RESUMO
Assessing the pathogenicity of missense mutations of MLH1 and MSH2 is critical to counsel patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC). Approximately 32% of all MLH1 mutations and 18% of MSH2 mutations are missense variants which often have an uncertain genetic significance. To assess the pathogenicity of four MLH1 missense mutations which were found in five patients with suspected HNPCC, P648S (CCC --> TCC), L559R (CTG --> CGG), K618A (AAG --> GCG), Y646C (TAT --> TGT), we studied their ability to disrupt MLH1 protein function and their relationship with all those clinical, genetic and pathological features which are typical of this syndrome. Our results indicated that the P648S and L559R mutations were probably pathogenic because they disrupted MLH1 protein interaction with its partner PMS2 in vitro and abolished MLH1 expression in HCT116 cells. In addition these variants were associated with features often found in HNPCC patients: in particular high microsatellite instability, occurrence of high grade tumours and, in one case, strong family history. The pathogenicity of the K618A and Y646C mutations was questionable as their correlation with features typical of HNPCC was low and the outcome of the functional analysis was ambiguous. These observations suggested that a clinically usable assessment of the pathogenicity of MLH missense variants can be achieved through the analysis of multiple mutation characteristics among which loss of protein function, occurrence of microsatellite instability and family history seemed to have a predominant role.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adulto , Idoso , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais Hereditárias sem Polipose/fisiopatologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Ligação Proteica , Análise de Sequência de DNA , TransfecçãoRESUMO
OBJECTIVE: To study how to identify patients with "triple negative" sporadic breast cancers (BCs) having BRCA1 silenced or down-regulated due to epigenetic BRCA1 inactivation. STUDY DESIGN: We selected, from our database, patients diagnosed with BC between 1995 and 2001 with tumors exhibiting the "triple negative" phenotype. "Triple positive" tumors were used as controls. BRCA1 protein expression was determined by immunohistochemistry. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing on genomic DNA were used to assess BRCA1 promoter methylation. BRCA1 m-RNA expression analysis was conducted by real-time PCR. RESULTS: Forty-four triple negative and 68 controls (triple positive) were eligible for our analysis. BRCA1 promoter methylation was present in 31.8% of triple negative and in 20.6% of triple positive cases. BRCA1 was inactivated (absent BRCA1 m-RNA expression and lack of BRCA1 protein) in 21.4% of tumors with BRCA1 promoter methylation, as compared with 6% of non-methylated ones (p = 0.0453). CONCLUSION: BRCA1 inactivation due to promoter methylation could play an important role in some sporadic BC cases. Patients with this signature could represent the basis for prospective studies aiming to compare clinical response to different drugs.