摘要
目的探讨深圳地区汉族人中NAT2基因分布特征,为开展NAT2基因与肿瘤相关性研究和药物代谢性疾病诊治提供依据。方法将DNA浓度为40ng/μl的标本进行1×10^0、1×10^1×10^2、1×10^3、1×104倍比梯度稀释,以验证实时荧光定量PCR检测NAT2基因的灵敏度。采用实时荧光定量PCR结合TaqMan探针技术检测554名深圳汉族健康人NAT2基因282、341、481、590和857突变位点,并进行基因分型。同时用直接测序法对47名健康人标本进行平行检测,以验证和评价荧光定量PCR检测NAT2基因的敏感度、特异度和准确性。结果经倍比梯度稀释试验验证实时荧光定量PCR检测NAT2基因的灵敏度可精确至10^-4ng/μl。采用实时荧光定量PCR从554名深圳汉族人中检出NAT2^*4、*5、*6、*7、*11等位基因频率分别为64.6%(358/554)、6.3%(35/554)、25.3%(140/554)、30.0%(166/554)、0.6%(3/554)。主要基因型NAT2*4/*6、*6/*7、*13/*13或*12/*12或*4/*4、*4/*7、*7/*13、*12、*6/*13(*12)频率依次为12.3%(68/554)、8.3%(46/554)、7.6%(42/554)、40.5%(224/554)、8.1%(45/554)、7.2%(40/554)、5.6%(31/554),7种基因型约占总基因型的89.6%(496/554)。深圳地区汉族人中NAT2基因主要等位基因为*4、*6、*7、*13,表型以快乙酰化型和中间型为主,分别占40.5%(224/554)、46.7%(259/554),慢乙酰化型仅占12.8%(71/554)。用荧光定量PCR和直接测序法平行检测47名健康人NAT2基因,与直接测序法比较,检测282、341、481、590、857位点的敏感度分别为88.2%(30/34)、87.5%(7/8)、80.0%(4/5)、100.0%(22/22)、93.8%(15/16),特异度分别为100.0%(13/13)、94.9%(37/39)、100.0%(42/42)、96.0%(24/25)、96.8%(30/31),准确性分别为91.5%(43/47)、93.6%(44/47)、97.6%(46/47)、97.9%(46/47)、95.7%(45/47)。结论实时荧光定量PCR检测NAT2基因的灵敏度、敏感度、特异度高,适用于临床及科学研究,深圳地区汉族人主要NAT2等位基因是*4、*6、*7,最常见慢乙酰化表型为*6/*7,这为与NAT2基因相关的代谢性疾病及肿瘤研究提供了数据。
Objective To explore and analyze the distribution of NAT2 gene in Han population in Shenzhen and provide the basis for treatment of NAT2-related metabolic diseases and cancer research. Method The study diluted the DNA sample(40 ng/μl) as follows : 1 × 10^0, 1× 10^1 , 1 × 10^2, 1 × 10^3 , 1 × 10^4, to verify the sensitivity of detection of NAT2 gene polymorphism by real-time PCR. Mutation locus of 282, 341, 481, 590 and 857 of NAT2 in 554 normal samples were detected and genotyped by realtime PCR with Taqman probes. Forty-seven cases among 554 healthy controls were detected by DNA sequencing to verify the sensitivity, specificity and accuracy of this assay. Results The lowest detection limit was 10 -4 ng/p,1. The frequencies of each allele of NAT2 were : * 4 allele 64. 6% (358/554) , * 5 allele 6. 3% (35/554), *6 allele 25.3% ( 140/554), *7 allele 30. 0% ( 166/554), * 11 allele 0. 6% (3/554) . The frequencies of main genotypes of NAT2 were as the follows : NAT2 * 4/* 6 12. 3% ( 68/554 ), * 6/* 7 8.3% (46/554), *13/*13 or *12/*12or *4/*4 7. 6% (42/554), *4/*7 8. 1% (45/554), *7/*13 12. 3% (68/554), * 12 7. 2% (40/554), *6/* 13( * 12)5. 6% (31/554). The samples with 7 genotypes account for 89. 6% (496/554) of the total cases. Rapid acetylation and Intermediate type accounting for 40.5 % (224/554) and 46.7 % (259/554) respectively were the main phenotypes of NAT2 in Han people in Shenzhen. In addition, the accuracy, specificity and sensitivity of the PCR were compared to direct DNA sequencing. The accuracy of 282, 341, 481, 590 and 857 were 88.2% (30/34), 87.5% (7/8), 80. 0% (4/5), 100. 0% ( 22/22 ) and 93.8% ( 15/16 ) respectively. The specificities were 100. 0% ( 13/13 ), 94. 9% (37/39), 1013. 0% ( 42/42 ), 96.0% ( 24/25 ) and 96. 8% ( 30/31 ) respectively. The sensitivities were 91.5% ( 43/47 ), 93.6% ( 44/47 ), 97.6% ( 46/47 ) 97.9% ( 46/47 ) and 95.7% ( 45/47 ), respectively. Conclusions The sensitivity, specificity and accuracy of real-time PCR established in this study is good. It can be widely used for clinical and scientific research. The major alleles of NAT2 of Han population in Shenzhen are " 4, * 6, * 7. The most common slow acetylation genotype is * 6/* 7. This study can provide a basis for NAT2-related metabolic diseases and cancer research.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2011年第9期842-848,共7页
Chinese Journal of Laboratory Medicine
关键词
芳基胺N-乙酰转移酶
多态性
单核苷酸
聚合酶链反应
Arylamine N-acetyltransferase
Polymorphism, single nucleotide
Polymerase chain reaction