人类白细胞抗原新等位基因A*2636的确认

背景：目前中华骨髓库主要采用分辨率较高的聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法检测人类白细胞抗原HLA-A、B、DRB1，该方法在提高分辨率的同时,有些检测结果难以一次性确定,可采用DNA测序技术确认。 目的：识别和确认一个人类白细胞抗原新等位基因。 设计、时间及地点：以DNA为观察对象的开放性实验。常规初检聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法于2007-11在河南省红十字血液中心中华骨髓库河南分库人类白细胞抗原组织配型实验室完成，测序实验于2008-02在戴诺生物技术(北京)有限公司人类白细胞抗原实验室完成。 材料：中国造血干细胞捐献者初检结果可疑为新基因的重采血样5 mL。 方法：采用聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法进行常规人类白细胞抗原分型，发现A位点反应格局异常，提示有2个无法解释的假阳性探针反应(10FP、88FP)和1个假阴性探针反应(58FN)、不能正常判读给出确切结果时，采用DNA测序技术测定 人类白细胞抗原A位点外显子2，3，4(Exon2，3，4)的核苷酸序列，并与已知相近等位基因进行序列对比分析。 主要观察指标：测序结果与已知人类白细胞抗原等位基因序列的比较分析。 结果：聚合酶链反应-寡核苷酸探针分型反向杂交荧光微珠法基因分析结果显示，受检者人类白细胞抗原基因分型为A*02xx, 26xx(10 FP、88FP 58FN)，B *15(75), 55xx ，DRB1 08xx, 11xx, A位点反应格局异常，提示有2个无法解释假阳性反应(10 FP、88FP)，有1个假阴性探针反应(58FN)，不能指定为任何人类白细胞抗原A位点等位基因。对本例人类白细胞抗原A基因第2，3，4外显子双向之直接测序结果分析显示：A *02010101, A *260101 292 S/C。分析测序结果峰图，292位置应为C，分析软件提示该基因改变在A *26new一侧。该序列与人类白细胞抗原A *260101进行比对，同源性99%，在第2外显子区域292位碱基发生了G→C，74位的密码子GAC>CAC最终导致了氨基酸的改变，即天冬氨酸(Asp)→组氨酸(His)。 结论：该序列为A位点的一个新等位基因，已在GenBank注册(注册号EU785343)，并于2008-04被WHO-HLA因子命名委员会正式命名为A *2636.(HWS10005530)。     

中国人群人类白细胞抗原A、B和DRB1基因测序分型中模棱两可等位基因的鉴别

背景：人类白细胞抗原基因测序分型中，当某些等位基因间的差异碱基位于测序范围外时，无法得到清晰的等位基因结果。 目的：分析中国人群人类白细胞抗原A、B、DRB1基因测序分型中模棱两可等位基因的分布规律，探讨其在中华骨髓库大样本人类白细胞抗原 A、B、DRB1基因的测序分型中的解决方案。 设计、时间及地点：采用随机抽样方法对研究样本进行人类白细胞抗原A、B、DRB1基因的测序分型，并对其中的模棱两可等位基因进行验证性实验，于2006-01/2008-06在深圳市血液中心完成。 材料：658名深圳骨髓库汉族供者乙二胺四乙酸盐抗凝血5 mL。 方法：采用聚合酶链式反应--测序分型方法对658名深圳骨髓库供者的人类白细胞抗原A、B和DRB1基因进行高分辨分型，并采用针对相应位点的高分辨聚合酶链式反应-序列特异性引物方法对其中由于碱基差异位于检测区外而产生的模棱两可等位基因进行鉴别。 主要观察指标：模棱两可等位基因的分布及确认。 结果：658份标本中，人类白细胞抗原A、B和DRB1三个基因座的模棱两可等位基因分别为9个(2种)、140个(5种)、406个(8种)，占等位基因总数的14.06%(555/3 948)。高分辨聚合酶链式反应-序列特异性引物鉴定结果显示，中国报道的DRB1*1401被全部确认为DRB1*1454；12例B*0705/B*0706中，有1例被确认为B*0706，其他13种等位基因的鉴定结果分别为A*6801、A*7402、B*2705、B*3501、B*4402、B*5801、DRB1*0101、DRB1*0406 、DRB1*0803、DRB1*1101、DRB1*1201、DRB1*1302 和DRB1*1502。 结论：在中华骨髓库大样本人类白细胞抗原基因的测序分型中可以应用直接鉴别的方法区分模棱两可等位基因，但在临床移植前的高分辨确认试验中则需要采用实验方法进行精确分型。     

