| N-acetyltransferase2 (NAT2) is one of the popular metabolic enzymes in the world. It catalyses acetyl radical from acetyl coA to mixed Anulus amines, which plays important role in drug metabolism as well as inactivation or activation of carcinogen. As the second enzyme of biotransformarion, its activity directly affects metabolic response of carcinogen in vivo and intensity of toxic reaction. NAT2 has M1,M2,M3 and M4 alleles due to mutation of different enzyme-cutting site. Allele in no mutation is WT. Different genotypes (WT/WT,WT/Mx,Mx1/Mx2)are composed of different NAT2 alleles. According to intensity of acetyl action, different genotype shows different phenotype (fast acetylator genotypes and slow acetylator genotypes). Genotype composed of any two mutation alleles(Mx1/Mx2)shows slow acetylator genotypes, while the individual consist of WT/WT,WT/Mx(X=1,2,3,4)shows faster acetylator genotypes. Both NAT2 phenotype and allele are remarkably different in different nationality and zone. Investigation of epidemiology showed that fatalness of canceration in different position is related to NAT2 polymorphism. The colorectal cancer is one of common tumor in the Hebei province. Besides behavior and environment, inheritance factor in the body become more important. Fast acetylation of NAT2 is commonly regarded as the dangerous factor in colorectal canceration. Researchers in America found that there were no difference in NAT2 alleles(for example WT,M1,M2,M3)and its genotypes among both patients with colorectal cancer and the healthy population. There is no report on the distributions, expressions and activities of NAT2 phenotype and genotype in colorectal caner and pericancer tissues. We studied the polymorphism of NAT2 of Hebei Chinese Han compared with colorectal cancer patients as control study as to discuss the susceptible factor or protective factor. We also assayed the level of NAT2 mRNA expression and enzymatic activity in different genotype patients. We investigated the relationship between NAT2 genotype and phenotype and incidence of colorectal cancer, which provide the experimental basis for early prevention, diagnosis and therapy. Polymorphism of N-acetyltransferase gene-2 (NAT2) in Hebei Chinese populationGene NAT2 located 8p22 and the whole length of it is 1093bp. Eleven kinds of single nucleotide polymorphism(SNPs) in the code of NAT2 have been identified and different SNPs may affect NAT2 enzymatic expression, stability and catalyzing activity. Cutting site of KpnI is located in code 481nt. If a mutation happened in the site, the cutting site disappeared and produced the allele M1; In gene NAT2 there are another three recognizing sites of enzymes. Mutation in code 590nt for TaqI produces the allele M2, mutation in code 857nt for BamHI produces M3, while mutation in code 191nt for MspI produces M4, no mutation is wild-type WT. In order to gain hereditary data on NAT2 of Hebei Chinese Han, we randomly extracted 237 peripheral blood from healthy population living over 10 years in Hebei. The mean age was 42.56 years old (ranging from 20 to 80 years old). Among them, 124 were men and 113 were women. DNA was extracted from peripheral leukocytes by using PEL-FREEZ reagent protocol. PCR performed in 40μl reaction mixture containing 50-200ng genomic DNA, 20mΜ each primer,　1.25mM dNTPs, 25mM MgCl2, 1(PCR buffer,5U DNA polymerase. Cycle parameters are 5 min at 95℃; 35 cycles of 1 min at 95℃, 1min at 57℃ and 1.5min at 72℃; followed by 5 min at 72℃. Following amplification, 15μl of the PCR-products, were digested with KpnI(M1 allele),TaqI(M2 allele),BamHI(M3 allele),MspI/AluI(M4 allele)in a 25μl final volume, then analysed on a 2% agrose gel(M1,M3)and 4% agrose gel(M2,M4)stained with ethidium bromide for 1.5 hours and visualized under ultraviolet transillumination. The results showed :1.1 We found four kinds of NAT2 alleles in 237 studied healthy population of Hebei Chinese Han, including wild-type gene WT and mutation gene M1, M2, M3. Among them... |
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