基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的 探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案.方法 人胚胎干细胞系SYSU-I、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotide probe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型.应用PCR-STR技术检测两株细胞系的基因遗传标记.结果 获得两株hESC细胞系的HLA高分辨分型和STR基因型.结论 可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案.     

新等位基因HLA-A*24:233与HLA-A*26:89的分析确认

背景：分子生物学新技术的发展和大量应用加快了中国发现HLA 新等位基因的步伐。新等位基因的发现不仅给HLA家族增加了新成员,同时也为研究民族或地区的优势基因或消失基因(不适合客观环境的基因)找到了突破点。 目的：确认2个新的HLA等位基因,并分析其核苷酸序列。 方法：应用PCR-SBT、GSSP测序基因分型技术对两份中华骨髓库供者样本进行HLA高分辨分型,发现2个样本HLA-A位点均为异常基因,与已知同源性最高的等位基因型进行序列比对,分析核苷酸序列的差异。 结果与结论：2个样本HLA-A位点与目前已知的相应HLA-A等位基因序列均不一致,样本1与其同源性最高的A*24:02:01的差异表现在第3外显子区域中的第360位碱基由G > C,导致第96位密码子由谷氨酰胺变为组氨酸,样本2与其同源性最高的A*26:01:01比较差异表现在第2外显子区域中第97位碱基由T>C,导致编码的第9位密码子由酪氨酸变为组氨酸。结果表明两个样本为HLA-A位点的新等位基因,已提交GenBank进行注册,并已被WHO HLA因子命名委员会正式命名为HLA-A*24:233与HLA-A*26:89。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人类白细胞抗原A和C座位基因重组二个家系分析

目的 探讨2个家系人类白细胞抗原(human leukocyte antigen,HLA)座位的重组情况.方法 采用聚合酶链反应-序列特异寡核苷酸探针技术检测2个家系成员HLA-A、-C、-B、-DRB1和-DQB1位点,应用测序分型方法进行HLA高分辨基因分型,然后通过家系遗传分析确定HLA基因重组相关位点,检测短串联重复序列位点确定其家系成员亲权关系.结果 2个家系HLA单倍型的重组发生在HLA-A和C位点之间,家系调查显示1例为父源、1例为母源HLA单倍型发生了交换后遗传给子代,短串联重复序列结果证实2个家系成员内具有高度的亲权关系.结论 发现了2个中国汉族人群HLA-A和C基因座位间的基因重组家系,为深入研究HLA的重组机制提供了基础.     

浙江汉族人群HLA-A、-B、-DRB1高分辨等位基因频率和单体型分析

研究表明HLA具有高度连锁不平衡的遗传特点,不同民族或地区人群基因或单体型频率分布存在明显的不同,可作为不同种群特征性的遗传标志,目前有关不同人群HLA-A、-B、-DRB1基因频率和单体型分布情况已有众多的报道,但大多研究采用低分辨基因分型方法[1-3].本实验采用高分辨基因分型技术检测了1100例浙江汉族人群的HLA-A、-B、-DRB1位点,分析了人群HLA高分辨等位基因分布、单体型频率及连锁不平衡特征,现将结果报告如下.     

高分辨熔解曲线技术鉴定多药耐药基因外显子单核苷酸多态性

目的高分辨熔解曲线技术（HRM）检测多药耐药基因（MDR1）外显子12单核苷酸多态性（SNP）。方法采用高分辨熔解技术对MDR1基因外显子12的SNP C1236T位点进行基因分型,以其-401C〉T位点为例设计PCR扩增引物,按PCR扩增效率和熔解曲线进行退火温度、升温速度等条件的优化,并用此优化体系基因分型20例外周血标本,以测序验证。结果 20例标本经测序与检测结果一致。结论高分辨熔解曲线技术检测SNP是一种低成本、简便易行、常规化,高通量的基因分型方法,能用于大规模临床筛查。     

广州4194份脐带血HLA-A、B、DRB1等位基因型和单倍型频率研究

目的 对广州脐血库10年来保存的脐血人类白细胞抗原(human leukocyte antigen,HLA)等位基因及单倍型分布特征进行分析.方法 采用单克隆板,序列特异引物聚合酶链反应,PCR序列特异性寡核苷酸探针和DNA测序分型方法对广州脐血库内4194份脐带血进行HIA-A、B、DRB1等位基因分型.用Arlequm软件计算HLA基因频率和单倍型频率.结果 在广州脐血库中,HLA-A、B、DRB1等位基因型分别有18,43,13种.累积频率>50%的显著高频率等位基因是:A*11,A*02,A*24,A*33,B*40,B*15,B*46,B*13,DRB1*12,DRB1*15,DRB1*09,DRB1*04;最常见的单倍型为:A2-B46、B6-DR9、A11-DR12、A2-B6-DR9.结论 广州脐血库脐血捐献者HIA-A、B、DRB1等位基因型及单倍型分布具有典型南方人群的特点,此资料有助于为临床移植寻找合适匹配的供受对.     

