中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2014年
4期
496-498
,共3页
高素青%王大明%徐筠娉%邓志辉
高素青%王大明%徐筠娉%鄧誌輝
고소청%왕대명%서균빙%산지휘
人类白细胞抗原%HLA-DQB1基因%测序分型%克隆和测序分析%等位基因丢失
人類白細胞抗原%HLA-DQB1基因%測序分型%剋隆和測序分析%等位基因丟失
인류백세포항원%HLA-DQB1기인%측서분형%극륭화측서분석%등위기인주실
Human Leukocyte Antigen%HLA-DQB1 gene%Sequence-based typing%Cloning and sequencing analysis%Allele dropout
目的 探讨HLA-DQB1基因测序分型时等位基因漏检和丢失的原因,以进一步提高HLA测序分型的准确性.方法 对2000份HLA高分辨组织配型血样,采用AlleleSEQR HLA-DQB1测序分型试剂盒进行常规检测.对序列峰图“异常”及无完全匹配结果的样本,进一步采用PCR-rSSO流式磁珠法复检,同时采用自行设计的特异性引物进行PCR产物分子克隆和测序,进行单倍型序列分析.结果 在2000份经AlleleSEQR HLA-DQB1测序分型的样本中,共发现2例样本序列峰图“异常”.经PCR-rSSO流式磁珠法和自行设计的引物测序复检,确认其为杂合型样本.PCR产物分子克隆和单倍型序列分析证实,第1份样本为第2外显子PCR扩增丢失HLA-DQB1*02:02等位基因序列.第2份样本为第2外显子PCR扩增丢失HLA-DQB1*02:01:01等位基因序列,未发现新的碱基突变.结论 HLA-DQB1基因测序分型时,因DQB1基因存在优势扩增现象,可导致HLA-DQB1第2外显子等位基因的漏检和丢失.上述发现将为HLA精确分型提供借鉴.
目的 探討HLA-DQB1基因測序分型時等位基因漏檢和丟失的原因,以進一步提高HLA測序分型的準確性.方法 對2000份HLA高分辨組織配型血樣,採用AlleleSEQR HLA-DQB1測序分型試劑盒進行常規檢測.對序列峰圖“異常”及無完全匹配結果的樣本,進一步採用PCR-rSSO流式磁珠法複檢,同時採用自行設計的特異性引物進行PCR產物分子剋隆和測序,進行單倍型序列分析.結果 在2000份經AlleleSEQR HLA-DQB1測序分型的樣本中,共髮現2例樣本序列峰圖“異常”.經PCR-rSSO流式磁珠法和自行設計的引物測序複檢,確認其為雜閤型樣本.PCR產物分子剋隆和單倍型序列分析證實,第1份樣本為第2外顯子PCR擴增丟失HLA-DQB1*02:02等位基因序列.第2份樣本為第2外顯子PCR擴增丟失HLA-DQB1*02:01:01等位基因序列,未髮現新的堿基突變.結論 HLA-DQB1基因測序分型時,因DQB1基因存在優勢擴增現象,可導緻HLA-DQB1第2外顯子等位基因的漏檢和丟失.上述髮現將為HLA精確分型提供藉鑒.
목적 탐토HLA-DQB1기인측서분형시등위기인루검화주실적원인,이진일보제고HLA측서분형적준학성.방법 대2000빈HLA고분변조직배형혈양,채용AlleleSEQR HLA-DQB1측서분형시제합진행상규검측.대서렬봉도“이상”급무완전필배결과적양본,진일보채용PCR-rSSO류식자주법복검,동시채용자행설계적특이성인물진행PCR산물분자극륭화측서,진행단배형서렬분석.결과 재2000빈경AlleleSEQR HLA-DQB1측서분형적양본중,공발현2례양본서렬봉도“이상”.경PCR-rSSO류식자주법화자행설계적인물측서복검,학인기위잡합형양본.PCR산물분자극륭화단배형서렬분석증실,제1빈양본위제2외현자PCR확증주실HLA-DQB1*02:02등위기인서렬.제2빈양본위제2외현자PCR확증주실HLA-DQB1*02:01:01등위기인서렬,미발현신적감기돌변.결론 HLA-DQB1기인측서분형시,인DQB1기인존재우세확증현상,가도치HLA-DQB1제2외현자등위기인적루검화주실.상술발현장위HLA정학분형제공차감.
Objective To explore the reason for HLA-DQB1 allele dropout during routine sequence-based typing(SBT) in order to improve the accuracy of typing.Methods Two thousand samples derived from HLA high-resolution typing laboratory were typed for HLA-DQB1 locus using an AlleleSEQR HLA-DQB1 SBT kit.Non-conclusive results and "abnormal" sequencing samples were retyped using a LABType rSSO HD HLA-DQB1 kit and further analyzed with both sequence-specific primers and group-specific primers and sequenced for haplotype analysis.Results Among the 2000 samples,2 samples with no conclusive result were identified.The heterozygosity was confirmed with both the LAB Type SSO HD HLA-DQB1 kit and PCR-SBT in house method.Subsequent HLA-DQB1 cloning and haplotype sequencing have elucidated that HLA-DQB1 * 02:02 dropped out at exon 2 for the first sample and HLA-DQB1 * 02:01:01 dropped out at exon 2 for the second sample during PCR amplification.No novel nucleotide mutation was found.Conclusion Our results indicated that preferential amplification at exon 2 of DQB1 may result in allele dropout in exon 2 sequences during HLA-DQB1 SBT test.This may provide useful information for HLA genotyping.
