Development And Application Of Quantitative Multiplex PCR Of Short Fluorescent Fragment Method For Detecting Of Alpha-globin Gene Deletions In Alpha-Thalassemia

Objective: α-Thalassemia is one of the most common autosomalrecessive inherited diseases. The main cause of this disease involvse thedeletion of the α-globin encoding genes (α1and α2). The diagnosis mainlyrelies on genetic analysis. Common methods like multiplex gap-PCR arelimited to the most common types of deletions. For investigating rare anduncharacterized large deletions, a new method is urgently needed. In thisstudy, we established a sensitive method of multiplex PCR for shortfluorescent fragment that can detect not only the presence of the commondeletions, but also the unusual or unknown α-globin gene deletions ofα-thalassemia. We also investigated its application in the diagnosis ofα-thalassemia.Methods:1. Based on the principle of Fluorescent Fragment analysis,and for the development of the method for α-globin gene deletions, we selected50persons with no deletion of α-globin gene as negative controls,50patients with α-globin gene heterozygous deletion and50with α-globingene homozygous deletion as positive controls of deletional α-thalassemia.The relative amounts of the α-globin gene dosage (Rα) were gained byintra-sample and inter-sample normalization.2. A total number of579samples from the individuals of Guangdongor Guangxi population were simultaneously analyzed using this assay,along with the multiplex Gap polymerase chain reaction (Gap-PCR, for themost common--SEA、-α3.7and-α4.2deletions in Chinese population). Theinconsistent results were further analyzed by multiplex ligation-dependentprobe amplification (MLPA) and followed-up analysis.Results:1.This method was capable of detecting α-globin genedeletions. The cut-off value of the Rαas showed by mean (SD, range) of thenegative controls of deletional α-thalassemia was1.00(0.14,0.77-1.31), andthe heterozygous deletion was0.60(0.07,0.40-0.75), the homozygousdeletion was0.00(0.00,00.0-0.00).2. Comparition between the results of the579subjects obtained bythis assay with multiplex Gap polymerase chain reaction showed: In572out of the579unrelated subjects, this newly method and multiplexGap-PCR gave consistent results, In7cases which were finally proved tobe composed of1rare deletion--Thai/-α3.7,1novel deletion--SEA/-α2.8,4αααanti3.7and1αααanti4.2triplications, this newly method showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to givethe correct diagnosis.Conclusions: This technique is reliable and precise in the detection ofrare and uncharacterized α-globin gene deletions as well as duplications inα-Thalassemia. It can be used as a α-thalassemia carrier screeningtechnique with the improvement of carrier detection and prenatal diagnosis,especially in those laboratories in which testing is restricted to a panel ofcommon deletions.