In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83). CONCLUSION: Our results confirm that BRCA1*R1699Q confers an intermediate risk for BC and OC. Breast surveillance for female carriers based on mammogram annually from age 40 is advised. Bilateral salpingo-oophorectomy should be considered based on family history.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença , Mutação/genética , Neoplasias Ovarianas/genética , Segregação de Cromossomos , Feminino , Humanos , Fatores de Risco
7.
Nat Genet ; 49(5): 680-691, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28346442

RESUMO

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC.


Assuntos
Loci Gênicos/genética , Predisposição Genética para Doença/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Alelos , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Metanálise como Assunto , Mutação , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Proteínas de Ligação a Telômeros/genética
8.
Int J Mol Sci ; 18(2)2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-28146134

RESUMO

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/diagnóstico , Neoplasias/genética , Biomarcadores Tumorais , Ensaios Clínicos como Assunto , Biologia Computacional , Exoma , Predisposição Genética para Doença , Testes Genéticos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Farmacogenética , Prognóstico , Reprodutibilidade dos Testes
9.
Breast Cancer Res Treat ; 161(1): 117-134, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27796716

RESUMO

PURPOSE: Cis-acting regulatory SNPs resulting in differential allelic expression (DAE) may, in part, explain the underlying phenotypic variation associated with many complex diseases. To investigate whether common variants associated with DAE were involved in breast cancer susceptibility among BRCA1 and BRCA2 mutation carriers, a list of 175 genes was developed based of their involvement in cancer-related pathways. METHODS: Using data from a genome-wide map of SNPs associated with allelic expression, we assessed the association of ~320 SNPs located in the vicinity of these genes with breast and ovarian cancer risks in 15,252 BRCA1 and 8211 BRCA2 mutation carriers ascertained from 54 studies participating in the Consortium of Investigators of Modifiers of BRCA1/2. RESULTS: We identified a region on 11q22.3 that is significantly associated with breast cancer risk in BRCA1 mutation carriers (most significant SNP rs228595 p = 7 × 10-6). This association was absent in BRCA2 carriers (p = 0.57). The 11q22.3 region notably encompasses genes such as ACAT1, NPAT, and ATM. Expression quantitative trait loci associations were observed in both normal breast and tumors across this region, namely for ACAT1, ATM, and other genes. In silico analysis revealed some overlap between top risk-associated SNPs and relevant biological features in mammary cell data, which suggests potential functional significance. CONCLUSION: We identified 11q22.3 as a new modifier locus in BRCA1 carriers. Replication in larger studies using estrogen receptor (ER)-negative or triple-negative (i.e., ER-, progesterone receptor-, and HER2-negative) cases could therefore be helpful to confirm the association of this locus with breast cancer risk.


Assuntos
Alelos , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/etiologia , Genes BRCA1 , Genes BRCA2 , Heterozigoto , Mutação , Biomarcadores Tumorais , Cromossomos Humanos Par 11 , Feminino , Expressão Gênica , Predisposição Genética para Doença , Variação Genética , Humanos , Locos de Características Quantitativas , Risco
10.
Hum Mutat ; 38(2): 226-235, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27767231

RESUMO

With the recent introduction of Poly(ADP-ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue, we have developed a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach. Our smMIP-based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin-induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation-negative results. Multiplex ligation-dependent probe amplification (MLPA) and Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.


Assuntos
Tomada de Decisão Clínica , Genes BRCA1 , Genes BRCA2 , Aconselhamento Genético , Neoplasias Ovarianas/genética , Alelos , Substituição de Aminoácidos , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Gerenciamento Clínico , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Genótipo , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Reprodutibilidade dos Testes
11.
Am J Med Genet A ; 170(12): 3172-3179, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27530205

RESUMO

Kabuki syndrome is a multiple congenital malformation syndrome with a spectrum of clinical features including short stature. Since there is no growth data on Kabuki syndrome patients with a proven KMT2D gene mutation, further research on growth and growth patterns is indicated. Data for this growth study on subjects with Kabuki syndrome were collected from referring clinicians. Subjects were eligible for inclusion in the study if the following criteria were met: a genetically confirmed diagnosis of Kabuki syndrome and no current treatment with growth hormones or other drugs that could influence growth. We present a report on growth data (n = 39) in Kabuki syndrome patients. The data showed that postnatal growth retardation is a clinical feature in all cases. All Kabuki syndrome subjects showed a growth deflection during childhood and a diminution of the pubertal growth spurt. A genotype-phenotype correlation was not observed. Further research is required in order to determine whether a defect in the growth hormone/IGF-I axis and estrogen receptor plays a role in the growth retardation. © 2016 Wiley Periodicals, Inc.


Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/genética , Face/anormalidades , Doenças Hematológicas/genética , Deficiência Intelectual/genética , Proteínas de Neoplasias/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/fisiopatologia , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/fisiopatologia , Face/fisiopatologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Doenças Hematológicas/fisiopatologia , Humanos , Fator de Crescimento Insulin-Like I , Deficiência Intelectual/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , Doenças Vestibulares/fisiopatologia , Adulto Jovem
12.
J Med Genet ; 53(8): 548-58, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27060066

RESUMO

BACKGROUND: BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations.


Assuntos
Processamento Alternativo/genética , Proteína BRCA2/genética , RNA Mensageiro/genética , Proteína BRCA1/genética , Neoplasias da Mama/genética , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Humanos , Células MCF-7 , Mutação/genética , Neoplasias Ovarianas/genética , Sítios de Splice de RNA/genética
13.
Oncotarget ; 6(38): 41108-22, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26517685

RESUMO

BACKGROUND: The risk to develop colorectal and endometrial cancers among subjects testing positive for a pathogenic Lynch syndrome mutation varies, making the risk prediction difficult. Genetic risk modifiers alter the risk conferred by inherited Lynch syndrome mutations, and their identification can improve genetic counseling. We aimed at identifying rare genetic modifiers of the risk of Lynch syndrome endometrial cancer. METHODS: A family based approach was used to assess the presence of genetic risk modifiers among 35 Lynch syndrome mutation carriers having either a poor clinical phenotype (early age of endometrial cancer diagnosis or multiple cancers) or a neutral clinical phenotype. Putative genetic risk modifiers were identified by Next Generation Sequencing among a panel of 154 genes involved in endometrial physiology and carcinogenesis. RESULTS: A simple pipeline, based on an allele frequency lower than 0.001 and on predicted non-conservative amino-acid substitutions returned 54 variants that were considered putative risk modifiers. The presence of two or more risk modifying variants in women carrying a pathogenic Lynch syndrome mutation was associated with a poor clinical phenotype. CONCLUSION: A gene-panel is proposed that comprehends genes that can carry variants with putative modifying effects on the risk of Lynch syndrome endometrial cancer. Validation in further studies is warranted before considering the possible use of this tool in genetic counseling.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias do Endométrio/genética , Estrogênios/metabolismo , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias do Endométrio/metabolismo , Feminino , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Humanos , Imuno-Histoquímica/estatística & dados numéricos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco
14.
J Med Genet ; 52(4): 224-30, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25643705

RESUMO

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.


Assuntos
Proteína BRCA1/genética , Biologia Computacional/métodos , Gráficos por Computador , Internet , Mutação de Sentido Incorreto , Software , Neoplasias da Mama/genética , Análise Mutacional de DNA , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Neoplasias Ovarianas/genética
15.
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
16.
Breast Cancer Res ; 15(2): R29, 2013 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-23566419

RESUMO

INTRODUCTION: Cyclooxygenase-2 (COX-2) is frequently over-expressed in primary breast cancer. In transgenic breast cancer models, over-expression of COX-2 leads to tumour formation while COX-2 inhibition exerts anti-tumour effects in breast cancer cell lines. To further determine the effect of COX-2 inhibition in primary breast cancer, we aimed to identify transcriptional changes in breast cancer tissues of patients treated with the selective COX-2 inhibitor celecoxib. METHODS: In a single-centre double-blind phase II study, thirty-seven breast cancer patients were randomised to receive either pre-operative celecoxib (400 mg) twice daily for two to three weeks (n = 22) or a placebo according to the same schedule (n = 15). Gene expression in fresh-frozen pre-surgical biopsies (before treatment) and surgical excision specimens (after treatment) was profiled by using Affymetrix arrays. Differentially expressed genes and altered pathways were bioinformatically identified. Expression of selected genes was validated by quantitative PCR (qPCR). Immunohistochemical protein expression analyses of the proliferation marker Ki-67, the apoptosis marker cleaved caspase-3 and the neo-angiogenesis marker CD34 served to evaluate biological response. RESULTS: We identified 972 and 586 significantly up- and down-regulated genes, respectively, in celecoxib-treated specimens. Significant expression changes in six out of eight genes could be validated by qPCR. Pathway analyses revealed over-representation of deregulated genes in the networks of proliferation, cell cycle, extracellular matrix biology, and inflammatory immune response. The Ki-67 mean change relative to baseline was -29.1% (P = 0.019) and -8.2% (P = 0.384) in the treatment and control arm, respectively. Between treatment groups, the change in Ki-67 was statistically significant (P = 0.029). Cleaved caspase-3 and CD34 expression were not significantly different between the celecoxib-treated and placebo-treated groups. CONCLUSIONS: Short-term COX-2 inhibition by celecoxib induces transcriptional programs supporting anti-tumour activity in primary breast cancer tissue. The impact on proliferation-associated genes is reflected by a reduction of Ki-67 positive cells. Therefore, COX-2 inhibition should be considered as a treatment strategy for further clinical testing in primary breast cancer. TRIAL REGISTRATION: ClinicalTrials.gov NCT01695226.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Inibidores de Ciclo-Oxigenase/uso terapêutico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Celecoxib , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Eur J Hum Genet ; 21(12): 1361-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23531862

RESUMO

Preimplantation Genetic Diagnosis (PGD) is a method of testing in vitro embryos as an alternative to prenatal diagnosis with possible termination of pregnancy in case of an affected child. Recently, PGD for hereditary breast and ovarian cancer caused by BRCA1 and BRCA2 mutations has found its way in specialized labs. We describe the route to universal single-cell PGD tests for carriers of BRCA1/2 mutations. Originally, mutation-specific protocols with one or two markers were set up and changed when new couples were not informative. This route of changing protocols was finalized after 2 years with universal tests for both BRCA1 and BRCA2 mutation carriers based on haplotyping of, respectively, 6 (BRCA1) and 8 (BRCA2) microsatellite markers in a multiplex PCR. Using all protocols, 30 couples had a total of 47 PGD cycles performed. Eight cycles were cancelled upon IVF treatment due to hypostimulation. Of the remaining 39 cycles, a total of 261 embryos were biopsied and a genetic diagnosis was obtained in 244 (93%). In 34 of the 39 cycles (84.6%), an embryo transfer was possible and resulted in 8 pregnancies leading to a fetal heart beat per oocyte retrieval of 20.5% and a fetal heart beat per embryonic transfer of 23.5%. The preparation time and costs for set-up and validation of tests are minimized. The informativity of microsatellite markers used in the universal PGD-PCR tests is based on CEPH and deCODE pedigrees, making the tests applicable in 90% of couples coming from these populations.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Mutação/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Adulto , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Linhagem , Gravidez , Diagnóstico Pré-Implantação/métodos , Diagnóstico Pré-Natal/métodos
18.
Hum Mutat ; 33(11): 1526-37, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22753008

RESUMO

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56-80% for breast cancer and 15-60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.


Assuntos
Genes BRCA1 , Variação Genética , Proteína BRCA1/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Bactérias , Neoplasias da Mama/genética , Feminino , Humanos , Masculino , Oligopeptídeos , Neoplasias Ovarianas/genética , Domínios e Motivos de Interação entre Proteínas , Tolerância a Radiação/genética , Fatores de Risco , Fatores de Transcrição , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo
19.
Clin Cancer Res ; 18(10): 2828-37, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22452945

RESUMO

PURPOSE: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. DESIGN: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics. RESULTS: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine. CONCLUSIONS: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Mutação em Linhagem Germinativa , Paraganglioma/genética , Feocromocitoma/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
20.
Mol Biol Rep ; 39(7): 7429-33, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22350158

RESUMO

A rapid and easy method to screen for aberrant cDNA would be a very useful diagnostic tool in genetics since a fraction of the DNA variants found affect RNA splicing. The currently used RT-PCR methods require new primer combinations to study each variant that might affect splicing. Since MLPA is routinely used to detect large genomic deletions and successfully detected exon skipping events in Duchenne muscular dystrophy in cDNA, we performed a pilot study to evaluate its value for BRCA1 cDNA. The effect of puromycin, DNase I and two different DNA cleaning protocols were tested in the RNA analysis of lymphocyte cultures. We used two samples from unrelated families with two different BRCA1 exon deletion events, two healthy unrelated controls and six samples from hereditary breast/ovarian cancer syndrome (HBOC) patients without BRCA1/2 mutations. Using RNA treated with DNase I and cleaned in a column system from puromycin-treated fractions, we were able to identify the two BRCA1 deletions. Additional HBOC patients did not show additional splice events. However, we were not able to get reproducible results. Therefore, the cDNA-MLPA technique using kit BRCA1 P002 is in our hands currently not reliable enough for routine RNA analysis and needs further optimization.


Assuntos
Proteína BRCA1/genética , DNA/análise , Genes BRCA1 , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Células Cultivadas , Primers do DNA , Éxons , Feminino , Humanos , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , RNA/genética , Reprodutibilidade dos Testes , Deleção de Sequência
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