单分子实时测序在α珠蛋白基因三联体及其复合变异等位基因鉴定中的临床应用  

Clinical application of the single-molecule real-time technology for identification of triplicatedα-globin genes and compound variant alleles

作　　者：张宇[1,2] 方艳平[1] 朱碧青[1] 梁丽仪 周万军[3] 江凌晓[1] ZHANG Yu;FANG Yanping;ZHU Biqing;LIANG Liyi;ZHOU Wanjun;JIANG Lingxiao(Department of Laboratory Medicine,Zhujiang Hospital,Southern Medical University,Guangzhou,Guangdong 510282,China;Department of Laboratory Medicine,People′s Hospital of Gaoming district,Foshan,Guangdong 528500,China;Department of Medical Genetics,Southern Medical University,Guangzhou,Guangdong 510515,China)

机构地区：[1]南方医科大学珠江医院检验医学部,广东广州510282 [2]佛山市高明区人民医院检验科,广东佛山528500 [3]南方医科大学医学遗传学教研室,广东广州510515

出　　处：《国际检验医学杂志》2025年第1期32-37,43,共7页International Journal of Laboratory Medicine

基　　金：国家自然科学基金项目(81972008)。

摘　　要：目的探究单分子实时测序(SMRT)技术在α珠蛋白基因三联体(简称α三联体)及其复合变异等位基因鉴定中的临床应用效果。方法收集α三联体阳性样本36例,其中经PCR-导流杂交技术确认28例、经高通量测序(NGS)技术确认8例。36例样本包含α三联体复合变异顺式或反式排列未明样本ααα^(anti4.2)复合α^(CS)α2例,ααα^(anti4.2)复合-α^(3.7)10例,HKαα/--SEA型待确证2例,均采用SMRT技术进行地中海贫血(简称地贫)基因检测。另招募一个基因型为ααα^(anti4.2)复合-α^(3.7)变异病例的家系,包含先证者(Ⅱ-1)和其父亲(Ⅰ-1)、母亲(Ⅰ-2),采用PCR-导流杂交和SMRT技术进行地贫基因检测。结果SMRT技术检测结果显示,36例样本中共检出35例α三联体,1例αααα^(anti4.2)型α珠蛋白基因四联体(简称α四联体)。2例ααα^(anti4.2)复合α^(CS)α变异样本中ααα^(anti4.2)与α^(CS)α均为反式排列,基因型为ααα^(anti4.2)/α^(CS)α,10例ααα^(anti4.2)复合-α^(3.7)变异样本中ααα^(anti4.2)与-α^(3.7)为顺式排列样本9例,基因型为HKαα/αα,ααα^(anti4.2)与-α^(3.7)为反式排列样本1例,基因型为ααα^(anti4.2)/-α^(3.7)。相较PCR-导流杂交技术,SMRT技术多检出1例大片段缺失型β珠蛋白基因变异和2例未知变异,阳性检出率提高10.71%(3/28)。家系分析表明,先证者(Ⅱ-1)ααα^(anti4.2)和-α^(3.7)变异均遗传自母亲(Ⅰ-2),其基因型为HKαα/αα,与SMRT技术检测结果一致。结论SMRT技术不仅能够准确检测α三联体、α四联体及其复合变异等位基因,而且具有高准确性、一步到位识别2种变异顺式或反式排列、地贫基因变异覆盖全等优势,具有良好的临床应用价值。Objective To assess the clinical utility of single-molecule real-time technology(SMRT)in identifying triplicatedα-globin genes and compound variant alleles.Methods A total of 36 samples with triplicatedα-globin genes were collected.Among them,28 samples were confirmed by PCR flow-through hybridization and 8 samples were confirmed by Next Generation Sequencing(NGS).These 36 samples included triplicatedα-globin genes compound variants with cis or trans arrangements unknown,such asααα^(anti4.2)compoundα^(CS)α(2 cases),ααα^(anti4.2)compound-α^(3.7)(10 cases),and HKαα/--SEA pending confirmation(2 cases),SMRT technology was employed to detect thalassemia gene variants.Additionally,a pedigree with the genotype ofααα^(anti4.2)compound-α^(3.7)variant was recruited,including the proband(Ⅱ-1),its father(Ⅰ-1),and mother(Ⅰ-2).PCR fl ow-through hybridization and SMRT were employed to detect thalassemia gene variants.Results SMRT detected 35 out of 36 samples with triplicatedα-globin genes,and 1 sample with quadrupllcatedα-globin genes(αααα^(anti4.2)).Among the 2ααα^(anti4.2)compoundα^(CS)αvariant samples,bothααα^(anti4.2)andα^(CS)αwere arranged in trans,with a genotype ofααα^(anti4.2)/α^(CS)α.Among the 10ααα^(anti4.2)compound-α^(3.7)variant samples,9 samples hadααα^(anti4.2)and-α^(3.7)in a cis arrangement,with a genotype of HKαα/αα,and 1 sample hadααα^(anti4.2)and-α^(3.7)in a trans arrangement,with a genotype ofααα^(anti4.2)/-α^(3.7).Compared with PCR flow-through hybridization,SMRT detected one case of a large segment deletion in theβ-globin gene and two unknown variants,which led to an increase in the positive detection rate of approximately 10.71%(3/28).The pedigree analysis showed that the proband(Ⅱ-1)inheritedααα^(anti4.2)and-α^(3.7)variants from his mother(Ⅰ-2),with a genotype of HKαα/αα,consistent with the SMRT detection results.Conclusion SMRT can accurately detect triplicated or quadrupllcatedα-globin genes,and compound variant alleles.It o

关 键 词：α珠蛋白基因三联体 HKαα 单分子实时测序 PCR-导流杂交技术 高通量测序 

分 类 号：R556.61[医药卫生—血液循环系统疾病]