A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia

• Published in: The Journal of molecular diagnostics : JMD Vol. 15; no. 5; pp. 642 - 651
• Main Authors: Zhou, Wanjun, Wang, Ge, Zhao, Xuefeng, Xiong, Fu, Zhou, Shaoxiong, Peng, Jianming, Cheng, Youming, Xu, Shun, Xu, Xiangmin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2013
• Subjects: alpha-Globins - genetics; alpha-Thalassemia - diagnosis; alpha-Thalassemia - genetics; Gene Dosage; Gene Order; Genotype; Humans; Mass Screening; Multilocus Sequence Typing - methods; Multiplex Polymerase Chain Reaction - methods; Pathology; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/j.jmoldx.2013.05.007

The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes ( HBA2 , HBA1 , and HBZ or HBPA1 ) versus a control gene ( CREBBP ). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2−ΔΔCq method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.