中国组织工程研究与临床康复 第 15 卷 第 44 期 2011–10–29 出版
Journal of Clinical Rehabilitative Tissue Engineering Research October 29, 2011 Vol.15, No.44
P.O. Box 1200, Shenyang 110004 cn.zglckf.com
8308
1
Immunogentics and
Histocompatibility
Testing Laboratory,
Shenzhen Blood
Center, Shenzhen
518035, Guangdong
Province, China;
2
Department of
Laboratory Medicine,
Dalian Medical
University, Dalian
116044, Liaoning
Province, China
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*
Received: 2011-04-15
Accepted: 2011-07-19
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
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
中图分类号:R318
文献标识码:B
文章编号:1673-8225
(2011)44-08308-05
收稿日期:2011-04-15
修回日期:2011-07-19
20110215003/D·S
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
Application of complete exon2/3 sequence to resolve ambiguous alleles of HLA-DRB1 locus
Li Zhen
1
, Yang Juan
2
, Cheng Liang-hong
1
, Zou Hong-yan
1
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.
Li Z, Yang J, Cheng LH, Zou HY. Application of complete exon2/3 sequence to resolve ambiguous alleles of HLA-DRB1 locus.
Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(44): 8308-8312.
[http://www.crter.cn http://en.zglckf.com]
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
doi:10.3969/j.issn.1673-8225.2011.44.035
李桢,杨鹃,程良红,邹红岩. 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[J].中国组织工程研究与
临床康复,2011,15(44):
8308-8312. [http://www.crter.org http://cn.zglckf.com]
0 引言
人类白细胞抗原(human leukocyte
antigen,HLA)是至今所知人类最复杂的免疫遗
传多态性系统。准确的HLA分型不仅被广泛用
于器官移植和造血干细胞移植中寻找HLA相匹
配的供者,而且是研究群体遗传学、亲子鉴定、
个体识别的基本数据,且有许多文献报导
HLA-DRB1基因与疾病的关联
[1-3]
。
聚合酶链式反应(polymerase chain
reaction,PCR)-基因测序分型(sequence based