A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and β-Thalassemia Carriers
A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and β-Thalassemia Carriers
复制标题
DOI：
10.1016/j.jmoldx.2020.05.004
复制
发表时间：
2020-08-01
影响因子：
4.1
通讯作者：
Cao, Hua
中科院分区：
文献类型：
--
作者：
Xu, Liangpu;Mao, Aiping;Cao, Hua
目前需要多种分子检测来准确检测地中海贫血的携带者。因此，长分子测序 (LMS) 被评估为 PacBio Sequel 平台上用于对 α 地中海贫血或 β 地中海贫血携带者进行基因分型的替代方案。使用多重长 PCR 生成 α (HBA1/2) 和 β (HBB) 基因位点的代表性扩增子。 LMS 之后，循环一致性测序读数与 hg19 参考基因组和使用 FreeeBayes 软件版本 1.2.0 调用的变体进行比对。在对 64 个已知携带者样本的盲法研究中，LMS 检测到的所有 HBA1/2 和 HBB 变异均与靶向 PCR 检测独立分配的变异一致。对于 HBA1/2 载体样本，LMS 准确检测到常见的东南亚、-α 3.7 和 -α 4.2 缺失以及四种不同的罕见单核苷酸变异 (SNV)。对于HBB携带者样本，LMS准确检测到最常见的中国插入和缺失变异c.126_129delCTTT和14种不同的SNV/插入和缺失，并且可以区分复合杂合SNV（反式配置）并识别与良性SNP（顺式配置）相关的变异。总体而言，LMS 显示了可扩展、准确且经济高效的基因分型方法的标志。随着进一步的测试覆盖范围还包括检测其他临床上显着的 HBA1/2 拷贝数变异，例如泰国、地中海和菲律宾缺失，LMS 最终可能成为大规模地中海贫血携带者筛查的综合方法。
Multiple molecular tests are currently needed for accurate carrier testing for thalassemia. Therefore, longmolecule sequencing (LMS) was evaluated as an alternate on the PacBio Sequel platform for genotyping carriers of alpha-thalassemia or beta-thalassemia. Multiplex long PCR was used to generate representative amplicons for the alpha (HBA1/2) and beta (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using FreeBayes software version 1.2.0. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common South East Asian, -alpha 3.7, and -alpha 4.2 deletions and four different rare single-nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the most common Chinese insertion and deletion variant c.126_129delCTTT and 14 different SNVs/insertions and deletions and could discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate, and cost-effective genotyping method. With further test coverage to additionally include detection of other clinically significant HBA1/2 copy number variations, such as the Thai, Mediterranean, and Filipino deletions, LMS may eventually serve as a comprehensive method for large-scale thalassemia carrier screening.