HLA-B等位基因间重组产生新等位基因B^(*)35:186的序列分析和蛋白分子三维结构分析  

Sequence and protein three-dimensional structure analysis of a novel inter-allelic recombination allele B∗35∶186 of HLA-B locus

作　　者：章旭 林凤秋 李晓丰[1] 李剑平[1] ZHANG Xu;LIN Fengqiu;LI Xiaofeng;LI Jianping(Institute of Transfusion Medicine,Shenyang Central Blood Station(Liaoning Blood Center),Shenyang 110044,China)

机构地区：[1]沈阳中心血站/辽宁省血液中心输血医学研究所,辽宁沈阳110044

出　　处：《中国输血杂志》2025年第3期322-326,332,共6页Chinese Journal of Blood Transfusion

基　　金：沈阳市科技创新平台建设计划项目(21-104-0-15)。

摘　　要：目的 研究HLA-B等位基因座位上的重组交换,探讨HLA新等位基因产生的分子遗传机制,预测分析重组氨基酸残基改变对编码蛋白分子三维空间结构的影响。方法 应用基于Luminex平台的聚合酶链式反应-序列特异性寡核苷酸探针(polymerase chain reaction-sequence specific oligonucleotide probes, PCR-SSOP)方法对先证者进行HLA基因分型,采用直接DNA测序方法和基因克隆方法进行外显子1~4序列分析。将该基因序列经IMGT/HLA database中“Alignment”程序进行比对,以确定B等位基因重组发生的位置。用Swiss-Model软件进行同源建模后,采用Swiss Pdb Viewer软件对分子间三维结构进行模拟,FATCAT在线软件对分子间三维结构进行差异比对。结果 PCR-SSOP基因分型显示先证者HLA-B位点反应格局异常,疑为HLA新等位基因。基因克隆后测序结果显示,其中1个等位基因为B^(*)13∶02,另1个等位基因序列与所有已知HLA等位基因不同,与同源性最高的HLA-B^(*)35∶01∶01∶01基因序列相比在第2外显子c.259-c.299位置发生12个碱基替换,导致了8氨基酸改变。该序列外显子1、3、4及内含子1、2、3和外显子2在c.74-c.258序列与HLA-B^(*)35∶01∶01∶01序列一致,外显子2的c.259-c.343序列与HLA-B^(*)46∶01∶01序列完全一致。蛋白分子三维空间结构分析变异等位基因与B^(*)35∶01∶01∶01和B^(*)46∶01∶01结构相似,重组部位氨基酸p.63-p.79局部氢键发生改变。结论 本研究在中国汉族人群发现了1例B等位基因间重组形成的HLA新等位基因,被世界卫生组织HLA因子命名委员会正式命名为HLA-B^(*)35∶186,阐明了新等位基因产生的来源,为深入研究基因重组及HLA多态性形成机制提供了理论依据。Objective To study the inter-allelic recombination event occurring in the HLA-B locus,and to evaluate the molecular genetic mechanisms of a novel HLA allele,predict and analyze the impact of its amino acid residue changes on the three-dimensional structure.Methods HLA typing was taken with polymerase chain reaction-sequence specific oligonucle-otide probes(PCR-SSOP)by Luminex.Sequence-based typing(SBT)and gene clone were used to analyze exons 1-4 se-quences of HLA-B allele.In order to determine the exact site of inter-allelic recombination event occurring in the HLA-B lo-cus,sequences of the HLA-B alleles were compared with the IMGT/HLA database by the program“Alignment”.After ho-mology modeling using the Swiss-Model software,the three-dimensional structure of the molecules was simulated using the Swiss Pdb Viewer software,and the FATCAT online software was used to compare the differences in the three-dimensional structures of the molecules.Results HLA typing indicated the PCR-SSOP pattern did not match with any known HLA-B al-leles,suspected to be a new HLA allele.The genetic clone sequencing results showed HLA-B alleles of the proband were B^(*)13∶02 and a novel allele.The HLA-B exon2 nucleotide sequence of the novel allele was different from any other known al-leles.The novel allele has 12 nucleotides replaced when compared with the closest matching B^(*)35∶01∶01∶01 allele from c.259 to c.299,which result in 8 amino acids changes.The sequence was identical in B^(*)35∶01∶01∶01 in exon 1,exon 3,exon 4,intron 1,intron 2,intron 3 and at c.74 to c.258 in exon 2,and c.259 to c.343 sequence in exon 2 was identical in B^(*)46∶01∶01 by blast search.The structure of the mutant alleles was similar to that of B^(*)35∶01∶01∶01 and B^(*)46∶01∶01,and the local hydrogen bonds of amino acids p.63-p.79 were changed at the recombination site.Conclusion This study demonstrates a rare inter-allelic recombination event occurring in the HLA-B locus.It has been officially designated as HLA-B^(*)35∶18

关 键 词：HLA-B抗原 B~*35∶186 基因重组 序列分析 蛋白三维结构分析 

分 类 号：R446[医药卫生—诊断学]