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1.
Oncotarget ; 9(45): 27882-27894, 2018 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-29963245

RESUMO

Azacitidine is the first drug to demonstrate a survival benefit for patients with MDS. However, only half of patients respond and almost all patients eventually relapse. Limited and conflicting data are available on predictive factors influencing response. We analyzed 128 patients from two institutions with MDS or AML treated with azacitidine to identify prognostic indicators. Genetic mutations in ASXL1, RUNX1, DNMT3A, IDH1, IDH2, TET2, TP53, NRAS, KRAS, FLT3, KMT2A-PTD, EZH2, SF3B1, and SRSF2 were assessed by next-generation sequencing. With a median follow up of 5.6 years median survival was 1.3 years with a response rate of 49%. The only variable with significant influence on response was del(20q). All 6 patients responded (p = 0.012) but survival was not improved. No other clinical, cytogenetic or molecular marker for response or survival was identified. Interestingly, patients from poor-risk groups as high-risk cytogenetics (55%), t-MDS/AML (54%), TP53 mutated (48%) or relapsed after chemotherapy (60%) showed a high response rate. Factors associated with shorter survival were low platelets, AML vs. MDS, therapy-related disease, TP53 and KMT2A-PTD. In multivariate analysis anemia, platelets, FLT3-ITD, and therapy-related disease remained in the model. Poor-risk factors such as del(7q)/-7, complex karyotype, ASXL1, RUNX1, EZH2, and TP53 did not show an independent impact. Thus, no clear biomarker for response and survival can be identified. Although a number of publications on predictive markers for response to AZA exist, results are inconsistent and improved response rates did not translate to improved survival. Here, we provide a comprehensive overview comparing the studies published to date.

2.
Biol Blood Marrow Transplant ; 24(11): 2337-2343, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29753838

RESUMO

Overexpression of the Wilms' tumor 1 (WT1) gene is informative in many patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS) and is measurable in peripheral blood (PB). Despite these advantages, WT1 has not broadly been established as a marker for minimal residual disease (MRD) monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to limited patient numbers, differing sample sources, and nonstandardized in-house methods. To estimate the value of WT1 as an MRD marker, we serially quantified PB WT1 expression using a standardized European LeukemiaNet-certified assay in 59 patients with AML and MDS after allo-HSCT. We compared its performance with routine methods such as chimerism, XY-fluorescence in situ hybridization (FISH), disease-specific cytogenetic, and molecular analyses, which were accessible in 100%, 34%, 68%, and 37%, respectively. Twenty-four patients (41%) relapsed within a median of 126 days after allo-HSCT, and 20 of them showed at least 1 elevated WT1 value above the validated cutoff. The other 35 patients (59%) remained in complete remission, and only 1 patient had a transient increase in WT1 expression. This reflects a sensitivity of 83% and a specificity of 97% for WT1 and appears to be favorable compared with the sensitivities and specificities observed for chimerism (33% and 91%), XY-FISH (67% and 73%), cytogenetic (33% and 77%), and molecular (78% and 85%) analyses. Further supporting its predictive impact, elevated WT1 expression prompted an earlier BM biopsy and consecutively the diagnosis of relapse in 62% of patients. The results of this real-life experience imply that PB WT1 expression is measurable by a standardized assay and predicts imminent relapse after allo-HSCT with high sensitivity and specificity in most patients with AML and MDS.

