RESUMO
Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.
Assuntos
Proteína BRCA1/química , Proteína BRCA1/genética , Mutação da Fase de Leitura/genética , Mutação de Sentido Incorreto/genética , Deleção de Sequência/genética , Proteína BRCA1/metabolismo , Western Blotting , Linhagem Celular , Biologia Computacional , Simulação por Computador , Éxons/genética , Feminino , Humanos , Masculino , Modelos Genéticos , Modelos Moleculares , Linhagem , Estrutura Terciária de Proteína , TransfecçãoRESUMO
A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.
Assuntos
Substituição de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/genética , Mutação de Sentido Incorreto/genética , Fosfopeptídeos/metabolismo , Adulto , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Segregação de Cromossomos , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Evolução Molecular , Proteínas de Grupos de Complementação da Anemia de Fanconi , Feminino , Humanos , Funções Verossimilhança , Lisina/genética , Masculino , Metionina/genética , Pessoa de Meia-Idade , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Hibridização de Ácido Nucleico , Linhagem , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/metabolismoRESUMO
Increasingly, the molecular genetics laboratory has to assess the biological significance of changes (variants) in a DNA sequence. Using the large genes BRCA1 and BRCA2 as examples, some approaches used to determine the biological significance of DNA variants are described. These include the characterization of the variant through a review of the literature and the various databases to assess if it has previously been described. Potential difficulties with the various databases that are available are described. Other considerations include the co-inheritance of the variant with other DNA changes, and its evolutionary conservation. Determining the possible effect of the variant on protein function is described in terms of the Grantham assessment as well as identifying functional domains. Studies looking at the distribution of the variant in both the population and the family can also help in assessing its significance. Loss of the variant in a tumor sample would imply that it is not deleterious. Ultimately, it is not any single parameter that helps determine the DNA variants biological significance. Usually this requires multiple lines of evidence.