Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.
Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing.
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DOI：
10.21037/atm-22-4309
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发表时间：
2022-10
影响因子：
--
通讯作者：
Zhu, Chunjiang
中科院分区：
文献类型：
--
作者：
Li, Jun;Ye, Guimei;Zeng, Dan;Tian, Baodong;Wang, Wenjuan;Feng, Qiao;Zhu, Chunjiang
Hong Kongαα (HKαα) 等位基因是 α-珠蛋白基因的复杂结构重排，包含 -α3.7 和 αααanti 4.2 交叉连接。临床上，使用传统的地中海贫血基因检测技术经常会误诊或漏诊携带HKαα等位基因的个体。本研究旨在利用第三代测序（TGS）技术识别和验证不同的 HKαα 地中海贫血亚型。 2015年1月至2021年6月期间，32名疑似患有HKαα地中海贫血的患者被纳入本研究。提取基因组DNA，采用间隙聚合酶链式反应（PCR）、两轮巢式PCR、多重连接依赖性探针扩增（MLPA）和TGS进行地中海贫血基因检测。使用gap-PCR方法，HKαα/αα和HKαα/-α3.7的结果与-α3.7/αα相似。两轮巢式PCR可验证HKαα基因，但无法区分HKαα地中海贫血的亚型。采用MLPA法检测α珠蛋白基因拷贝数的变化，但无法判断-α3.7和αααanti 4.2是顺式还是反式。长读长TGS技术可准确检测HKαα等位基因，无需进行谱系分析即可区分HKαα/αα、HKαα/-α3.7、HKαα/-α4.2、HKαα/--SEA的基因型。使用 TGS 方法检测 HKαα 等位基因的连续序列。这项研究还表明，具有 HKαα/αα 和 βN/βN 基因型的个体往往具有正常的血液学表型。长读长TGS是准确检测HKαα地中海贫血的可靠、高效的方法，可广泛应用于临床实践。 HKαα地中海贫血的准确分子诊断将有利于临床遗传咨询和产前诊断。
The Hong Kongαα (HKαα) allele is a complex structural rearrangement of the α-globin gene containing -α3.7 and αααanti 4.2 crossover junctions. Clinically, individuals carrying the HKαα allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HKαα thalassemia subtypes using third-generation sequencing (TGS) technology. Between January 2015 and June 2021, 32 patients suspected of having HKαα thalassemia were included in this study. Genomic DNA was extracted, and gap-polymerase chain reaction (PCR), two-round nested PCR, multiplex ligation-dependent probe amplification (MLPA), and TGS were used for thalassemia gene detection. The results of HKαα/αα and HKαα/-α3.7 were similar to -α3.7/αα using the gap-PCR method. Two-round nested PCR could be used to verify the HKαα gene, but could not distinguish the subtypes of HKαα thalassemia. The MLPA assay was used to detect the change in the copy number of the α-globin gene, but it could not determine whether -α3.7 and αααanti 4.2 were in cis or in trans. Long-read TGS technology could accurately detect the HKαα allele and distinguish the genotypes of HKαα/αα, HKαα/-α3.7, HKαα/-α4.2, and HKαα/--SEA without pedigree analysis. The contiguous sequence of the HKαα allele was detected using the TGS approach. This study also demonstrated that individuals with HKαα/αα and βN/βN genotypes tended to have normal hematological phenotypes. Long-read TGS is a reliable and efficient approach for accurate detection of HKαα thalassemia, which can be widely used in clinical practice. Accurate molecular diagnosis of HKαα thalassemia will benefit clinical genetic counseling and prenatal diagnosis.
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