Somatic mutation analysis of p53 and ST7 tumor suppressor genes in gastric carcinoma by DHPLC.
OBJECTIVE
To verify the effectiveness of denaturing high-performance liquid chromatography (DHPLC) in detecting somatic mutation of p53 gene in gastric carcinoma tissues. The superiority of this method has been proved in the detection of germline mutations, but it was not very affirmative with respect to somatic mutations in tumor specimens. ST7 gene, a candidate tumor suppressor gene identified recently at human chromosome 7q31.1, was also detected because LOH at this site has also been widely reported in stomach cancer.


METHODS
DNA was extracted from 39 cases of surgical gastric carcinoma specimen and their correspondent normal mucosa. Seven fragments spanning the 11 exons were used to detect the mutation of p53 gene and the four exons reported to have mutations in ST7 gene were amplified by PCR and directly analyzed by DHPLC without mixing with wild-type allele.


RESULTS
In the analysis of p53 gene mutation, 9 aberrant DHPLC chromatographies were found in tumor tissues, while their normal-adjacent counterparts running in parallel showed a normal shape. Subsequent sequencing revealed nine sequence variations, 1 polymorphism and 8 mutations including 3 mutations not reported before. The mutation rate of p53 gene (21%) was consistent with that previously reported. Furthermore, no additional aberrant chromatography was found when wild-type DNA was added into the DNA of other 30 tumor samples that showed normal shapes previously. The positivity of p53 mutations was significantly higher in intestinal-type carcinomas (40%) than that in diffuse-type (8.33%) carcinomas of the stomach. No mutation of ST7 gene was found.


CONCLUSIONS
DHPLC is a very convenient method for the detection of somatic mutations in gastric carcinoma. The amount of wild type alleles supplied by the non-tumorous cells in gastric tumor specimens is enough to form heteroduplex with mutant alleles for DHPLC detection. ST7 gene may not be the target gene of inactivation at 7q31 site in gastric carcinoma.
Lu
Chong
C
Laboratory of Medical Genomics, Oncology Department, the First Affiliated Clinical College, China Medical University, Shenyang, 110001, Liaoning Province, China.
Xu
Hui-Mian
HM
Ren
Qun
Q
Ao
Yang
Y
Wang
Zhen-Ning
ZN
Ao
Xue
X
Jiang
Li
L
Luo
Yang
Y
Zhang
Xue
X
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002897:Chromosomes, Human, Pair 7; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D016147:Genes, Tumor Suppressor; D016158:Genes, p53; D018095:Germ-Line Mutation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D004273:DNA, Neoplasm; D008565:Membrane Proteins; D016159:Tumor Suppressor Protein p53