中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (44): 8308-8312.doi: 10.3969/j.issn.1673-8225.2011.44.035

• 移植与基因 transplantation and gene • 上一篇    下一篇

应用完整外显子2/3序列解决HLA-DRB1座位基因分型的歧义结果

李  桢1，杨  鹃2，程良红1，邹红岩1   

  1. 1深圳市血液中心免疫遗传研究室，广东省深圳市  518035
    2大连医科大学检验医学系，辽宁省大连市 116024
  • 收稿日期:2011-04-15 修回日期:2011-07-19 出版日期:2011-10-29 发布日期:2011-10-29
  • 作者简介:李桢，女，1969年生，北京市人，汉族，1993年兰州医学院毕业，主任技师，主要从事免疫遗传学研究。 sontony@yahoo.cn
  • 基金资助:

    深圳市科技计划项目(200902120)，课题名称：获得HLA-DRB1基因外显子2和3完整序列的测序新策略。

Application of complete exon2/3 sequence to resolve ambiguous alleles of HLA-DRB1 locus

Li Zhen1, Yang Juan2, Cheng Liang-hong1, Zou Hong-yan1   

  1. 1Immunogentics and Histocompatibility Testing Laboratory, Shenzhen Blood Center, Shenzhen  518035, Guangdong Province, China
    2Department of Laboratory Medicine, Dalian Medical University, Dalian  116044, Liaoning Province, China
  • Received:2011-04-15 Revised:2011-07-19 Online:2011-10-29 Published:2011-10-29
  • About author:Li Zhen, Chief technician, Immunogentics and Histocompatibility Testing Laboratory, Shenzhen Blood Center, Shenzhen 518035, Guangdong Province, China sontony@yahoo.cn
  • Supported by:

    Science and Technology Plan Program of Shenzhen City, No. 200902120*

PDF

424

被引次数

6

摘要：

背景：人类白细胞抗原基因测序分型中，当等位基因的差异碱基位于测序范围之外或不同等位基因对的杂合序列相同时，无法得到清晰的结果。
目的：通过完整外显子2/3序列的测定，解决常规HLA-DRB1基因分型中的高比例歧义结果。
方法：初次分型采用常规的测序方法检测320份样本的HLA-DRB1外显子2第一高变区以外的序列，测序反应设置codon86。后期采用一次性扩增外显子2/3，测序反应针对外显子2设置组特异性引物：DRB1*04/07/09为一组，其它基因家族为一组，设置conden86，对初次分型后为歧义结果的样本重新分型。
结果与结论：初次分型有180份样本为歧义结果，占总样本数的56.25 %。其中A类为差异碱基位于测序范围之外，共114例；B类为等位基因对的杂合序列相同，共17例；C类为两种情况同时存在，有49例。3种类别的歧义等位基因数分别为119个、34个、98个，占等位基因总数的33.06%、9.44 %、27.22%。完整外显子2/3序列的测定使歧义结果比例从56.25%下降到14.37%，其中A类103例、B类8例、C类23例样本的等位基因得到确认。此次研究中发现了一个新等位基因，与跟它最相近的等位基因DRB1*110101相比，其外显子3的第381位碱基G>T，导致第98位氨基酸AAG(赖氨酸 Lys)>AAT(天冬酰氨 Asn)。序列已提交Genbank，编号HM807583，2010-08被世界卫生组织HLA因子命名委员会命名为HLA-DRB1*1197(编号HWS10010999)。提示，完整外显子2/3序列的测定能大幅降低歧义分型结果的比例。

关键词: 人类白细胞抗原, 聚合酶链式反应-基因测序分型, 外显子, 歧义等位基因, 组序列特异性引物

Abstract:

BACKGROUND: Some alleles could not be confirmed when different bases located outside the sequencing region or allele pairs have same heterozygous sequence during the human leukocyte antigen genes genotyped by sequencing method.
OBJECTIVE: To resolve a high proportion of ambiguous genotyping results of HLA-DRB1 locus through complete exon2/3 sequence determination.
METHODS: Using the routine sequence-based typing which only sequences the 124-360 nucleotide of exon2 for the first typing of 320 samples, the sequence reaction included codon 86. Then the ambiguous samples were determined by complete exon2/3. The method designed group-specific primers on exon2 (DRB1*04/07/09 as a group and other gene family as a group, conden86). Ambiguity and the confirm results were calculated by direct counting method, and calculation was performed twice for the homozygotes.
RESULTS AND CONCLUSION: 180 samples of the initial typing were ambiguous results, accounting for 56.25% of the total number of samples. “A” means the ambiguity result which is caused by the different sequences located outside the sequencing region in 114 cases, accounting for 63.33% of the total ambiguous results. “B” means the ambiguity result which is caused by the same heterozygous sequence in 17cases, accounting for 9.44% of the total ambiguous results. “C” means the two types simultaneously existing in 49 cases, accounting for 27.22% of the total ambiguous results. The number of ambiguous alleles was 119, 34 and 98 in three types, accounting for 33.06% (119/360), 9.44% (34/360), and 27.22% (98/360) of ambiguous samples, respectively. Complete exon2/3 sequencing made the ratio of the ambiguous results decrease from 56.25% to 14.37%, of which 103 cases of “A”, 8 cases of “B”, 23 cases of “C” were confirmed. A novel allele was discovered, and its sequences were identical to DRB1*110101 except for a single nucleotide substitution at nt381 where G>T, codon98 Lys (AAG)> Asn (AAT). The sequence was submitted to Genbank and the accession number was HM807583. The name HLA-DRB1*1197 had been officially assigned by the WHO Nomenclature Committee in August 2010(Accession Number HWS10010999). These showed that the complete exon2/3 of sequence analysis can significantly reduce the proportion of ambiguous typing results.

中图分类号: