Abstract 228: A comprehensive study to functionally classify the BRCA2 missense mutations found in Fanconi anemia patients

Abstract

Abstract Biallelic mutations in breast cancer susceptibility gene 2 (BRCA2) cause a severe Fanconi Anemia (FA) phenotype that is a rare recessive disorder and reflects the cellular and phenotypic consequences of genetic instability. A number of mutations found in FA patients are missense mutations in BRCA2 and classified as variants of unknown clinical significance. This study is aimed towards the evaluation of functional significance of a few BRCA2 unclassified variants. In this study, six of the human mutations (L2510P, R2336H, I2490T, W2626C, K2729N and IVS7+2T>G) were generated in human BRCA2 gene cloned in a bacterial artificial chromosome vector. The effect of these variants on BRCA2 function was examined by using a mouse embryonic stem (ES) cell-based assay. We have shown the ability of wild-type human BRCA2 and neutral BRCA2 variants to rescue the lethality of Brca2-null mouse ES cells and functionally complement the loss of endogenous mouse Brca2. W2626C and IVS 7+2T>G mutations failed to restore the viability of ES cell colonies after deletion of the endogenous mouse Brca2. The K2729N BRCA2 rescued the ES cell lethality and appeared similar to wild-type BRCA2 in various functional assays. In contrast, L2510P and R2336H BRCA2 exhibited reduced ES cell viability. In addition, ES cells expressing L2510P BRCA2 are sensitive to DNA damaging agents. Furthermore, L2510P BRCA2 mutant ES cells are slow growing, have increased genomic instability, defective in G1-S checkpoint control and show upregulation of p53 and p21. These mutant cells are defective in homologous recombination mediated DNA repair. IVS7+2T>G and R2336H mutations result in exon-skipping leading to premature protein truncation. Alterations in L2510P, I2490T, W2626C and K2729N map to a highly conserved region of BRCA2 and the homology based molecular modeling of the mutant proteins supports our ES-cell results. L2510P and W2626C mutations disrupt the BRCA2 structure and the interaction of BRCA2 with a highly conserved protein DSS1, whereas I2490T and K2729N mutations do not affect the BRCA2 structure and are neutral in nature. It is possible that the patients with the variant alleles that are neutral in this assay have other undetected mutation in that allele or have mutations in some other FA genes. In conclusion, this study provides functional classification of a few variants of unknown clinical significance that should help in counseling individuals carrying these alleles. . Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 228. doi:10.1158/1538-7445.AM2011-228