Voorter Christina E M, van den Berg-Loonen Ella M
Tissue Typing Laboratory, University Hospital Maastricht, Maastricht, The Netherlands.
Hum Immunol. 2006 Sep;67(9):756-63. doi: 10.1016/j.humimm.2006.01.002. Epub 2006 Jun 23.
Typing of DQA1 by sequencing has been a challenge because of a 3-nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but need additional exon information. With the sequencing strategy presented here the complete exons 1-4 are sequenced heterozygously, enabling identification of all DQA1 alleles by sequence-based typing (SBT). Exons 1-4 were amplified and sequenced separately, the combined sequences were used for automated allele assignment. The method was validated by typing 21 individuals with all possible different allele group combinations. In addition 26 quality control samples were correctly typed by this method. To determine the phenotype frequencies 155 unrelated Dutch Caucasian individuals were DQA1 typed. In total 15 known and two new DQA1 alleles were identified. DQA1*0103 and *0505 were the most frequent alleles with phenotype frequencies of 30% and 29%, respectively. The SBT method presented here is an improvement compared to already existing protocols in that the complete exon sequence is obtained for all coding exons, using identical polymerase chain reaction conditions. Furthermore, all exons are sequenced heterozygously, facilitating allele assignment and reducing the number of amplification reactions.
由于一半的等位基因在外显子2中有一个3核苷酸缺失，通过测序对DQA1进行分型一直是一项挑战。此外，28个等位基因中的19个不能仅基于外显子2来鉴定，而是需要额外的外显子信息。采用本文介绍的测序策略，外显子1 - 4以杂合子形式进行测序，从而能够通过基于序列的分型（SBT）鉴定所有DQA1等位基因。外显子1 - 4分别进行扩增和测序，合并后的序列用于自动等位基因分型。通过对21个具有所有可能不同等位基因组组合的个体进行分型，验证了该方法。此外，用该方法对26个质量控制样本进行了正确分型。为了确定表型频率，对155名不相关的荷兰白种人个体进行了DQA1分型。总共鉴定出15个已知的和2个新的DQA1等位基因。DQA10103和0505是最常见的等位基因，表型频率分别为30%和29%。本文介绍的SBT方法与现有方案相比有改进，因为在相同的聚合酶链反应条件下获得了所有编码外显子的完整外显子序列。此外，所有外显子均以杂合子形式测序，便于等位基因分型并减少扩增反应的数量。
