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1.
Clin Epigenetics ; 11(1): 171, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31779681

RESUMO

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

2.
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.

3.
Int J Cancer ; 145(10): 2682-2691, 2019 11 15.
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.

4.
J Med Genet ; 56(8): 521-525, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30580288

RESUMO

IMPORTANCE: Genetic testing of hereditary cancer using comprehensive gene panels can identify patients with more than one pathogenic mutation in high and/or moderate-risk-associated cancer genes. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS), which has been potentially linked to more severe clinical manifestations. OBJECTIVE: To determine the prevalence and clinical features of MINAS in a large cohort of adult patients with hereditary cancer homogeneously tested with the same gene panel. PATIENTS AND METHODS: A cohort of 1023 unrelated patients with suspicion of hereditary cancer was screened using a validated panel including up to 135 genes associated with hereditary cancer and phakomatoses. RESULTS: Thirteen (1.37%) patients harbouring two pathogenic mutations in dominant cancer-predisposing genes were identified, representing 5.7% (13/226) of patients with pathogenic mutations. Most (10/13) of these cases presented clinical manifestations associated with only one of the mutations identified. One case showed mutations in MEN1 and MLH1 and developed tumours associated with both cancer syndromes. Interestingly, three of the double mutants had a young age of onset or severe breast cancer phenotype and carried mutations in moderate to low-risk DNA damage repair-associated genes; two of them presented biallelic inactivation of CHEK2. We included these two patients for the sake of their clinical interest although we are aware that they do not exactly fulfil the definition of MINAS since both mutations are in the same gene. CONCLUSIONS AND RELEVANCE: Genetic analysis of a broad cancer gene panel identified the largest series of patients with MINAS described in a single study. Overall, our data do not support the existence of more severe manifestations in double mutants at the time of diagnosis although they do confirm previous evidence of severe phenotype in biallelic CHEK2 and other DNA repair cancer-predisposing genes.

5.
Br J Cancer ; 119(8): 978-987, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30283143

RESUMO

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Epigênese Genética/genética , Predisposição Genética para Doença/genética , Proteína 1 Homóloga a MutL/genética , Sequência de Bases , Neoplasias Colorretais/epidemiologia , Ilhas de CpG/genética , Feminino , Haplótipos/genética , Humanos , Masculino , Mutação/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA
6.
Int J Cancer ; 141(7): 1365-1380, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28577310

RESUMO

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , DNA Glicosilases/genética , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Molécula de Adesão da Célula Epitelial/genética , Exodesoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Perda de Heterozigosidade , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética
7.
Fam Cancer ; 16(4): 501-507, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28365877

RESUMO

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.


Assuntos
Neoplasias Colorretais/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Mutação de Sentido Incorreto , Idade de Início , Estudos de Casos e Controles , Reparo de Erro de Pareamento de DNA , Feminino , Frequência do Gene , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Humanos , Masculino , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Linhagem
8.
Sci Rep ; 7: 37984, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28050010

RESUMO

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes.


Assuntos
Predisposição Genética para Doença , Testes Genéticos/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Benchmarking , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento Completo do Exoma/métodos
9.
Sci Rep ; 7: 39348, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051113

RESUMO

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.


Assuntos
Detecção Precoce de Câncer/métodos , Testes Genéticos/métodos , Técnicas de Diagnóstico Molecular/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Sensibilidade e Especificidade
10.
Breast Cancer Res Treat ; 155(2): 253-60, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26780556

RESUMO

Germline inactivating mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome (HBOCS). Genetic testing of these genes identifies a significant proportion of variants of uncertain significance (VUS). Elucidation of the clinical impact of these variants is an important challenge in genetic diagnostics and counseling. In this study, we assess the RNA effect of 28 BRCA1 and BRCA2 VUS identified in our set of HBOCS families with the aim of gaining insight into their clinical relevance. mRNA was extracted from VUS carriers and controls lymphocytes cultured for 5-6 days and treated with puromycin. RNA was reverse transcribed to perform transcriptional analysis for the study of splicing aberrations. In silico prediction tools were used to select those variants most likely to affect the RNA splicing process. Six out of the 28 variants analyzed showed an aberrant splicing pattern and could therefore be classified as probably pathogenic mutations. Reclassification of VUS improves the genetic counseling and clinical surveillance of carriers of these mutations and highlights the importance of RNA studies in routine diagnostic laboratories.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Mutação em Linhagem Germinativa/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , RNA Mensageiro/genética , Feminino , Humanos , Processamento de RNA/genética , Transcrição Genética/genética
11.
Hum Mutat ; 35(3): 271-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24227591

RESUMO

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.


