| The genetic polymorphism of drug metabolizing enzyme, transporter and target greatly influence the individual difference of drug efficiency and toxicity. N-acetyltransferase 2 (NAT2) metabolizes over 20 drugs in human. The capacity of metabolizing is influenced by genetic factors. The target of this research is to establish convenient and reliable method to study the genotype of NAT2 in Chinese people. Through the study of NAT2 genotype and INH plasma concentration, we want to find out the relationship between NAT2 polymorphism and rational drug therapy.Part I. An one-step allele specific amplificaton for genotyping of NAT2 in Chinese subjectsPoor metabolizers (PM) of NAT2 are mainly caused by *5, *6 and *7 alleles. The purpose of this part is to establish a simplified one-step allele specific PCR amplification (ASA), and study the distribution of alleles of NAT2 polymorphism in Chinese subjects. A reformed phenol/chloroform method was used to extract DNA in blood. Allele specific primers (P1~P6) were designed to pair wild type and mutant template. One of the allele specific primers and public primer (P7) were added to a tube. After the PCR, the result was detected by electrophresis. Genotypes of 345 healthy Chinese subjects were detected by this method. The fragment of *5, *6 and *7 alleles were 803, 554 and 287 bp respectively. The NAT2 allele frequencies of *5, *6 and *7 were 2.9%, 22.6% and 10.3%, respectively. The NAT2 genotypes distribution for all detected combinations of NAT2 alleles in 345 Chinese subjects were in consistent with Hardy-Weinberg equilibrium. Homozygous wildtype, heterozygous mutant and homozygous mutant subjects were 144(41.7%), 155(44.9%) and 46(13.3%), respectively. One-step ASA was proved to be accurate and convenient, it provides a basis for the use in rational drug therapy. Part II. A Reverse Dot Blot method for Genotyping of NAT2 in Chinese SubjectsThe principle of reverse dot blot (RDB) is similar to gene chip, and it can be used to detect many mutations simutaneously. The objective of this part is to establish a RDB method to detect five NAT2 mutations simultaneously. Bio-11-dUTP was added besides other reagents in a PCR system to obtain a biotin labeled NAT2 fragment. PolyT tailed allele specific oligonucleotide probes was obtained under terminal deoxynucleotidyl transferase. Probes conjugated to the nylon membrane by UV radiation. The labeled DNA fragment hybridized to the membrane and further conjugated to the horseradish peroxidase linked with streptavidin. Finally, a nonradioactive colorimetric reaction was used to detect five mutants of NAT2. NAT2 genotypes of 46 patients with pulmonary tuberculosis were detected with RDB. The results obtained from RDB were in consistent with those from allele specific amplification (ASA). The NAT2 allele frequencies of *5, *6, *7 were 1.09%, 20.6% and 17.4%, respectively. Homozygous wildtype, heterozygous mutant and homozygous mutant subjects were 37.0%, 47.8% and 15.2%, respectively. RDB method was proved to be accurate and convenient. On the basis of this study, we will further design RDB method to detect mutations of various enzymes and receptors simultaneously. We think RDB can be used in rational drug therapy.Part III. The influence of genotypes of NAT2 on the plasma concentration of isoniazid and its metabolitesIsoniazid (INH) is widely used in the treatment of tuberculosis. PM of the NAT2 is an important reason of inter-individual difference of the plasma concentration of INH and its metabolite acetyl-isoniazid (AcINH). The purpose of this part is to determine plasma concentration of INH and AcINH in pulmonary tuberculosis patients by high performance liquid chromatograph (HPLC). The relationship of NAT2 genotype and concentration will be studied.1. The establishment of HPLC method20% trichloroacetic acid was added to plasma to precipitate protein. Supernatant was divided in two parts: one part was added 20 (l 6mol/L HCl and incubated at 80(C for 1 hour. The other part was added 20(l water. Each of the... |
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