等位基因特异荧光PCR检测ALDH2基因多态性

doi:10.3969/j.issn.1673-8640.2017.04.005

摘要/Abstract

摘要：

目的了解人群乙醛脱氢酶2（ALDH2）基因多态性的分布,评价等位基因特异荧光聚合酶链反应（PCR）检测ALDH2基因应用于临床的可行性。方法采用等位基因特异荧光PCR和DNA测序法同时检测375例患者外周血ALDH2基因型,结果不一致的标本采用基因芯片技术进行复检。结果 375例标本中,荧光PCR检出ALDH2* 1/ 1型248例（66.1%）、ALDH2*1/ 2型107例（28.5%）、ALDH2* 2/ 2型20例（5.4%）,而DNA测序法则分别检出246例（65.6%）、108例（28.8%）和21例（5.6%）,2种方法检测ALDH2基因型差异无统计学意义（χ²=1.33,P=0.392）,且一致性较好（κ=0.978,P<0.001）。2种方法基因型检测结果一致率为 98.9%（371/375）。4例结果不一致的标本经基因芯片法复检,结果与等位基因特异荧光PCR一致。结论等位基因特异荧光PCR检测ALDH2基因型简便、可靠,能满足临床对ALDH2基因型检测的要求。

关键词: 乙醛脱氢酶2, 基因多态性, 等位基因特异荧光聚合酶链反应

Abstract:

Objective To investigate the distribution of acetaldehyde dehydrogenase 2 （ALDH2） gene polymorphism,and to evaluate the feasibility of allele specific fluorescence polymerase chain reaction （PCR）for ALDH2 gene polymorphism. Methods Allele specific fluorescence PCR and DNA sequencing were used for ALDH2 genotyping in peripheral blood of 375 patients. Gene chip method was adopted to determine the specimens with inconsistent results by the 2 methods. Results Among the 375 specimens,248 cases （66.1%） of ALDH2*1/ 1,107 cases （28.5%） of ALDH2*1/ 2 and 20 cases （5.4%） of ALDH2*2/ 2 were determined by allele specific fluorescence PCR,while 246 cases （65.6%） of ALDH2*1/ 1,108 cases （28.8%） of ALDH2*1/ 2 and 21 cases （5.6%） of ALDH2*2/ 2 were determined by DNA sequencing,respectively. There was no statistical significance for ALDH2 genotyping between allele specific fluorescence PCR and DNA sequencing （χ²=1.33,P=0.392）,with good consistency（κ=0.978,P<0.001）. The coincidence rate was 98.9% （371/375）between the 2 methods. For 4 cases with inconsistent results,the results of gene chip method was in agreement with those by allele specific fluorescence PCR. Conclusions Allele specific fluorescence PCR for ALDH2 genotyping is feasible,and could meet the requirements of clinical ALDH2 gene determination.

Key words: Acetaldehyde dehydrogenase 2, Gene polymorphism, Allele specific fluorescence polymerase chain reaction

中图分类号:

R446.1

孙宇晶, 线海鹏, 刘向祎, 刘畅, 龙彦, 孙媛媛, 赵晓涛. 等位基因特异荧光PCR检测ALDH2基因多态性[J]. 检验医学, 2017, 32(4): 267-271.

SUN Yujing, XIAN Haipeng, LIU Xiangyi, LIU Chang, LONG Yan, SUN Yuanyuan, ZHAO Xiaotao. Polymorphism of ALDH2 gene with allele specific fluorescence polymerase chain reaction[J]. Laboratory Medicine, 2017, 32(4): 267-271.

