Preparation and analysis of cSNP chip on hepatocellular carcinoma-related genes.
BACKGROUND
The understanding of cSNPs of cancer-related genes harboring in high frequency loss regions of tumor chromosomes can advance the disclosure of genetic and variant mechanisms of tumorigenesis, and the investigation of cancer susceptibility. In preparing a gene chip for detecting polymorphisms on coding region of genes in hepatocellular carcinoma tissues, some cSNPs are of interest for their potential links with phenotype.


METHODS
The genes harboring in loss regions with high frequency of hepatocellular carcinoma (HCC) were selected, the related information of cSNP sequences was obtained from the SNP database (dbSNP) of the National Center for Biotechnology Information (NCBI). Then appropriate primers and oligonucleotide probes were designed according to the SNP sites, and a gene chip for the detection of SNPs was constructed. The chip included 48 cSNPs of 25 hepatocellular carcinoma-related genes. The PCR products labeled by Dig-dUTP were hybridized with the cSNP chip.


RESULTS
The sensitivity, influence by probe concentration, and reiteration of the chip were detected, with a high sensitivity of 6X10(-3) ng/mul. The signal of hybridization was reduced with a lower concentration of probe. Seven polymorphisms of caspase 9 (rs2308941)C-->T and DOK2(rs2242241) T-->G, 6 of polymorphisms of EGFL3 (rs947345)A -->G, caspase 9 ( rs2308938) C-->G and PHGDH(rs1801955)T-->A, 5 of polymorphisms of E2F2(rs3218170) G-->A,4 of polymorphisms of MUTYH(rs1140507)T-->C and BNIP3L(rs1055806)G-->T, and 1 of polymorphism of TNFRSF1B (rs1061622)T-->G were detected by the chip in the tissues of 10 HCC. Samples of caspase 9 (rs2308941G) and (rs2308941A) were verified by PCR-SSCP and sequencing.


CONCLUSIONS
The cSNP chip of hepatocellular carcinoma-related genes can accelerate the discovery of polymorphic markers on hepatocellular carcinoma.
Wang
Juan
J
College of Life Sciences, Shenzhen University, Shenzhen 518060, China. wangjuannk@eyou.com
Ni
Hong
H
Chen
Li
L
Liu
Yan-Xue
YX
Chen
Cheng-Bin
CB
Song
Wen-Qin
WQ
D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D004077:Digoxin; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D015335:Molecular Probes; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results
D015335:Molecular Probes; D004077:Digoxin