CAJ | 학술논문

目的探讨无关供-受者器官移植人类白细胞抗原(human leukocyte antigen,HLA)高分辨分型确认实验中,HLA测序分型模棱两可结果的种类、比例及高效解决方法.方法采用美国Atria公司的HLA测序分型试剂盒,对650人份无关供-受者样本的HLA-A、B、C基因第2、3和4外显子,HLA-DRB1第2外显子及HLA-DQB1第2、3外显子进行常规检测,统计常见模棱两可等位基因种类及比例,再应用PCR序列特异性引物(squence specific primer,PCR-SSP)高分辨试剂盒或增加常规检测区外的外显子进行再测序分型(sequencing-based typing,SBT),以获得精确的HLA高分辨分型结果.结果 650人份供-受者的HLA-A、B、C、DRB1及DQB1基因的直接测序分型出现模棱两可比例分别为76.31％ (496/650),91.08％ (592/650),97.69％ (635/650),88.62％ (576/650)和43.38％ (141/650).共发现常规检测区内模棱两可等位基因型组合36种,常规检测区外模棱两可等位基因组合22种.依据中国常见及确认的(common and well-documented,CWD) HLA等位基因表(1.01版),排除罕见等位基因后,共有9种模棱两可CWD组合,除了HLA-B、C位点包括等3种外,其余的3个位点各出现1种;检测区外模棱两可等位基因组合减少为10种,其中HLA-A、B位点各1种,HLA-C、-DRB1位点各4种.所有模棱两可结果采用高分辨PCR-SSP或PCR-SBT方法进行必要的复核确认.结论分析了HLA 5个位点测序分型中模棱两可结果的常见种类,采用相应的解决策略,可以达到高效、低耗、精确分型的目的.
목적 탐토무관공-수자기관이식인류백세포항원(human leukocyte antigen,HLA)고분변분형학인실험중,HLA측서분형모릉량가결과적충류、비례급고효해결방법.방법 채용미국Atria공사적HLA측서분형시제합,대650인빈무관공-수자양본적HLA-A、B、C기인제2、3화4외현자,HLA-DRB1제2외현자급HLA-DQB1제2、3외현자진행상규검측,통계상견모릉량가등위기인충류급비례,재응용PCR서렬특이성인물(squence specific primer,PCR-SSP)고분변시제합혹증가상규검측구외적외현자진행재측서분형(sequencing-based typing,SBT),이획득정학적HLA고분변분형결과.결과 650인빈공-수자적HLA-A、B、C、DRB1급DQB1기인적직접측서분형출현모릉량가비례분별위76.31％ (496/650),91.08％ (592/650),97.69％ (635/650),88.62％ (576/650)화43.38％ (141/650).공발현상규검측구내모릉량가등위기인형조합36충,상규검측구외모릉량가등위기인조합22충.의거중국상견급학인적(common and well-documented,CWD) HLA등위기인표(1.01판),배제한견등위기인후,공유9충모릉량가CWD조합,제료HLA-B、C위점포괄등3충외,기여적3개위점각출현1충;검측구외모릉량가등위기인조합감소위10충,기중HLA-A、B위점각1충,HLA-C、-DRB1위점각4충.소유모릉량가결과채용고분변PCR-SSP혹PCR-SBT방법진행필요적복핵학인.결론 분석료HLA 5개위점측서분형중모릉량가결과적상견충류,채용상응적해결책략,가이체도고효、저모、정학분형적목적.
Objective To investigate the number and ratio of ambiguous allele combinations from human leukocyte antigen (HLA) confirmatory test by sequencing-based typing for unrelated donor marrow transplantation,and to establish an efficient strategy for identifying such ambiguities.Methods A total of 650 donor-receipt samples were genotyped for 5 loci of the HLA gene using an Atria SBT commercial kit.Exons 2,3 and 4 of HLA-A,-B and-C,exon 2 of HLA-DRB1 and exons 2 and 3 of HLA-DQB1 were tested by routine HLA genotyping.The ratio of usual ambiguous allele combination was calculated.The ambiguities were subjected to further confirmatory test by PCR-SSP or PCR-SBT retest at outside of the routine sequencing region.Results Among the 650 tested samples,the ratio of ambiguity at HLA-A,B,C,DRB1 and DQB1 were 76.31％ (496/650),91.08％ (592/650),97.69％ (635/650),88.62％ (576/650) and 43.38％ (141/650),respectively.A total of 36 ambiguous allele combinations inside the routine sequencing region and 22 ambiguous allele combinations outside of the routine sequencing region were discovered.After removing rare alleles based on the Chinese common and well-documented (CWD) Allele Table (Version 1.01),9 ambiguous CWD allele combinations inside the routine sequencing region,including 3 located in HLA-B,HLA-C and 1 located in other three HLA loci were found.Ten ambiguous CWD allele combinations outside of the routine sequencing region,including 4 located in HLA-C,-DRB1 and 1 in HLA-A,-B respectively were determined.All samples with ambiguous CWD allele combinations could be distinguished by high-resolution PCR-SSP commercial kits or PCR-SBT retest at outside of the routine sequencing region.Conclusion The common and well-documented allele combinations in sequencing-based typing at five HLA loci have been analyzed.Our strategy may provide valuable information for more efficient,low-cost and accurate method for high-resolution genotyping of HLA genes.