摘要
目的对1例血型血清学检测为RHD变异型的样本进行基因分型,并进行家系调查分析。方法运用血型血清学检测方法对先证者及其家系样本进行RHD确认及RH血型抗原表位检测,用序列特异性引物PCR(sequence specific primer PCR,PCR-SSP)法分析RHD基因外显子的表达,运用Sanger和SMRT(single molecule real-timesequencing)测序法对先证者及其家系样本进行RHD基因的1~10外显子测序分析。通过AlphaFold模拟构建蛋白质三级结构,将突变前后的蛋白质结构进行叠合以观察结构内分子间相互作用力的改变。结果先证者为RHD弱表型,其他抗原为Ccee,直接抗人球蛋白试验、抗体筛查、抗体鉴定试验均为阴性,PCR-SSP初步分型为RHD阳性。其父母血型血清学及PCR-SSP结果均为RHD阳性。SMRT测序结果显示先证者母亲为RHD^(+)/RHD^(-)杂合子。Sanger测序结果显示,先证者父亲携带弱D型54等位基因。AlphaFold建模预测揭示,p.Ser122Leu突变不能与146位GLU形成氢键相互作用。结论该样本为弱D型54,p.Ser122Leu突变导致氨基酸内部分子间作用力发生改变,从而引起突变后蛋白质结构和功能的部分改变。
Objective To detect the genotype of a blood sample which was demonstrated as RHD variant by serological method and conduct the investigation and analysis for the pedigree.Methods The blood group serology assay was performed to confirm this RHD type and detect other D epitomes of RH blood group for the proband and the members of this family.Sequence specific primer-PCR(PCR-SSP)method was established to identify the exons of RHD gene to screen RHD preliminary.Sanger and single molecule real-time(SMRT)sequencing method were used to analyze the exons 1 to 10 of RHD gene in the smaples from proband and his family.The tertiary structure of the proteins was constructed by Alpha-fold simulation.The altered protein structures before and after mutation were overlapped to observe the changes of inter-molecular interaction within the protein structure.Results The proband showed RHD weak phenotype serologically,and other antigens of RH blood group were Ccee.The direct anti-human globulin test,antibody screening and antibody identification tests were all negative.The results of PCR-SSP indicated that his parents and all members in the family were RHD^(+).SMRT sequencing revealed that the proband′s mother was RHD^(+)/RHD^(-)heterozygote.The results of Sanger sequencing suggested that the proband′s father carried weak D type 54 allele.AlphaFold modeling prediction for the tertiary structure of protein,revealedthat no interaction between p.Ser122Leu mutation and hydrogen bond formed by GLU146.Conclusion The blood group of this sample was identified as weak D type 54.p.Ser122Leu mutation triggered the change of internal force among amino acid molecules and ultimately lead to partial alteration of structure and function in the protein after mutation.
作者
芮雪
张钰
蔡杰
许纪玲
傅强
何成涛
RUI Xue;ZHANG Yu;CAI Jie;XU Jiling;FU Qiang;HE Chengtao(Nanjing Red Cross Blood Center,Nanjing 210003,Jiangsu,China)
出处
《临床检验杂志》
CAS
2023年第3期186-190,共5页
Chinese Journal of Clinical Laboratory Science
基金
南京市医学科技发展项目(YKK21173)
江苏省输血协会爱康生物科研基金(JSAK2022004)。
关键词
弱D型
血型血清学
序列特异性引物PCR
外显子测序
家系研究
weak D phenotype
blood group serology
sequence specific primer-PCR
exon sequencing
pedigree analysis
