Ou-Yang H, Hua L, Mo Q H, Xu X M
Department of Medical Genetics, First Military Medical University, Guangzhou 510515, Guangdong, PR China.
J Clin Pathol. 2004 Feb;57(2):159-63. doi: 10.1136/jcp.2003.011130.
To develop an alternative assay for specific genotyping of the -alpha(4.2) thalassaemia deletion based on the DNA sequence features surrounding the breakpoint.
The 5' and 3' ends of the breakpoint regions of the -alpha(4.2) allele and the normal homologous segments were sequenced in Chinese individuals. A sequence haplotype composed of four single nucleotide variations within the X2/X1 box of the -alpha(4.2) breakpoint region was found in all of the 10 Chinese -alpha(4.2) thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the -alpha(4.2) allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the alpha globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system.
The three major genotypes (-alpha4.2/alphaalpha, -alpha(4.2)/--SEA, and alphaalpha/alphaalpha) could be distinguished through the characteristic chromatograms generated by the WAVE system. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterised by Southern blotting or Gap-PCR.
This study validates the PCR/DHPLC approach as a simple, rapid, highly accurate, and cost effective method, potentially adaptable for use in epidemiological surveys, genetic screening, and diagnosis of silent alpha+ thalassaemia and Hb H disease.
基于断点周围的DNA序列特征，开发一种用于-α(4.2)地中海贫血缺失特异性基因分型的替代检测方法。
对中国个体的-α(4.2)等位基因断点区域的5'和3'端以及正常同源片段进行测序。在所研究的10个中国-α(4.2)地中海贫血等位基因中，均发现了由-α(4.2)断点区域的X2/X1框内四个单核苷酸变异组成的序列单倍型。基于这些发现，开发了一种新型聚合酶链反应（PCR）/变性高效液相色谱（DHPLC）检测方法，用于-α(4.2)等位基因的快速基因分型，以替代传统的Southern印迹法或缺口PCR。该方法包括扩增包含这四个多态性位点的α珠蛋白靶序列，然后使用转基因WAVE DNA片段分析系统进行部分变性HPLC分析。
通过WAVE系统产生的特征色谱图可区分三种主要基因型（-α4.2/αα、-α(4.2)/--SEA和αα/αα）。对该技术的准确性进行了盲法评估，结果与先前通过Southern印迹法或缺口PCR鉴定的基因型100%（40/40）一致。
本研究验证了PCR/DHPLC方法是一种简单、快速、高度准确且经济高效的方法，可能适用于流行病学调查、基因筛查以及静止型α+地中海贫血和Hb H病的诊断。