中国汉族人群人类白细胞抗原新等位基因A*1155的确认

背景：在国内发现的新等位基因中，大部分都是先采用人类白细胞抗原分型低分辨方法进行分型，如发现反应格局异常后，再进一步用基因测序或基因克隆方法进行确认，这样有可能导致一些新等位基因未被发现。基因测序分型方法被认为是人类白细胞抗原分型的金标准，它可得出精确的核苷酸序列。 目的：直接采用基因测序分型方法对中华骨髓库标本进行检测，识别和确认中国汉族人群中新发现的等位基因。 方法：应用中国造血干细胞捐献者测序结果可疑为新等位基因的重采血样5 mL，采用基因测序分型技术，发现一样本HLA-A位点的杂合结果HLA-A*030101/A*110101在外显子上有1个位置与数据库不符，为了区别哪一个为新等位基因，采用基因克隆(TOPO TA Cloning)技术对两个等位基因1-8外显子进行DNA双向测序，发现1个与HLA-A*110101序列相近的新等位基因，分析两者核苷酸序列的差异。并将测序结果与已知人类白细胞抗原等位基因序列比较分析。 结果与结论：通过克隆分离杂合等位基因，再进行测序确认发现新的序列与A*110101相比，在第2外显子第330碱基位置上C→G，密码子86AAC→AAG，发生了氨基酸的改变，由天冬酰氨变为赖氨酸。提示该等位基因为新的HLA-A等位基因，2009-10-31被世界卫生组织人类白细胞抗原因子命名委员会正式命名为HLA-A*1155。     

造血干细胞移植56例人类白细胞抗原基因和单倍型分析

背景：收集56例欲行造血干细胞移植的供受者，均为无血缘关系的江西省汉族人群。了解个体的人类白细胞抗原基因型和单倍型。 目的：分析56例造血干细胞移植供受者的人类白细胞抗原基因频率，单倍型频率。 方法：收集56例欲行造血干细胞移植的供受者，均无血缘关系的江西省汉族人群。应用PCR-SSP的方法进行人类白细胞抗原(HLA)-A、B、DRB1基因分型，计算出HLA-A、B、DRB1各位点的基因频率和单倍型频率。 结果与结论：56例供受者测出HLA-A位点等位基因8种，HLA-B位点等位基因19种，HLA-DRB1位点等位基因13种，呈现出丰富的基因多态性。56例供受者两位点共224条等位基因中，A﹡02-B﹡46、A﹡11-B﹡40、B﹡46-DRB1﹡09单倍型的频率高于0.10。有10种A-B单倍型，4种B-DRB1单倍型呈现出显著的连锁不平衡。提示江西省汉族人群人类白细胞抗原基因具有较丰富的基因多态性。     

中国人群HLA-DRB1*1454等位基因和HLA-DRB1第3外显子鉴定意义

目的：在中国人群中鉴定人类白细胞抗原（human leukocyte antigen,HLA）DRB1*1454等位基因,分析供受者HLA-DRB1第3外显子DNA序列信息对器官移植、细胞移植及人类遗传学研究的重要现实意义。 方法：应用聚合酶链反应-直接测序分型方法对58例等待造血干细胞移植供受者进行人类白细胞抗原测序分型,1 268例广东地区随机健康供者采用聚合酶链式反应-序列特异寡核苷酸探针反向杂交法进行HLA-DRB1中高分辨基因分型,部分模棱两可结果采用聚合酶链式反应-序列特异引物PCR-SSPHLA-DRB*14高分辨方法分型法。 结果：在1 268例广东地区随机健康人群中检出HLA-DRB1*1403,1406,1410,1412,1418,1425和1454等位基因,HLA-DRB*1401/1434/1454等位基因模棱两可结果的8例样本被确认为HLA-DRB1*1454等位基因,DRB1*1454等位基因可能是广东人群最为常见的HLA-DRB1*14基因。中国人群HLA-DRB1*14第3外显子存在多态性。 结论：中国人群HLA-DRB1*1454等位基因确认了DRB1等位基因第3外显子多态性的存在,进一步明确了开展中国汉族人群及少数民族HLA-DRB1第3外显子检测的意义。     

HLA新等位基因HLA-B*5145的确认

背景：作者在用美国onelamba 试剂公司生产的HLA低分辨试剂进行分型实验时,将反应结果导入HLA数据分析软件,发现HLA-B位点反应格局异常,且阴性磁珠和阳性磁珠反应情况良好,但是分析软件并未给出相应结果,怀疑有新基因的可能。 目的：发现和认定1个HLA新等位基因。 方法：采集中华骨髓库造血干细胞捐献者血样,应用PCR-SSO基因分型技术筛选可能的新等位基因,PCR产物测序和DNA基因克隆测序,分析基因序列。 结果与结论：PCR-SSO基因分型显示,供者HLA-A位点基因型为 A*02XX,A*33XX ;HLA-DRB1位点基因型为DRB1*1202,DRB1*1302,HLA-B位点反应格局异常,不能指定任何HLA-B位点的等位基因,提示B*44XX,B*53XX;90FN,91FP,调整不合理,故进一步用其他试剂(Dynal,PCR-SSO) 分型方法进行分析,也显示无法判定的结果。等位基因HLA-B*5145是HLA-B*5106的变异体,该基因和B*5106的差异在第3 外显子的339位碱基A→G,引起相应编码氨基酸113位上的N(天冬酰胺,Asn)→D(天冬氨酸,Asp),346位碱基A→C,引起相应编码氨基酸115位上的Y(酪氨酸,Tyr)→S(丝氨酸,Ser),362位碱基A→G,则不引起相应编码氨基酸120位上的K(赖氨酸,Lys)的变化。该基因为新等位基因,2006-10被世界卫生组织HLA因子命名委员会正式命名HLA-B*5145。     

河南省骨髓库汉族捐献者HLA-A、B、DR高分辨基因多态性调查分析

背景：2009年开始,中华骨髓库加大HLA高分检测的力度,可缩短配型检索周期,提高配型成功率。 目的：统计分析河南省骨髓捐献者HLA-A、B、DRB1高分辨基因多态性。 方法：河南省汉族造血干细胞志愿捐献者血样3 874人份,志愿捐献静脉血,EDTA抗凝。采用SSO HD 荧光微珠流式高分辨HLA-A、B、DRB1基因分型检测方法,对3 874份骨髓捐献志愿者血样进行高分辨分型检测,用PCR-SSP高分辨检测、SBT检测解决存在的模棱两可问题。等位基因计数法计算等位基因的基因频率。 结果与结论：共检出A 34个,B 84个,DRB1 50个,A位点基因频率处于前5位的有A*0201(0.160 9)、 A*1101(0.162 7)、 A*2402(0.160 2)、A*3001(0.080 8)、 A*3303(0.078 9);B位点处于前5位的是B*1302(0.077 7)、B*4001(0.072 2)、B*5101(0.062 8)、B*4601(0.061 2)和B*0702(0.050 7);DRB1位点处于前5位的是DRB1*1501(0.146 9)、DRB1*0901(0.131 8)、DRB1*0701(0.121 1)、DRB1*1202(0.061 2)、DRB1*0405(0.058 2)。     

中国南方汉族人群HLA-A，B，DRB1基因测序分型模棱两可结果的分布及其解决方案

背景：聚合酶链式反应-直接测序分型技术被誉为HLA分型的金标准，在临床移植配型和骨髓库供者的大规模HLA分型中逐渐被广泛采用，但该方法的最大缺点是存在较高比例的模棱两可分型结果，故亟待寻找并建立适合中华骨髓库大规模供者HLA-测序模棱两可分型结果的解决方案。 目的：调查中国南方汉族人群HLA-A，B，DRB1基因测序分型中模棱两可结果的分布状况，评估其可能的解决方案。 设计、时间及地点：观察测量实验，2007-08/2008-08在深圳市血液中心输血医学研究所免疫遗传重点实验室完成。 对象：选择深圳骨髓库的汉族骨髓供者416名，供者民族和籍贯按照自述原则确定。 方法：采用聚合酶链反应-直接测序分型方法对416名汉族供者的HLA-A，B，DRB1基因进行测序分型，分析3个基因座模棱两可分型结果的分布情况，并分别采用高分辨聚合酶链反应-序列特异性引物法和杂合性模棱两可引物分离法进行解决；采用直接计数法统计基因频率，计算真实等位基因的相对概率。 主要观察指标：416名供者HLA-A，B，DRB1基因直接测序分型结果的分布。HLA-A，B，DRB1基因测序模棱两可分型结果的分类及分布。序列特异性引物法及杂合性模棱两可引物分离法对模棱两可分型结果的解决能力。 结果：80.29%的标本其HLA-A，B，DRB1基因出现模棱两可结果。80%左右的HLA-A，B基因和不足40%的HLA-DRB1基因模棱两可分型结果可以采用杂合性模棱两可引物分离法解决，其他则需要高分辨聚合酶链反应-序列特异性引物法解决。根据等位基因频率计算553个模棱两可等位基因组合中，75%的真实等位基因组合的相对概率大于98%。 结论：杂合性模棱两可引物分离法和高分辨聚合酶链反应-序列特异性引物法分别对位于HLA-A，B和HLA-DRB1基因检测区内、外的模棱两可分型结果具有较高的解决能力，二者互为补充；在大规模供者HLA分型中应用频率数据解决模棱两可结果有一定的应用价值。     

94例缺血性脑卒中患者HLA基因表达分析

目的 探讨人类白细胞抗原(HLA)基因遗传与缺血性脑卒中发病的关联.方法 采用聚合酶链反应-直接测序分型技术(PCR-SBT)对广东医学院附属南山医院2008年收治的94例缺血性脑卒中患者和同期122例正常对照者进行HLA-A、B、DRB1位点的等位基因分型.结果 缺血性脑卒中患者表达HLA-A位点16个等位基因,HLA-B位点32个等位基因,HLA-DRB1位点25个等位基因.患者组HLA-A*1102基因频率较对照组明显降低,HLA-A*1102与缺血性脑卒中呈负相关(RR=0.06,P=0.019).结论 JLA-A*1102与缺血性脑卒中呈易感负相关,提示HLA等位基因与缺血性脑卒中的发生存在遗传免疫关联,对该病具有临床预测意义.

Abstract：

Objective To discuss the relationship between human leukocyte antigen (HLA) gene heredity and morbidity of cerebral infarction by a random survey on the allele expression of HLA-A, B and DRB1 seats of patients with cerebral infarction. Methods The genotypes of HLA-A, B and DRB1 alleles in 94 patients with cerebral infarction and 122 healthy blood donors were detected by polymerase chain reaction-sequencing based typing (PCR-SBT) method. Results Sixteen alleles in HLA -A locus,32 alleles in HLA -B locus and 25 alleles in HLA -DRB1 locus expressed themselves in these patients with cerebral infarction. The gene frequency of HLA -A*1102 in patients was lower than that in healthy controls, and negative association was found between HLA -A* 1102 allele and cerebral infarction (RR=0.06,P=0.019). Conclusion The research reveals susceptibility association of HLA -A*1102 with patients having cerebral infarction, displaying close genetic immunity correlation between HLA alleles and pathogenesis of cerebral infarction. So, the research in this paper is useful in the clinical prediction of this disease.     

亲属活体肾移植人类白细胞抗原基因和单型分析36例

背景：了解个体的人类白细胞抗原单型和基因型在同种异体器官移植中有重要意义。 目的：分析36例亲属活体肾移植供受者的人类白细胞抗原基因频率、单型频率和特点。 设计、时间及地点：基因多态性分析,于2007-01/2008-06在广西壮族自治区中医学院附属瑞康医院移植配型实验室完成。 参试者：36例行亲属活体肾移植的供受者,均为有血缘关系的广西壮族自治区汉族人。 方法：应用单克隆抗体干板进行供受者人类白细胞抗原-Ⅰ分型,应用PCR-SSP的方法进行人类白细胞抗原-Ⅱ基因分型,计算人类白细胞抗原-A、B、DRB1各位点基因频率和单型频率。 主要观察指标：广西壮族自治区人群人类白细胞抗原基因型和单型。 结果：36例供受者中共检测出人类白细胞抗原-A位点抗原11种,人类白细胞抗原-B位点抗原24种,人类白细胞抗原-DRB1位点抗原13种,呈现出丰富的多态性。144条单型中,A2-B46、A11-B60、B46-DRB1*09、B60-DRB1*15、A2-B46- DRB1*09等5条单型的频率高于0.05,共有10种A-B单型和9种B-DRB1单型呈现出显著的连锁不平衡。36例供受者与其他地区汉族人群的人类白细胞抗原-A-B单型频率比较,发现广西汉族A-B单型的分布与湖南、台湾等南方人相似,但也有其自身的特殊性。 结论：广西壮族自治区汉族人群人类白细胞抗原基因具有较为丰富的多态性及地区性遗传特征。     

假肥大型肌营养不良症患者HLA-A、B、DR基因表达研究

目的 研究南方假肥大型肌营养不良症(duchenne muscular dustrophy，DMD)患者HLA-A、B、DR基因多态性，探讨免疫遗传因素在DMD发病中的作用，并为临床骨髓移植提供基础性资料。方法 采用PCR反向序列特异性寡核苷酸杂交技术与美国骨髓库编码软件(National marrow donor program，NMDP)，对29例DMD患者HLA-A、B、DR等位基因多态性进行研究。结果 DMD组HLA-A24等位基因频率9.03％，与对照组22.16％相比有所降低(P=0.017)；DMD组HLA-B13等位基因频率16.95％，与对照组6.75%相比显著增高(P=0.007)。但校正后P值差异均无显著性(P值分别为0.255和0.231)。结论 DMD患者HLA-A、B、DR基因表达与正常对照组差异无显著性，HLA遗传易感性与DMD发病无明显相关，但尚需要扩大样本量或进行高分辨加深研究。     

Optic neuritis, multiple sclerosis and human leukocyte antigen: results of a 4-year follow-up study

In the present study the relation between human leukocyte antigen (HLA), optic neuritis (ON) and multiple sclerosis (MS) has been investigated in 56 Iranian patients (46 females and 10 males). HLA-A and -B typing by microlymphocytotoxicity method and HLA-DRB, DQA and DQB by polymerase chain reaction based on sequence specific primers method was performed for the selected patients with ON. The diagnosis of clinically defined MS (CDMS) was confirmed in 15 of them (26.7%) during their follow-up. HLA-A24 was significantly higher in ON patients, whilst A23, A26, and A30 showed a significant decrease in these patients. HLA-A10 and A26 were absent in CDMS patients and A2 and A11 were significantly decreased in ON and CDMS patients. HLA-B5, B51, B38, B27, and B35 were significantly increased in ON patients compared with control subjects. HLA-B44, B16 and B38 alleles were not present in CDMS patients. Regarding DR locus, the frequency of HLA-DRB1*15 and DRB1*04 has been increased in CDMS patients, whilst the frequency of HLA-DRB1*07 and *11 was much higher in ON patients. In DQA region, the most frequent allele in the MS patients was DQA1*0102, which was significantly higher than ON patients, and control group. The frequency of DQA1*0103 was significantly increased in both patients group. In DQB1, the frequency of DQB1*0602 increased significantly in the MS patients. In conclusion existence of common genetic basis for early manifestations of MS could be suggested.     

Genetic predictors of Stevens–Johnson syndrome and toxic epidermal necrolysis induced by aromatic antiepileptic drugs among the Chinese Han population

BackgroundPrevious studies suggested that one or more HLA alleles participate in the pathogenesis of AED-induced SJS/TEN, but most of these studies focused only on the HLA-B alleles.PurposeThe aim of this study was to investigate the pathogenesis of AED-induced SJS/TEN across a broader spectrum of HLA alleles, including the HLA-A, -B and -DRB1 alleles, to further explore the association between each HLA allele and SJS/TEN induced by aromatic AEDs.MethodsA total of 27 patients exhibiting AED-induced SJS/TEN (16 CBZ-SJS/TEN, seven LTG-SJS/TEN, two PHT-SJS/TEN, and two PB-SJS/TEN patients) and 64 patients who exhibited tolerance to AEDs were recruited. High-resolution HLA genotyping was performed to estimate the prevalence of the HLA-A, -B and -DRB1 alleles for each subject.ResultsFifteen subjects in the SJS/TEN group (12 exhibiting CBZ-SJS/TEN, two exhibiting LTG-SJS, and one exhibiting PB-SJS) carried the HLA-B*15:02 allele, whereas only 4/64 subjects in the AED-tolerant group carried this allele; the carrier rate of HLA-B*15:02 was significantly different between the groups (P < 0.001). Nine patients in the SJS/TEN group carried the HLA-DRB1*15:01 allele, while 12/64 subjects in the tolerant group carried this allele; considering that two patients in the SJS/TEN group (one exhibiting LTG-SJS and one exhibiting PB-SJS) were homozygous for this allele, the prevalence of HLA-DRB1*15:01 expression between the two groups was significantly different (P = 0.041). Furthermore, the carrier rates of HLA-A*33:03, HLA-B*58:01, and HLA-DRB1*03:01 were lower in the SJS/TEN group compared with the AED-tolerant group. The carrier rates of these alleles between the two groups were significantly different (P = 0.009, 0.016, and 0.009, respectively).ConclusionsThe HLA-DRB1*15:01 allele may represent a risk factor for AED-induced SJS/TEN among Han Chinese. The HLA-A*33:03, HLA-B*58:01, and HLA-DRB1*03:01 alleles may be “protectors” against AED-induced SJS/TEN, especially CBZ-SJS/TEN.     

Human leukocyte antigen alleles in patients with bipolar disorder in the Korean population

We performed an association study between human leukocyte antigen (HLA) alleles and bipolar disorder to evaluate the potentiality of HLA as a genetic marker in bipolar disorder. HLA class I and class II allele frequencies were assessed in 87 bipolar patients and were compared with those of 206 normal controls in the Korean population. HLA class I typing was performed using the microlymphocytotoxicity method, whereas class II (DRB1 and DQB1) genotyping was performed with polymerase chain reaction-sequence specific oligonucleotide probes. When the allele frequency of HLA in bipolar patients was compared with that in normal controls, there were some significant differences. Bipolar patients showed statistically significant increased allele frequencies of HLA-A29 and B54. Allele frequencies of HLA-B51 and DRB1*02 were significantly higher in normal controls. However, these results were no longer significant after correcting for the number of alleles. The results of the present study suggest that HLA alleles may not confer susceptibility to bipolar disorder in the Korean population. To clarify the genetic influence of HLA on bipolar disorder, we should conduct a consecutive study with a larger cohort of subjects.     

Association study of HLA-A gene and schizophrenia in Han Chinese from Taiwan

Dysregulation of the immune response has been proposed as a precipitating factor of schizophrenia, and human leukocyte antigens (HLA) play a critical role in regulating the cascade of immunological reaction. Hence, many studies have investigated the relationship between the HLA system and schizophrenia. HLA is a complex gene family that contains several highly polymorphic genes, while the HLA-A gene is the most often studied gene to be associated with schizophrenia in the literature. A recent study reported that the interaction of the HLA-A10 allele and Chlamydial infection was highly associated with schizophrenia in a German population, which prompted us to investigate whether the HLA-A gene was also associated with schizophrenia in our population. Using a sequencing-based HLA typing method, we determined the HLA-A genotypes in 377 Han Chinese patients with schizophrenia (214 males, 163 females) and 321 non-psychotic Han Chinese control subjects (164 males, 157 females) from Taiwan. In total, 26 DNA-defined HLA-A alleles were identified in this sample. However, no significant differences of these allelic frequencies were found between the patients and the control subjects, suggesting that the HLA-A gene was unlikely a major risk factor of schizophrenia in this sample. As different populations have different HLA polymorphisms, an examination of the relationship of other HLA genes and schizophrenia in our population, with a larger sample size, is warranted in the future.