3.
Am J Med Genet A ; 161A(6): 1453-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23633430

RESUMO

We describe a female patient with mild lissencephaly (pachygyria), severe intellectual disability, and facial dysmorphisms with an inverted 1.4 Mb microduplication of chromosome 17p13.3. The 17p13.3 microduplication syndrome is associated with mild intellectual disabiltiy and contains, among others, the PAFAH1B1 (LIS1) gene, whereas microdeletions of the same segment cause Miller-Dieker syndrome (MDS) with severe to profound retardation. The duplication identified in our patient encompasses 29 genes, including CRK and YWHAE. The proximal breakpoint of the duplication is located in the first intron of the PAFAH1B1 gene. Analysis of total RNA showed that only one PAFAH1B1 allele is expressed. Therefore, this patient has a unique alteration: a duplication including YWHAE and CRK and haploinsufficiency of PAFAH1B1. Overexpression of YWHAE is associated with macrosomia, mild developmental delay, autism and facial dysmorphisms, and deletion of PAFAH1B1 alone leads to isolated lissencephaly (ILS). The patient described here shares features with MDS, but she is affected to a lesser degree. Her facial features are similar to MDS, and she has manifestations seen in other cases with YWHAE duplication.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Proteínas 14-3-3/genética , Transtornos Cromossômicos/genética , Duplicação Cromossômica/genética , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Lisencefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Malformações do Sistema Nervoso/genética , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/diagnóstico por imagem , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/diagnóstico , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/diagnóstico por imagem , Hibridização Genômica Comparativa , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/diagnóstico por imagem , Feminino , Haploinsuficiência , Humanos , Hibridização in Situ Fluorescente , Lactente , Deficiência Intelectual/diagnóstico , Íntrons/genética , Lisencefalia/diagnóstico , Lisencefalia/diagnóstico por imagem , Hipotonia Muscular , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/diagnóstico por imagem , Fenótipo , RNA/genética , Radiografia , Análise de Sequência de DNA , Inversão de Sequência/genética
4.
PLoS One ; 6(6): e20464, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21674048

RESUMO

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99-1.67; P(value) = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06-1.75; P(value) = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12-1.61; P(value) = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.


Assuntos
Neoplasias Colorretais/genética , Grupo com Ancestrais do Continente Europeu/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas-G/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Controle de Qualidade
5.
Fam Cancer ; 10(2): 273-84, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21404117

RESUMO

Missense mutations of the DNA mismatch repair gene MLH1 are found in a significant fraction of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) and their pathogenicity often remains unclear. We report here all 88 MLH1 missense variants identified in families from the German HNPCC consortium with clinical details of these patients/families. We investigated 23 MLH1 missense variants by two functional in vivo assays in yeast; seven map to the ATPase and 16 to the protein interaction domain. In the yeast-2-hybrid (Y2H) assay three variants in the ATPase and twelve variants in the interaction domain showed no or a reduced interaction with PMS2; seven showed a normal and one a significantly higher interaction. Using the Lys2A (14) reporter system to study the dominant negative mutator effect (DNE), 16 variants showed no or a low mutator effect, suggesting that these are nonfunctional, three were intermediate and four wild type in this assay. The DNE and Y2H results were concordant for all variants in the interaction domain, whereas slightly divergent results were obtained for variants in the ATPase domain. Analysis of the stability of the missense proteins in yeast and human embryonic kidney cells (293T) revealed a very low expression for seven of the variants in yeast and for nine in human cells. In total 15 variants were classified as deleterious, five were classified as variants of unclassified significance (VUS) and three were basically normal in the functional assays, P603R, K618R, Q689R, suggesting that these are neutral.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Células Cultivadas , Reparo de Erro de Pareamento de DNA , Humanos , Proteína 1 Homóloga a MutL , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genética
6.
Hum Mutat ; 32(4): 407-14, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21309036

RESUMO

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.


Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Variação Genética , Mutação em Linhagem Germinativa/genética , Deleção de Sequência/genética , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Moléculas de Adesão Celular/metabolismo , Metilação de DNA , Molécula de Adesão da Célula Epitelial , Modelos Genéticos , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Países Baixos , Regiões Promotoras Genéticas , Recidiva
8.
BMC Gastroenterol ; 10: 112, 2010 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-20920174

RESUMO

BACKGROUND: Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), has two major functions: the stimulation of the growth hormone production and the stimulation of food intake. Accumulating evidence also indicates a role of ghrelin in cancer development. METHODS: We conducted a case-control study to examine the association of common genetic variants in the genes coding for ghrelin (GHRL) and its receptor (GHSR) with colorectal cancer risk. Pairwise tagging was used to select the 11 polymorphisms included in the study. The selected polymorphisms were genotyped in 680 cases and 593 controls from the Czech Republic. RESULTS: We found two SNPs associated with lower risk of colorectal cancer, namely SNPs rs27647 and rs35683. We replicated the two hits, in additional 569 cases and 726 controls from Germany. CONCLUSION: A joint analysis of the two populations indicated that the T allele of rs27647 SNP exerted a protective borderline effect (Ptrend = 0.004).


Assuntos
Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Grelina/genética , Polimorfismo Genético , Receptores de Grelina/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Criança , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/metabolismo , República Tcheca/epidemiologia , Feminino , Predisposição Genética para Doença , Alemanha/epidemiologia , Grelina/metabolismo , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Receptores de Grelina/metabolismo , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
9.
J Cancer Res Clin Oncol ; 136(1): 123-34, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19669161

RESUMO

Hereditary non-polyposis colorectal cancer, an autosomal dominant predisposition to colorectal cancer and other malignancies, is caused by inactivating mutations of DNA mismatch repair genes, mainly MLH1 and MSH2. Missense mutations affect protein structure or function, but may also cause aberrant splicing, if located within splice sites (ss) or cis-acting sequences of splicing regulatory proteins, i.e., exonic splicing enhancers or exonic splicing silencers. Despite significant progress of ss scoring algorithms, the prediction for the impact of mutations on splicing is still unsatisfactory. For this study, we assessed ten ss and nine missense mutations outside ss in MLH1 and MSH2, including eleven newly identified mutations, and experimentally analyzed their effect at the RNA level. We additionally tested and compared the reliability of several web-based programs for the prediction of splicing outcome for these mutations.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Sítios de Splice de RNA/genética , Algoritmos , Sequência de Bases , Neoplasias Colorretais Hereditárias sem Polipose/genética , Biologia Computacional/métodos , Análise Mutacional de DNA , Éxons/genética , Humanos , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Processamento de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Int J Cancer ; 124(7): 1727-35, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19115204

RESUMO

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real-time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down-regulation in 59% and 61% of the analyzed tissues, respectively. This down-regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57-62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down-regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease-free (p = 0.037) and overall survival (p = 0.032), particularly in those node-positive patients who received adjuvant anthracycline-based chemotherapy, but not in those treated by either cyclophosphamide-methotrexate-5-fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metilação de DNA/genética , Proteínas da Matriz Extracelular/biossíntese , Neovascularização Patológica/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Análise Mutacional de DNA , Epigênese Genética , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos
11.
Hum Mutat ; 29(7): 948-58, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18431737

RESUMO

We present a comprehensive analysis of 1,506 German families for large genomic rearrangements (LGRs) in the BRCA1 gene and of 450 families in the BRCA2 gene by the multiplex ligation-dependent probe amplification (MLPA) technique. A total of 32 pathogenic rearrangements in the BRCA1 gene were found, accounting for 1.6% of all mutations, but for 9.6% of all BRCA1 mutations identified in a total of 1,996 families, including 490 with small pathogenic BRCA1/2 mutations. Considering only high risk groups for hereditary breast/ovarian cancer, the prevalence of rearrangements is 2.1%. Interestingly, deletions involving exon 17 of the BRCA1 gene seem to be most frequent in Germany. Apart from recurrent aberrations like del ex17, dupl ex13, and del ex22, accounting for more than 50% of all BRCA1 LGRs, we could fully characterize 11 novel deletions. Moreover, one novel deletion involving exons 1-7 and one deletion affecting the entire BRCA1 gene were identified. All rearrangements were detected in families with: 1) at least two breast cancer cases prior to the age of 51 years; 2) breast and ovarian cancer cases; 3) ovarian cancer only families with at least two ovarian cancer cases; or 4) a single breast cancer case prior to the age of 36 years, while no mutations were detected in breast cancer only families with no or only one breast cancer case prior to the age of 51 years. Analysis for gross rearrangements in 412 high-risk individuals, revealed no event in the BRCA2 gene and only two known CHEK2 mutations. However, in an additional 38 high-risk families with cooccurrence of female breast/ovarian and male breast cancer, one rearrangement in the BRCA2 gene was found. In summary, we advise restricting BRCA1 MLPA screening to those subgroups that revealed LGRs and recommend BRCA2 MLPA screening only for families presenting with cooccurrence of female and male breast cancer.


Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Deleção de Sequência , Idade de Início , Éxons , Família , Feminino , Rearranjo Gênico , Genes BRCA2 , Testes Genéticos , Alemanha , Humanos , Masculino , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase
12.
Eur J Hum Genet ; 16(7): 804-11, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18301449

RESUMO

Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCR-screening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Sanguíneas/patologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Metilação de DNA , Padrões de Herança/genética , Mutação/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Alelos , Sequência de Bases , Ilhas de CpG/genética , Análise Mutacional de DNA , DNA Complementar/genética , DNA de Neoplasias/genética , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Fenótipo , Sulfitos/metabolismo
13.
Int J Cancer ; 122(7): 1476-82, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18059028

RESUMO

The CapG protein, a Gelsolin-related actin-binding protein, is expressed at higher levels in breast cancer, especially in metastasizing breast cancer, than in normal breast epithelium. Furthermore, it is known that an increased expression of the CapG protein triggers an increase in cell motility. According to in vitro experiments, it was supposed that it is the nuclear fraction of the protein, which causes the increase in cell motility. Here, we examined the dynamical distribution of the CapG protein within the living cell, i.e. the import of the CapG protein into the nucleus. The nuclear import kinetics of invasive, metastasizing breast cancer cells were compared to the import kinetics of non-neoplastic cells similar to normal breast epithelium. FRAP kinetics showed a highly significant increase in the recovery of photobleached CapG-eGFP in the cancer cells, so that a differentiation of invasive, metastasizing cells and non-invasive, non-metastasizing cells on the basis of transport processes of the CapG protein between the nucleus and the cytoplasm seems to be possible. Comprehension of the mobility and compartmentalization of the CapG protein in normal and in cancer cells in vivo could constitute a new basis to characterize the invasiveness and metastasizing potential of breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Recuperação de Fluorescência Após Fotodegradação , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Invasividade Neoplásica , Proteínas Nucleares/genética , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
14.
Int J Cancer ; 121(3): 547-54, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17415710

RESUMO

Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome-wide methylation analysis of five sporadic breast carcinomas we identified a hypermethylated CpG island within the first intron of the prospero related homeobox gene 1 (PROX1). We, therefore, investigated CpG island methylation of PROX1 in a series of 33 pairs of primary breast cancer and corresponding normal tissue samples by bisulfite sequencing and COBRA analyses. Seventeen of these (52%) breast cancer samples revealed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Frequent methylation was also observed in brain metastases from primary breast cancer (21/37 = 57% of cases). Secondary, we analysed 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression of PROX1 compared to normal breast tissue (p < 0.001) and primary breast carcinomas (p < 0.05), respectively. Additionally, treatment of breast cancer cell lines with demethylating agents could reactivate PROX1 transcription. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Epigênese Genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Azacitidina/farmacologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Ilhas de CpG , DNA de Neoplasias/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Genética , Células Tumorais Cultivadas
15.
Br J Haematol ; 133(2): 188-97, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16611311

RESUMO

Fanconi anaemia (FA) is a rare recessive DNA repair disorder clinically characterised by congenital malformations, progressive bone marrow failure and a high propensity for developing malignancies at an early age, predominantly acute myeloid leukaemia (AML) and squamous cell carcinoma. It is conceivable that a number of patients with hypomorphic mutations are not diagnosed as FA until severe complications in the treatment of a malignancy occur. Here, we report on a patient with FA-A, diagnosed only at the age of 49 years due to persistent pancytopenia and myelodysplastic syndrome/AML induced by a first cycle of chemotherapy for bilateral metachronic breast cancer. This exceptional case clearly demonstrates that, in instances of long-lasting mild pancytopenia or development of malignancies, especially at an unusually young age, FA should be ruled out, irrespective of the patient's age and features, especially before inflicting severe genotoxic stress.


Assuntos
Anemia de Fanconi/diagnóstico , Síndromes Neoplásicas Hereditárias/diagnóstico , Adulto , Fatores Etários , Sequência de Aminoácidos , Neoplasias da Mama/etiologia , Anemia de Fanconi/complicações , Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Feminino , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/etiologia , Síndromes Neoplásicas Hereditárias/genética , Pancitopenia/etiologia
16.
J Pathol ; 205(1): 21-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15586368

RESUMO

The identification of novel disease-associated genes in gynaecological tumours has important implications for understanding the process of tumourigenesis and the development of novel treatment regimens. cDNA libraries from disease tissues may represent a valuable source to identify such genes. Recently, a bio-informatic procedure based on an 'electronic Northern' approach was established to screen expressed sequence tag (EST) libraries for genes differentially expressed in tumour and normal tissues, and identified 450 candidate genes differentially expressed in breast and ovarian cancer. In this report, the validation of an initial set of 40 candidate genes, which were selected due to their localization in chromosomal regions frequently altered in gynaecological tumours, is described. Differential expression of 29 of these genes, including three uncharacterized novel genes, was confirmed by applying cancer profiling arrays with 106 matched pairs of tumour/normal cDNAs and quantitative reverse transcription-polymerase chain reaction (RT-PCR) on 60 clinical specimens. The majority of these differentially expressed genes have not been described previously in the context of breast and ovarian cancer, and may constitute novel diagnostic markers for these tumour entities.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Carcinoma Ductal de Mama/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Etiquetas de Sequências Expressas , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
17.
Hum Mutat ; 23(6): 612-20, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15146466

RESUMO

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.


Assuntos
Neoplasias da Mama/genética , Cromatografia Líquida de Alta Pressão/métodos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Humanos , Masculino , Dados de Sequência Molecular , Sensibilidade e Especificidade
18.
Int J Cancer ; 110(3): 320-5, 2004 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15095295

RESUMO

To establish the importance of CHEK2 mutations for familial breast cancer incidence in the German population, we have screened all 14 of the coding exons in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. We found 12 distinct variants in 30 unrelated patients (5.81%), including 5 that are novel and an additional 4 found for the first time in breast cancer. These aberrations were evaluated in 500 healthy women aged over 50 years and in the case of the 2 exon 10 mutations, 1100delC and 1214del4bp, in 1315 randomized healthy controls. According to our results, a statistically significant association for the exon 10 mutations was observed (p = 0.006). The prevalence of the 1100delC mutation in the German population, however, is significantly lower than those reported for other Caucasian populations both in familial breast cancer patients (1.6%) and controls (0.5%), and shows independent segregation with breast cancer in 2 of 4 families analyzed. The remaining 10 variants were more abundant in patients (21) compared to the controls (12) although the difference was not statistically significant. Interestingly, we found no increased breast cancer risk associated with the splice site mutation IVS2+1G-->A or the most common missense mutation I157T, which account for more than half (12/21) of the variants observed in patients. The low prevalence and penetrance of the exon 10 deletion mutations together with no, or an uncertain elevation in risk for other CHEK2 mutations suggests a limited relevance for CHEK2 mutations in familial breast cancer. Further evaluation of the unique variants observed in breast cancer is required to determine if they may play a role in a polygenic model of familial breast cancer. Nevertheless, it seems premature to include CHEK2 screening in genetic testing.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Quinase do Ponto de Checagem 2 , Éxons , Feminino , Deleção de Genes , Genes BRCA1 , Genes BRCA2 , Humanos , Masculino , Mutação , Mutação de Sentido Incorreto , Neoplasias Ovarianas/genética , Linhagem , Reação em Cadeia da Polimerase
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