单管多重PCR快速检测中国人三种常见缺失型α-地中海贫血基因

目的建立一种简单快速、准确可靠、经济实用的检测常见缺失型α-地中海贫血(α-地贫)突变(--^SEA、-α^3.7和-α^4.2)的单管多重PCR技术以用于α-地贫的分子筛查和产前诊断。方法采用gap—PCR方案设计4组PCR引物并对PCR反应条件进行系统优化以扩增代表这3种地贫和α2基因存在的特异性DNA片段。此外，还设计1对引物扩增LIS1基因3′-端非翻译区片段作为整个PCR体系扩增成功与否的内在对照。采用盲法分析对该技术的特异性、准确性和可靠性进行评价并应用于产前分子诊断中。结果所建的单管多重PCR技术成功地检出这3种常见缺失型α-地贫突变的纯合子、杂合子和双重杂合子。正常人出现LIS1和α2基因特异扩增片段，而--^SEA、-α^3.7和-α^4.2杂合子除了出现正常人的2条扩增带外，还分别出现1条指示这些突变存在的特异性扩增片段。盲法分析结果显示，该技术对α-珠蛋白基因型经过Southern印迹法或DNA直接测序确证的DNA标本的诊断准确性达到100％，并被成功地应用于9个重症α-地贫高风险家庭的产前诊断中。结论该技术可准确、快速地检测--^SEA、-α^3.7和-α^4.2 3种常见缺失型α-地贫突变，且具有经济和实用的优点，非常适合于α-地贫的大人群分子筛查和临床样品的基因诊断。

Rapid detection of three common deletional α-thalassemias in Chinese by single-tube multiplex PCR

OBJECTIVE: To develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population. METHODS: Four groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation. RESULTS: Homozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families. CONCLUSION: The single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics.