五例样本HLA-C基因测序分型中等位基因丢失及其原因分析  

An analysis of the reason for HLA-C allele dropout in five samples by sequence-based typing

作　　者：曾健强[1] 徐筠娉[1] 王大明[1] 邹红岩[1] 邓志辉[1] 杨宝成[1] 

机构地区：[1]广东省深圳市血液中心,深圳市组织配型与免疫遗传重点实验室,518035

出　　处：《中华医学遗传学杂志》2009年第5期562-566,共5页Chinese Journal of Medical Genetics

基　　金：基金项目：深圳市科技计划项目（200802116）

摘　　要：目的探讨HLA-C基因测序分型时等位基因漏检和丢失的原因，以提高HLA测序分型的成功率和准确性。方法620份随机选择的深圳健康捐血者血样，采用AlleleSEQRHLA—Cplus测序分型试剂盒进行检测，对无完全匹配、分型结果“异常”的标本，采用分子克隆和测序方法进行全长单倍体序列分析；对未检出新的碱基点突变的样本，进一步采用自行设计的PCR引物和AlleleSEQR试剂盒中的测序引物进行再测序，分析测序分型结果“异常”的原因。结果620份经AlleleSEQRHLA—C测序分型的样本中，发现5例样本无完全匹配的基因型，与之最接近的多种等位基因型均存在单个碱基的不匹配；并且在第4外显子出现碱基杂合，但第2和第3外显子区域内无杂合碱基。经分子克隆和单倍体测序，以及采用自行设计的PCR引物和AlleleSEQR试剂盒中的测序引物再测序，证实了5例标本均存在Cw＊0706等位基因漏检和丢失现象，未发现新的碱基点突变。结论HLA-C基因测序分型时，因PcR引物与模板DNA不匹配会导致等位基因的漏检和丢失。根据中国人群HLAC分子全长序列特点，开发适合于中国人群的测序分型试剂十分必要。Objective To analyze the possible reason for HLA-C allele dropout in routine sequencebased typing （SBT） and improve the accuracy of HLA-C SBT test. Methods A total of 620 randomly selected samples from healthy voluntary blood donors in Shenzhen were typed at HLA-C locus by sequencebased typing using the AlleleSEQR HLA-C plus sequence-based typing kit. Samples with no full match result were subjected to cloning and haplotype sequencing of the full-length HLA-C gene. If no novel mutations were found, samples were then retyped, using our selbdesigned PCR primer pair and PCR conditions replacing the AlleleSEQR HLA-C PCR reagents in the PCR set-up procedure so as to analyze the potential reasons for causing ＂abnormal＂ SBT result. Results In the 620 samples typed at HLA-C locus using the AlleleSEQR HLA-C SBT commercial kit, 5 samples with no full match result were identified. The closest genotype showed one nucleotide mismatch with many different allele groups at different nueleotide position. Based on the PCR-SBT nucleotide sequence, heterozygous nucleotides were determined only in exon 4, whereas the nucleotides in exon 2 and 3 were all homozygotes. The results showed that HLA-Cw 0706 allele dropout existed in all the 5 samples with ＂abnormal＂ SBT results initially identified by AlleleSEQR HLA-C SBT kit, no novel mutation was found. Conclusion The results indicate that the PCR primer pair incompatible with DNA template may result in allele dropout in HLA C SBT test. Based on the characterization of HLA-C full-length, it is essential to develop HLA-C SBT kit suitable for Chinese population in the future.

关 键 词：人类白细胞抗原 HLA-C基因 测序分型 克隆 测序分析 等位基因丢失 

分 类 号：R686[医药卫生—骨科学]