Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (44): 8308-8312.doi: 10.3969/j.issn.1673-8225.2011.44.035

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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*

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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.

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