图/表 5

图1 ALDH2 1510位核苷酸G或A多态性测序图

图2 ALDH2 1510位核苷酸G或A多态性荧光PCR结果

表1 real-time ASA PCR和DNA测序法检测ALDH2 基因型的比较

方法		DNA测序法			合计
方法		1/1	1/2	2/2	合计
real-time	1/1	245	2	1	248
ASA PCR	1/2	1	106	0	107
	2/2	0	0	20	20
合计		246	108	21	375

图3 ALDH2 1510位核苷酸G或A多态性基因芯片结果图

表2 基因芯片法检测real-time ASA PCR与DNA测序法结果不一致的标本

编号	性别	real-time ASA PCR	DNA测序法	基因芯片法
138	女	1/1	2/2	1/1
283	女	1/1	1/2	1/1
285	男	1/2	1/1	1/2
286	女	1/1	1/2	1/1

参考文献 15

[1]	NOSE J,SAITO A,KAMATANI N.Statistical analysis of the associations between polymorphisms within aldehyde dehydrogenase 2 (ALDH2),and quantitative and qualitative traits extracted from a large-scale database of Japanese single-nucleotide polymorphisms (SNPs)[J]. J Hum Genet,2008,53(5):425-438.
[2]	GOEDDE H W,AGARWAL D P,HARADA S,et al.Population genetic studies on aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity[J]. Am J Hum Genet,1983,35(4):769-772.
[3]	CHEN Z,FOSTER M W,ZHANG J,et al.An essential role for mitochondrial aldehyde dehy-drogenase in nitroglycerin bioactivation[J]. Proc Natl Acad Sci U S A,2005,102(34):12159-12164.
[4]	LI Y,ZHANG D,JIN W,et al.Mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys polymorphism contributes to the variation in efficacy of sublingual nitroglycerin[J]. J Clin Invest,2006,116(2):506-511.
[5]	CUI R,KAMATANI Y,TAKAHASHI A,et al.Functional variants in ADH1B and ALDH2 coupled with alcohol and smoking synergistically enhance esophageal cancer risk[J]. Gastroenterology,2009,137(5):1768-1775.
[6]	YOKOYAMA A,YOKOYAMA T,OMORI T.Past and current tendency for facial flushing after a small dose of alcohol is a marker for increased risk of upper aerodigestive tract cancer in Japanese drinkers[J]. Cancer Sci,2010,101(11):2497-2498.
[7]	PARK J Y,MATSUO K,SUZUKI T,et al.Impact of smoking on lung cancer risk is stronger in those with the homozygous aldehyde dehydrogenase 2 null allele in a Japanese population[J]. Carcinogenesis,2010,31(4):660-665.
[8]	GUO L K,ZHANG C X,GUO X F.Association of genetic polymorphisms of aldehyde dehy-drogenase-2 and cytochrome P450 2E1-RsaI and alcohol consumption with oral squamous cell carcinoma[J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2012,34(4):390-395.
[9]	CAO H X,LI S P,WU J Z,et al.Alcohol dehydrogenase-2 and aldehyde dehydrogenase-2 genotypes,alcohol drinking and the risk for stomach cancer in Chinese males[J]. Asian Pac J Cancer Prev,2010,11(4):1073-1077.
[10]	BERNARD P S,LAY M J,WITTWER C T.Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves[J]. Anal Biochem,1998,255(1):101-107.
[11]	李志祥,谢桂岚. ALDH2基因第12外显子(G/A)多态性在北京汉族人群的检测[J]. 中国现代医学杂志,2005,15(3):457-458.
[12]	YARDE F,MAAS A,FRANX A,et al.Serum AMH levels in women with a history of preeclampsia suggest a role for vascular factors in ovarian aging[J]. J Clin Endocrinol Metab,2013,99(2):579-586.
[13]	袁青,薛亚东,郑雅萍,等. 乙醛脱氢酶 2 基因多态性及生活习惯与胃癌易感性的相关性分析[J]. 检验医学,2016,31(7):584-587.
[14]	LI Y,ZHANG D,JIN W,et al.Mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys polymorphism contributes to the variation in efficacy of sublingual nitroglycerin[J]. J Clin Invest,2006,116(2):506-511.
[15]	MIZUNO Y,HOKIMOTO S,HARADA E,et al.Variant aldehyde dehydrogenase 2 (ALDH2*2) is a risk factor for coronary spasm and ST-segment elevation myocardial infarction[J]. J Am Heart Assoc,2016,5(5):e003247.