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

• Autores: Wanjun Zhou, Ge Wang, Xuefeng Zhao, Fu Xiong, Shaoxiong Zhou, Jianming Peng, Youming Cheng, Shun Xu, Xiangmin Xu
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 15, Nº. 5, 2013, págs. 642-651
• Idioma: inglés
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• Resumen

  • The predominant determinants of ?-thalassemia are deletions in the human ?-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 ?-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 ?-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-??Cq 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 ?-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.