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10541 Background: Despite the identification of a large number of sequences in BRCA1/2 mutation analyses, many genetic alterations are classified as variants of unknown significance (VUS). Increasing data demonstrates ethnic variation of BRCA mutation. We report a novel BRCA2 VUS in a Chinese family with multiple breast cancers and characterized it as pathogenic by RNA analysis. Method: Peripheral blood was collected from the breast cancer proband and her family who were suspected to have a pathogenic mutation due the segregation of the disease phenotype and the young age of presentation of breast cancer. DNA and RNA was extracted from the blood. The entire coding regions and flanking introns of BRCA1/2 were screened for germline mutations using full gene sequencing and Multiplex Ligation -dependent Probe Amplification. Variant DNA alteration leading to an aberrant BRCA transcript was demonstrated by RT-PCR of the RNA and Polyacrylamide Gel Electrophoresis. Abnormal bands were cloned and direct sequencing conducted. Results: A germline mutation c.7806–9T>G was identified in the proband at the BRCA2 intron 16. This mutation was also found in 3 of her 4 sibling sisters with early breast cancer. The proband's mother harbored the same BRCA2 mutation in DNA extracted from archival gastric tumor tissues. Computational analyses showed that this mutation might give rise to a cryptic splice site for alternative RNA splicing. To confirm the pathogenicity, RT- PCR using specific primers flanking the cryptic splice site and sequencing was performed on RNA extracted from the proband's blood. Four different sizes of transcripts were found: wild type, r.7806_7874del , r.7806_7976del and r.7806–8_7806-lins. The latter 3 aberrant transcripts were not found in the 30 controls therefore polymorphism is unlikely. The r.7806–8_7806-lins transcript caused a frameshift which created a truncated protein; whereas the other two short transcripts produced shorter BRCA2 protein isoforms. Conclusion: We identified a novel BRCA2 VUS and classified it as pathogenic. Classification of VUS as neutral or pathogenic, particularly in ethnic groups where limited knowledge is known is a challenge. Research on the spectrum of mutations in diversed ethnic groups has important implications on management. [Genbank: DQ889340 ] No significant financial relationships to disclose.

Chemoresistance is a major problem in the treatment of hepatocellular carcinoma. Certain p53 mutants may enhance drug resistance in cancer cells. To determine whether two frequently occurring p53 mutants, R248Q and R273C, would increase the drug resistance of liver cancer cells, stable cell lines expressing these specific p53 mutants were established by transfecting the p53-null Hep3B cells with mutant p53 expression vectors, and then treating them with the anticancer drugs doxorubicin and paclitaxel. The cells expressing the p53 mutant, R248Q, but not R273C, displayed cross-resistance to both drugs, in contrast to the control cells expressing the vector alone. Moreover, both the expression and the activity of the multiple drug resistance gene product, P-glycoprotein, were elevated in p53 mutant R248Q-expressing cells. Reduced uptake of doxorubicin was also observed in the R248Q-expressing cells. These results suggest that expression of the p53 mutant, R248Q, in liver cancer cells may enhance their drug resistance and that upregulation of P-glycoprotein activity may contribute to this protective effect.