Development of bead-based suspension array technology for the diagnosis of thalassemia
Authors: Aihua Yin, Liang Zhang, Mingyong Luo, Tianwen He, Yan Zhang, Chang Liu, Li Du, Danqing Qin, Juqing Liang, Bing Li, Li Wu, Yuanzhu Ma, Hao Guo, Jicheng Wang, Tenglong Yuan, Yixia Wang, Yanxia Zhang, Xiaozhuang Zhang
Journal: American Journal of Hematology (2014)

To the Editor: Thalassemia is the most common recessive inherited single-gene disorders in the world, affecting approximately 5% of the world's population 1. Because of the high carrier rates in southern China and the limited treatments for thalassemia, carrier screening and prenatal diagnosis have been advocated by the Chinese government 2. However, few of routine diagnostic methods meet with the clinical diagnostic demands because of their limitations 3. Combining multiplex PCR and bead-based suspension array technology (BBSAT), we developed an assay to simultaneously detect 3 deletions of α-thalassemia, 3 point mutations of α-thalassemia, and 17 point mutations of β-thalassemia in a single assay within 5 hr. More than 200 previously genotyped DNA samples corresponding to 23 targeted mutations were detected by the BBSAT, to assess the performance of primers and probes, and to determine the cutoff values for mutation detection. The determination of cutoff values for point mutations utilizing the N/M ratios in combination with AUC calculation was established. The assessment results are partly shown in Figure 1. Moreover, the BBSAT was applied to screen thalassemia in 40,813 clinical samples collected from Guangdong Province to evaluate the clinical applicability of the new assay. We identified 6,774 samples (16.60%) carry thalassemia gene mutations, of which 4,963 samples (12.16%) carry α-thalassemia gene mutations, 1,549 samples (3.80%) carry β-thalassemia gene mutations, and 262 samples (0.64%) carry both α- and β-thalassemia gene mutations. To further validate the new assay, 2% of BBSAT detected mutated samples as well as 2% of wild type samples were randomly chose, genotyped by DNA sequencing and gap-PCR for point mutations and deletions, respectively. We found that the results of new BBSAT assay turned out to be fully consistent with those obtained by DNA sequencing and gap-PCR. The dot plots presented the specific signal ratios of wild type probe to mutation probe and also showed the mean ratio values with dash lines. A total of more than 200 clinical samples corresponding to 20 point mutations were detected by the BBSAT, to determine the cutoff values. In this study, the BBSAT technology was developed to diagnose point mutations and deletions in thalassemia genes in parallel. The BBSAT provided a simple and feasible detecting platform with distinct advantages. Firstly, this platform is theoretically capable of detecting up to 100 individual biomarkers in a single test, which significantly increases the detecting throughput 4. Secondly, the beads used in this platform are manufactured with biocompatible polymers, resulting in the highly stable chemical surface and superiorities of easy washing and separation. Thirdly, suspension array technology potentially avoids the majority of unsatisfactory conditions with solid microarray. Both probes and targets are mixed in the same liquid phase, and the hybridization dynamic becomes better, which significantly shorten hybridization time, increase reproducibility, and make it suitable for large scale production. Additionally, 23 mutations account for the majority (>98%) of thalassemia in Chinese population have been detected in the new assay, and the quantity of detection index can be easily expanded. Depending on the technical advantages of BBSAT, the new assay demonstrates outstanding extensibility for the requirement of various genetic mutations diagnosis. Furthermore, conventional molecular analysis of α- and β-thalassemia usually utilizes two or three kinds of commercial kits, and the costs of which are comparatively high. The BBSAT can detect point mutations and deletions in parallel for the diagnosis of thalassemia, and contribute to reduce the costs for 20%. The authors thank BioChain (Beijing) Science and Technology Inc. for technical assistance. Aihua Yin1,2,3, Liang Zhang2,4, Mingyong Luo1,2,3, Tianwen He1,2, Yan Zhang1,2,Chang Liu2,4, Li Du1,2, Danqing Qin1,2, Juqing Liang1,2, Bing Li5, Li Wu5,Yuanzhu Ma5, Hao Guo1,2, Jicheng Wang1,2, Tenglong Yuan1,2, Yixia Wang1,2,Yanxia Zhang1,2, and Xiaozhuang Zhang3,4* 1Medical Genetic Centre, Guangdong Women and Children Hospital, Guangzhou, Guangdong, People's Republic of China; 2 Maternal and Children Metabolic-Genetic Key Laboratory, Guangdong Women and Children Hospital, Guangzhou, Guangdong, People's Republic of China; 3 Guangdong Thalassemia Diagnostic Centre, Guangzhou, Guangdong, People's Republic of China; 4 Translational Medicine Research Center, Guangdong Women and Children Hospital, Guangzhou, Guangdong, People's Republic of China; 5 Department of Healthcare, Guangdong Women and Children Hospital, Guangzhou, Guangdong,10, People's Republic of China