Assuntos
Biologia Computacional/métodos , Genes Neoplásicos , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Internet , Genoma Humano , Genômica/métodos , Humanos , Interface Usuário-Computador
12.
J Med Genet ; 50(8): 552-63, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23709753

RESUMO

BACKGROUND AND AIM: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. METHODS: From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. RESULTS: Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. CONCLUSIONS: Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.


Assuntos
Adenosina Trifosfatases/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Estudos de Coortes , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA , Variação Genética , Células HEK293 , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Polimorfismo Genético , Transfecção
13.
Eur J Hum Genet ; 21(8): 864-70, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23249957

RESUMO

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Algoritmos , Análise Custo-Benefício , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Testes Genéticos/economia , Testes Genéticos/métodos , Humanos , Mutação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Breast Cancer Res Treat ; 132(3): 979-92, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21735045

RESUMO

Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls. We identified a splicing defect in 8 of the 26 newly studied variants and ruled out splicing alteration in the remaining 18 variants. The results for the four positive and the two negative control variants were consistent with results presented in the literature. Our results strongly suggest that the combination of RNA analysis and in silico programs is an important step towards the classification of VUS. The results revealed a very high correlation between experimental data and in silico programs when using tools for predicting acceptor/donor sites but a lower correlation in the case of tools for identifying ESE elements.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , RNA Mensageiro/genética , Processamento Alternativo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Sequência de Bases , Simulação por Computador , Feminino , Predisposição Genética para Doença , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
15.
Breast Cancer Res Treat ; 130(1): 341-4, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21761158

RESUMO

In this study, we present a novel complex rearrangement in the BRCA1 gene. The genomic rearrangement was identified using one of the two commercially available MLPA BRCA1 kits but was not confirmed with the other. In this report, we present the full characterization at the DNA and RNA levels of a new partial deletion of exon 20 of BRCA1. This is a complex deletion with four breakpoints which promotes aberrant splicing with partial deletion of exon 20 plus the insertion of a cryptic exon corresponding to a fragment of intron 20. The aberrant splicing generates an abnormal transcript with a frameshift that will result in a truncated BRCA1 protein.


Assuntos
Éxons , Genes BRCA1 , Neoplasias Ovarianas/genética , Deleção de Sequência , Adulto , Sequência de Bases , Feminino , Ordem dos Genes , Humanos , Dados de Sequência Molecular , Linhagem , Processamento de RNA , Alinhamento de Sequência
17.
Breast Cancer Res Treat ; 122(3): 733-43, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19894111

RESUMO

Large genomic rearrangements are estimated to account for approximately 5-10% of all disease-causing mutations in BRCA1 and BRCA2 genes in patients with hereditary breast and ovarian cancer syndrome (HBOC). We use MRC-Holland Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for such rearrangements in patients with HBOC and as a first step in our genetic testing workflow. The technique was applied to a set of 310 independent patients and detected eight different copy number alterations, corresponding to 2.6% of the studied samples. MLPA was also found to identify point mutations located in probe sequences. As commercial MLPA tests are not suitable for determining the specific breakpoints or for defining the exact extent of rearrangements, we applied a set of different complementary techniques to characterize these genetic alterations with greater precision. Long-range PCR amplification, RNA analysis, SNP-array chips, non-commercial MLPA probes, and FISH analysis were used to fully define the extent and mechanism of each alteration. In BRCA1, six rearrangements were characterized: deletion of E22, duplication of E9-E24, deletion of E16-E23, deletion of E1-E13, deletion of E1-E2 and duplication of E1-E2. In BRCA2, we studied a deletion of E15-E16 and a deletion of E1-E24. To the best of our knowledge, this is the most comprehensive study of the nature and underlying molecular causes of these mutational events in the BRCA1/2 genes.


Assuntos
Neoplasias da Mama/genética , Rearranjo Gênico , Genes BRCA1 , Genes BRCA2 , Genoma Humano , Neoplasias Ovarianas/genética , Adulto , Sequência de Bases , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Homologia de Sequência do Ácido Nucleico
18.
Am J Med Genet A ; 143A(10): 1108-13, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17431916

RESUMO

We report on a child with a small supernumerary marker chromosome (sSMC) causing partial trisomy 6p. The child showed a phenotype consisting of neonatal craniosynostosis, microcephaly, and borderline developmental delay. By molecular techniques the sSMC has been shown to contain approximately 16 Mb of genomic DNA from 6p21.1 to 6cen, being de novo and of maternal origin.


Assuntos
Cromossomos Humanos Par 6 , Craniossinostoses/genética , Marcadores Genéticos , Trissomia/genética , Bandeamento Cromossômico , Análise Mutacional de DNA , Genótipo , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Linhagem
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