Screen p53 mutations in hepatocellular carcinoma by FASAY: a novel splicing mutation.
OBJECTIVE
To establish a routine procedure for the detection of p53 mutations in hepatocellular carcinoma (HCC) surgical resections using the FASAY (functional analysis of separated alleles of p53 on yeast) procedure.


METHODS
p53 status was analyzed by FASAY and cDNA sequencing in 50 cases of HCC. After the extraction of RNA from the frozen tumor and corresponding normal tissues, reverse transcription RT-PCR was carried out using these samples. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies.


RESULTS
Of the 50 specimens, 29 (58%) were positive (mutant) by FASAY. Sequencing analysis confirmed that all 29 FASAY positive tumors harbored mutations, and that no mutations were detectable in any FASAY negative tumors. In 29 p53 mutations, 22 mutations were point missense mutation, 5 were deletions and 2 were splicing mutations. A novel splice mutation on splice donor of intron 6 was reported, which could produce two different mRNAs, respectively using the nearest upstream and downstream recessive splice donor sites.


CONCLUSIONS
FASAY is a sensitive method for detecting the various types of p53 mutations in HCC, suggesting that the yeast functional assay for the detection of p53 mutations may be essential for elucidating their clinical significance.
Wu
Xiao-mo
XM
Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Fu
Jing-geng
JG
Ge
Wang-zhong
WZ
Zhu
Jiang-yan
JY
Wang
Jun-yong
JY
Zhang
Wei
W
Qian
Wei
W
Huo
Ke-ke
KK
D017398:Alternative Splicing / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005787:Gene Frequency; D005820:Genetic Testing; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53