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。     

用低形态学评分D3胚胎建立人胚胎干细胞系

目的 寻找人胚胎干细胞(hESC)建系材料来源.方法 选用IVF低形态学评分的D3胚胎行序贯囊胚培养,用免疫外科的方法去除滋养细胞,将得到的内细胞团(ICM)接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞(MEFs)上培养5～8 d,每4～7 d传代1次,分别取不同代的hESC进行碱性磷酸酶(AKP)染色、转录因子OCT-4、阶段特异性胚胎抗原(SSEA)SSEA-4、SSEA-1、肿瘤排斥抗原(TRA)TRA-1-60、TAR-1-81、核型及体内外分化全能性鉴定.结果 130枚废弃的D3低形态学评分(评分＜16)的胚胎培养出囊胚19枚,获得原代克隆5个,成功培养出两株hESC系,它们具有hESC的共同的生物学特性.结论 部分低形态学评分的D3废弃胚胎可发育成囊胚.囊胚形成率与形态学评分相关,这些胚胎可作为建立hESC系的材料来源之一.     

建立一种实时荧光PCR对维生素D受体基因单核苷酸多态性快速分型

目的:建立实时荧光PCR快速检测维生素D受体(VDR)基因rs2228570、rs1544410、rs7975232位点多态性。方法:以VDR基因的rs2228570、rs1544410、rs7975232三个SNP位点的变异碱基分别设计并合成特异性引物。11例体检健康儿童血液标本作为检测样本,采用实时荧光PCR方法检测样本中VDR基因型,采用Sanger法对检测结果测序验证。结果:利用所设计的特异性引物对人全血基因组中VDR进行SNP位点特异性扩增,根据7500 FAST实时荧光PCR仪的分析结果得到基因型,与测序结果比对后发现,11个样本采用实时荧光PCR方法基因分型结果与测序结果均一致。结论:实时荧光PCR快速检测VDR基因rs2228570、rs1544410、rs7975232位点多态性的方法或可用于临床VDR基因SNP快速分型。     

山东地区汉族人群中HLA-A、-B、-DRB1等位基因与原因不明卵巢早衰相关性研究

目的探讨HLA-A,-B和-DRB1位点等位基因多态性与原因不明卵巢早衰(premature ovarian failure,POF)的相关性。方法利用毛细管电泳测序技术(Capillary Electrophoresis),对36例汉族原因不明POF患者进行HLA-A,-B,-DRB1基因分型,并以865例山东健康汉族个体造血干细胞分型资料作为对照,分析HLA等位基因频率在两组中的分布差异。结果 POF组中HLA-A*33、HLA-B*07、HLA-B*52和HLA-B*55等位基因频率显著高于对照组(P<0.05)。HLA-A*33的等位基因频率POF组为19.44%,而正常对照组为10.17%,RR=2.18;HLA-B*07的等位基因频率POF组为12.50%,而正常对照组为5.32%,RR=2.65;HLA-B*52的等位基因频率POF组为11.11%,而正常对照组为4.10%,RR=3.06;HLA-B*55的等位基因频率POF组为5.56%,而正常对照组为1.50%,RR=4.23。结论山东汉族人群中HLA-A*33、HLA-B*07、HLA-B*52和HLA-B*55等位基因可能是POF的易感基因。     

Human leukocyte antigen-A, -B, and -DRB1 haplotypes of cord blood units in the Tzu Chi Taiwan Cord Blood Bank

Cord blood (CB) is considered an alternative resource to bone marrow and peripheral blood stem cells (PBSC) for allogeneic stem cell transplantation. In this study, human leukocyte antigen (HLA)-A, -B, and -DRB1 high-resolution allele types were analyzed from a total of 710 CB units in the Tzu Chi Taiwan Cord Blood Bank. We observed 21 HLA-A alleles, 59 HLA-B alleles, and 28 HLA-DRB1 alleles, whereas 19 unique alleles were present in the CB units of 2,023 individuals selected for confirmatory testing in the Tzu Chi Taiwan Marrow Donor Registry (TCTMDR). The allelic associations between the HLA-A and -B locus were stronger than that of either the HLA-B and -DRB1 loci or the HLA-A and -DRB1 loci. The most common haplotype of CB units in the general Taiwanese population was A*3303-B*5801-DRB1*0301 (6.59%), followed by A*0207-B*4601-DRB1*0901 (3.47%) and then A*1101-B*4001-DRB1*0901 (2.11%). Moreover, two haplotypes, A*2402-B*5201-DRB1*1502 and A*0201-B*1301-DRB1*1202, existed uniquely in the CB units but were not observed in the data of TCTMDR. Although the number of CB units studied for high-resolution of HLA typing in the current study is small, we believe our data should provide useful information to increase the chances of obtaining acceptable HLA-A-, -B-, and -DRB1-matched CB units for patients.     

