A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia Wanjun Zhou, Ge Wang, Xuefeng.

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A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia Wanjun Zhou, Ge Wang, Xuefeng Zhao, Fu Xiong, Shaoxiong Zhou, Jianming Peng, Youming Cheng, Shun Xu, Xiangmin Xu The Journal of Molecular Diagnostics Volume 15, Issue 5, Pages 642-651 (September 2013) DOI: 10.1016/j.jmoldx.2013.05.007 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Diagram of the locations of quadruple qPCR amplicons designed for the assay and representative α-thalassemia deletions in the human α-globin gene cluster. The human α-globin gene cluster and the CREBBP gene at 16p13.3 are shown at the top. Blue and green bars indicate the extent of deletions; blank regions at the end of each bar, uncertain breakpoints. The common deletion types in southern China are green. Tested sites for target (ζ-, α2- , and α1-) and reference (CREBBP) genes are indicated by double vertical lines. Homologous regions in α2- and α1-globin genes (X, Y, and Z boxes) are at the bottom. Specific amplicons for α2- and α1-genes are in nonhomologous regions between the X and Y boxes. Chrom, chromosome. The Journal of Molecular Diagnostics 2013 15, 642-651DOI: (10.1016/j.jmoldx.2013.05.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Assessment of amplification efficiencies of target and reference genes. A: A plot of Cq versus gDNA quantity for PCR efficiency analysis. The formula in the upper right corner shows the efficiencies of the four PCRs were all near 100% for gDNA quantities ranging from 50 to 1400 ng. B: A plot of ΔCq versus gDNA quantity for amplification consistency analysis. The ΔCq values for α1, α2, and ζ for each gDNA quantity were calculated using the equations ΔCq(α1) = Cq(α1) − Cq(CREBBP), ΔCq(α2) = Cq(α2) − Cq(CREBBP), and ΔCq(ζ) = Cq(ζ) − Cq(CREBBP). The absolute values of the three slopes were close to 0, indicating similar efficiencies for the target and reference genes. The Journal of Molecular Diagnostics 2013 15, 642-651DOI: (10.1016/j.jmoldx.2013.05.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The 2−ΔΔCq values for ζ-, α2-, and α1-globin gene copy number quantitative determination (n = 858). Left panel, ζ-globin; middle panel, α2-globin; and right panel, α1-globin gene copy number quantifications. The ordinate is the 2−ΔΔCq value, and the abscissa is the gene copy number (gene dosage). The box is the average 2−ΔΔCq value with SD. The black line in the box indicates the average value. Vertical lines in boxes are maximum and minimum 2−ΔΔCq values. The Journal of Molecular Diagnostics 2013 15, 642-651DOI: (10.1016/j.jmoldx.2013.05.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Identification of two novel gross deletions by MLPA A: An MLPA profile of patient 1 showing an approximately 6.4-kb deletion removing the entire α1- gene and a 5′ section of the HBQ1 gene. B: An MLPA profile of patient 2 showing the large deletion removing the entire α-globin gene cluster. A and B, top graphs: Vertical black bars are relative dosage of the probing fragment; C, calibrator probes with a relative dosage of two copies. The α-globin gene cluster is below. Down arrows on the gene cluster show probe positions. Horizontal black bars are the deletion range of samples characterized in this study. The Journal of Molecular Diagnostics 2013 15, 642-651DOI: (10.1016/j.jmoldx.2013.05.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions