Positional cloning of a novel Fanconi anemia gene, FANCD2.
ABSTRACT Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.
Blood 01/2001; 98(13):3650-3657. DOI:10.1182/blood.V98.13.3650 · 9.78 Impact Factor
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ABSTRACT: DNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC). The methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing. In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated. Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76). We demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.12/2014; 6(1):22. DOI:10.1186/1868-7083-6-22
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ABSTRACT: Inherited germline mutations in the tumor suppressor gene BRCA1 predispose individuals to early onset breast and ovarian cancer . BRCA1 together with its structurally related partner BARD1 is required for homologous recombination and DNA double-strand break repair, but how they perform these functions remains elusive 2 and 3. As part of a comprehensive search for DNA repair genes in C. elegans, we identified a BARD1 ortholog. In protein interaction screens, Ce-BRD-1 was found to interact with components of the sumoylation pathway, the TACC domain protein TAC-1, and most importantly, a homolog of mammalian BRCA1. We show that animals depleted for either Ce-brc-1 or Ce-brd-1 display similar abnormalities, including a high incidence of males, elevated levels of p53-dependent germ cell death before and after irradiation, and impaired progeny survival and chromosome fragmentation after irradiation. Furthermore, depletion of ubc-9 and tac-1 leads to radiation sensitivity and a high incidence of males, respectively, potentially linking these genes to the C. elegans BRCA1 pathway. Our findings support a shared role for Ce-BRC-1 and Ce-BRD-1 in C. elegans DNA repair processes, and this role will permit studies of the BRCA1 pathway in an organism amenable to rapid genetic and biochemical analysis.Current Biology 01/2004; 14(1):33-39. DOI:10.1016/S0960-9822(03)00871-6 · 9.92 Impact Factor