通过长距离PCR和高通量测序对HLA-DRB1、-DRB3、-DRB4和-DRB5进行超高分辨率基因分型。
HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.
作者信息
Ozaki Y, Suzuki S, Shigenari A, Okudaira Y, Kikkawa E, Oka A, Ota M, Mitsunaga S, Kulski J K, Inoko H, Shiina T
机构信息
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Japan.
出版信息
Tissue Antigens. 2014 Jan;83(1):10-6. doi: 10.1111/tan.12258. Epub 2013 Nov 30.
Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.
超高分辨率单分子序列分型(SS-SBT)是一种人类白细胞抗原(HLA)DNA分型方法,可达到等位基因分辨率的第4级水平(以前称为八位分型),通过长距离聚合酶链反应(PCR)扩增和下一代测序(NGS)技术的结合,能够有效检测新的和无效等位基因,且不存在相位模糊问题。我们之前报道了SS-SBT方法在八个经典HLA基因座,即A、B、C、DRB1、DQA1、DQB1、DPA1和DPB1上的开发与应用。在本文中,我们描述了针对三个与DRB1连锁的基因座DRB3、DRB4和DRB5(DRB3/4/5)的SS-SBT方法的开发,以及DRB1-DRB3/4/5单倍型结构在第4级水平的特征分析。针对DRB3/4/5设计了基因座特异性PCR引物,以扩增从内含子1到外显子6[3'非翻译区(3'UTR)]的基因区域。使用从日本人中获得的19个DR51、DR52和DR53阳性基因组DNA样本,通过SS-SBT在不存在相位模糊的情况下确定了总共20个DRB1和13个DRB3/4/5等位基因序列,达到第4级水平。此外,通过SS-SBT方法将18个DRB1-DRB3/4/5单倍型估计到第4级水平,相比之下,传统方法将10个单倍型估计到第1级水平(以前称为两位分型)。因此,预计通过SS-SBT进行DRB1-DRB3/4/5单倍型分型可为医学研究、移植程序、HLA相关疾病研究和人类群体多样性研究中改进HLA匹配提供有用的数据。