Liu Hui, Du Yepei, Yang Yanting, Cui Di, Chen Libao, Zhou Cong, Wang Jing
Department of Medical Genetics/Prenatal Diagnostic Center, West China Second University Hospital, Sichuan University, Chengdu, China.
Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu, China.
Ann Hematol. 2025 Jul 12. doi: 10.1007/s00277-025-06488-7.
Various methods are available to detect common deletions and mutations of genes related to thalassemia, including gap-polymerase chain reaction (Gap-PCR), next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and quantitative real-time polymerase chain reaction (qRT-PCR). Unequal crossover during the recombination of α1 and α2 hemoglobin can be detected but hardly accurately defined by above-mentioned technologies. A couple with abnormal hematological test results arrived at our department for genetic consultation. Preliminary analysis using NGS revealed a 3.7 kb (chr16:223462-227311) heterozygote deletion in the wife and nearly six copies in the chr16:223462-227311 (GRCh37/hg19) region of the husband. Further Gap-PCR results for the wife were consistent with the NGS results. MLPA and qRT-PCR were performed to detect the potential extra copies of the α-globin gene of the husband. The analyses simultaneously showed that the copy numbers of HBA1/HBA2 genes were nearly six. A specialized primer of the α-globin gene was designed to elucidate the structure of the 4.2 or 3.7 kb repeats of the husband. Third-generation sequencing (TGS) revealed the existence of the four extra tandem duplications of 3.7 kb in DNA strand 1 (chr16:173302-177106, hg38) of the α-globin gene and the existence of a heterozygous c.301-31_301-24delinsG insertion and deletion (InDel) in DNA strand 2 in the HBA1 gene (chr16:177252-177259, hg38). These results were confirmed by Sanger sequencing. Integrative Genomics Viewer analysis of the BAM files of NGS detected a low-level (reference/alternative, 0.19%) heterozygous InDel (c.301-31_301-24delinsG) in HBA1. Compared to traditional methods, TGS was better able to detect variants accurately and find rare genotypes and rearrangements of the α-globin gene cluster.
有多种方法可用于检测与地中海贫血相关基因的常见缺失和突变，包括缺口聚合酶链反应（Gap-PCR）、下一代测序（NGS）、多重连接依赖探针扩增（MLPA）和定量实时聚合酶链反应（qRT-PCR）。α1和α2血红蛋白重组过程中的不等交换可以被检测到，但上述技术很难准确界定。一对血液学检查结果异常的夫妇来到我们科室进行遗传咨询。使用NGS进行的初步分析显示，妻子存在一个3.7 kb（chr16:223462-227311）杂合缺失，而丈夫chr16:223462-227311（GRCh37/hg19）区域有近六个拷贝。对妻子进一步的Gap-PCR结果与NGS结果一致。进行了MLPA和qRT-PCR以检测丈夫α珠蛋白基因潜在的额外拷贝。分析同时显示HBA1/HBA2基因的拷贝数接近六个。设计了α珠蛋白基因的特异性引物以阐明丈夫4.2或3.7 kb重复序列的结构。第三代测序（TGS）显示，α珠蛋白基因的DNA链1（chr16:173302-177106，hg38）存在四个3.7 kb的额外串联重复，HBA1基因的DNA链2（chr16:177252-177259，hg38）存在一个杂合的c.301-31_301-24delinsG插入缺失（InDel）。这些结果通过桑格测序得到证实。对NGS的BAM文件进行综合基因组浏览器分析，检测到HBA1中存在一个低水平（参考/变异，0.19%）的杂合InDel（c.301-31_301-24delinsG）。与传统方法相比，TGS能够更好地准确检测变异，并发现α珠蛋白基因簇的罕见基因型和重排。
