van der Vlies S A, Voorter C E, van den Berg-Loonen E M
Tissue Typing Laboratory, University hospital Maastricht, The Netherlands.
Tissue Antigens. 1999 Aug;54(2):169-77. doi: 10.1034/j.1399-0039.1999.540208.x.
HLA-C was shown to be a highly polymorphic gene which can be accurately typed for by sequencing methodologies. Most HLA-C sequence-based typing protocols described so far are based on analysis of sequence data of exons 2 and 3. Nonetheless, exons 1, 4 and 5 also contain nucleotide substitutions which contribute to the polymorphisms of the HLA-C locus. Ten alleles contain polymorphic positions in exons 1, 4 and 5, Cw0701/06, Cw1202112, Cw15051/2, Cw1701/02, and Cw1801/02. Here we describe a reliable solid-phase sequence-based typing strategy for sequencing exons 1, 4 and 5, which is an extended protocol of our previous HLA-C study. A panel of 16 individuals, carrying 27 different Cw-alleles, was typed for exons 1, 4 and 5 to check the newly designed primers. No allelic dropout or preferential amplification was noticed in these individuals. The panel was also sequenced in order to check the known polymorphisms present in exons 1, 4 and 5. For exon 5 the sequences of the alleles Cw0302, 0501 and 07011 did not correspond with the published data. In addition, exons 1, 4 and 5 were sequence-based typing typed in 28, 17 and 59 individuals, respectively. Two new alleles were detected which contain polymorphic positions outside exons 2 and 3, Cw07012 and Cw1703. The unknown sequence data of exons 1, 4 and 5 of the alleles Cw*02024, *0308, *1506 and *16041 were elucidated. The described high-resolution sequence-based typing protocol for sequencing exons 1, 4 and 5 will be a valuable tool to study the HLA-C locus for polymorphisms outside exons 2 and 3 and for identification of the presently known HLA-C alleles with polymorphic positions in these exons.
HLA - C被证明是一个高度多态性的基因，可通过测序方法准确分型。到目前为止描述的大多数基于HLA - C序列的分型方案都是基于对第2和第3外显子序列数据的分析。尽管如此，第1、4和5外显子也包含导致HLA - C基因座多态性的核苷酸替换。有10个等位基因在第1、4和5外显子中存在多态性位点，即Cw0701/06、Cw1202112、Cw15051/2、Cw1701/02和Cw1801/02。在此，我们描述了一种可靠的基于固相序列的分型策略，用于对第1、4和5外显子进行测序，这是我们之前HLA - C研究的扩展方案。选取了一组16名个体，他们携带27种不同的Cw等位基因，对其第1、4和5外显子进行分型以检验新设计的引物。在这些个体中未发现等位基因缺失或优先扩增现象。对该组个体也进行了测序，以检查第1、4和5外显子中已知的多态性。对于第5外显子，Cw0302、0501和07011等位基因的序列与已发表数据不符。此外，分别对28、17和59名个体的第1、4和5外显子进行了基于序列的分型。检测到两个新等位基因，它们在第2和第3外显子之外含有多态性位点，即Cw07012和Cw1703。阐明了Cw*02024、*0308、1506和16041等位基因第1、4和5外显子的未知序列数据。所描述的用于对第1、4和5外显子进行测序的高分辨率基于序列的分型方案，将成为研究HLA - C基因座在第2和第3外显子之外的多态性以及鉴定这些外显子中具有多态性位点的当前已知HLA - C等位基因的有价值工具。
