Genotyping of TNF-α, IL-6, IL-10 and IL-18 Genes in Septicemia Patients by Multiplex-Minisequencing

• Main Authors: Wei-Huan Lee, 李威寰
• Other Authors: Mu-Chiou Huang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/tvk75a

碩士 === 國立中興大學 === 動物科學系所 === 97 === The aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) with traits relative to inflammatory process in septicemia patients. Genotyping of TNF-α, IL-6, IL-10 and IL-18 genes were carried out by PCR-SSCP, PCR-RFLP, minisequencing and multiplex minisequencing. Specific primer pairs were designed by GCG sequence analysis software and each of the target genes was amplified. The results showed that the multiplex-minisequencing method was able to unambiguously and simultaneously genotyping many SNP loci in one microtubes. Genotypic and allelic frequencies of each gene were analyzed. The results revealed that the survival day and survival rate of GG type of TNF-α (G-308A), GG type of IL-6 (G-174C) were better than the other types in septic patients in the Nantou Hospital. The survival rate of AA type of IL-10 (G-1082A) was higher than GA type. Patients with both of CA type of IL-18 (C-607A) and GC type of IL-18 (G-137C) have the highest survival rate. According to above evidence revealed G allele of TNF-α (G-308A), G allele of IL-6 (G-174C) and A allele of IL-10 (G-1082A) were most advantageous for survival. Patients with GG type of TNF-α (G-308A), GG type of IL-6 (G-174C), and AA type of IL-10 (G-1082A) have a higher survival rate than other types (p <0.05). Patients with GA type of TNF-α (G-308A) have a higher serum IL-10 level at entry (p < 0.05). These patients also have doubled serum creatinine represents poor renal functions than GG type patients. It may be one reason for higher mortality happened to GA type patients revealed from previous studies. We have successfully developed a rapid, accuracy and cost-effective for genotyping many loci in the same capillary tube. These results could be useful for human body or animal physiological and pathological analysis.