Accurate detection of α-globin gene copy number variants with two reactions using droplet digital PCR

• Published in: Hematology (Luxembourg) Vol. 27; no. 1; pp. 198 - 203
• Main Authors: Bao, Xiuqin, Qin, Danqing, Ma, Jian, Zhou, Xiangcheng, Wang, Jicheng, Yao, Cuize, Zhang, Liang, Du, Li
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 31.12.2022; Taylor & Francis Group
• Subjects: alpha-Globins - genetics; alpha-Thalassemia - genetics; beta-Thalassemia - genetics; Copy number variants; deletion; DNA Copy Number Variations; droplet digital PCR; Gene Dosage; Humans; Polymerase Chain Reaction - methods; triplication; α-globin gene; triplication; deletion; droplet digital PCR; α-globin gene; Copy number variants
• ISSN: 1607-8454; 1607-8454
• DOI: 10.1080/16078454.2022.2030885

The α-thalassemia is a highly prevalent disease in tropical and subtropical regions, including southern China, and is mainly caused by deletion in α-globin genes (HBA1 and HBA2). The clinical manifestation of α-thalassemia is highly correlated with the copy number of α-globin genes. The decrease in copy number results in α-thalassemia, while duplication or triplication compounded with β-thalassemia may aggravate the clinical manifestation. However, the common methods used to measure the copy number variants can only detect the three common types: - SEA , -α 3.7 , and -α 4.2 , and may easily miss the rare deletional type and duplication or triplication cases. Therefore, a new method that allows the detection of different copy number variants in α-globin genes simultaneously and accurately needs to be established. A total of 428 peripheral-blood and fetal chorionic villus or amniotic fluid samples were used in this study. We employed a pair of primers and two probes, one for HBA1 and another for HBA2, to perform droplet digital polymerase chain reaction (ddPCR). Each reaction needed the ddPCR of RPP30 as a reference gene to calculate the copy number. We accurately detected the copy number variants in α-globin genes, including the common form α-thalassemia, triplications such as ααα anti4.2 , and trisomy 16, by performing only two reactions. The accuracy rate for detecting the copy number of α-globin genes was up to 100%. In conclusion, ddPCR served as an accurate and rapid method for detecting copy number variations in the clinical screening for α-thalassemia.