HLA-A, -B, -DR haplotype frequencies from DNA typing data of 26,266 Chinese bone marrow donors

HLA phenotypes of 26,266 Chinese individuals who were recruited as potential hematopoietic stem cell donors by the Shanghai Red Cross Marrow Donor Registry, part of the China Marrow Donor Program, were determined for HLA-A, -B, and -DRB1 alleles at low to intermediate resolution using DNA-based typing methods. The large sample size of the study allowed accurate calculation of the Chinese HLA haplotype frequencies. The observed alleles correspond to 19 HLA-A, 44 -B, and 13 -DR split antigens. The serologic equivalents of HLA-A36, -A80, -B78, and -DR18 alleles were not observed. A total of 2,241 distinct HLA-A, -B, -DRB1 haplotypes were identified. Three-locus haplotype frequency was estimated using the maximum likelihood method. The lowest haplotype frequency that can be reliably estimated at a 95% confidence level was 0.000057. Using this cutoff value, 1,220 haplotypes (54%) were statistically reliable and their cumulative haplotype frequency was 0.9730. The cumulative haplotype frequency of the remaining 1,021 haplotypes (46%) was 0.0270. A regression equation of p = 0.192 log N - 0.576 was derived to estimate the probability (p) of finding an HLA-A, -B, -DR split antigens-matched donor in a pool of N Chinese donors.     

西北地区汉族人群HLA-A、-B、-DRB1基因座单倍型分析

目的 分析西北地区汉族群体HLA-A、-B和-DRB1基因座等位基因频率和HIA-A-B、B-DRB1和A-B-DRB1单倍型，获得单倍型频率数据。方法 采用序列特异性寡核苷酸探针反向斑点杂交技术对西北地区62个家系和101个无关个体HLA-A、-B和-DRB1基因座进行基因分型，分析HLA单倍型。结果 在西北地区汉族人群中检出15个HLA-A等位基因，28个HLA-B等位基因，13个HLA-DRB1等位基因，A02、A11、A24、B13、B15、1340、DRB1＊04、DRB1＊07、DRB1＊09和DRB1＊15基因频率较高（〉10％），A02（0．3244）、B13（0．1200）和DRB1＊15（0．1400）等位基因频率最高。分析得出HLA-A-B、B-DRB1、A-B-DRB1单倍型分别有122、147和278种，83种A-B-DRB1单倍型有至少两条以上相同的单倍型，占总单倍型数的18．44％（83／450）。A30-B13-DRB1＊07、A02-B46-DRB1＊09、A01-B37-DRB1＊10、A24-B15-DRB＊15、A02-B46-DRB1＊08、A33-B58-DRB1＊03是最常见的单倍型。结论 西北地区汉族群体HLA单倍型多态性较为丰富，等位基因频率和单倍型频率数据可用于骨髓移植供者的选择、法医学亲权鉴定以及人类学研究。     

The HLA dictionary 2001: a summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens

This report presents the serologic equivalents of 123 HLA-A, 272 HLA-B, and 155 HLA-DRB1 alleles. The equivalents cover over 64 percent of the presently identified HLA-A, -B, and -DRB1 alleles. The dictionary is an update of the one published in 1999 (Schreuder GMTh, Hurley CK, Marsh SGE, Lau M, Maiers M, Kollman C, Noreen H. The HLA dictionary 1999: a summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens. Tissue Antigens 54:407, 1999) and also includes equivalents for HLA-C, DRB3, DRB4, DRB5, and DQB1 alleles. The data summarize information obtained by the WHO Nomenclature Committee for Factors of the HLA System, the International Cell Exchange (UCLA), the National Marrow Donor Program (NMDP), and individual laboratories. In addition, a listing is provided of alleles which are expressed as antigens with serologic reaction patterns that differ from the well-established HLA specificities. The equivalents provided will be useful in guiding searches for unrelated hematopoietic stem cell donors in which patients and/or potential donors are typed by either serology or DNA-based methods. These equivalents will also serve typing and matching procedures for organ transplant programs where HLA typings from donors and from recipients on waiting lists represent mixtures of serologic and molecular typings. The tables with HLA equivalents and a questionnaire for submission of serologic reaction patterns for poorly identified allelic products will also be available on the WMDA web page: www.worldmarrow.org.     

Diversities of HLA-A, -B, -C, -DRB1 and -DQB1 loci in Chinese Kazak population and its genetic relatedness dissection with multiple populations: a comparative study

Studying the allele and haplotype distributions of human leukocyte antigen (HLA) loci at 2nd-field level in different populations was important. Allele and haplotype frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 loci in 110 unrelated healthy Kazak individuals living in Xinjiang (China) were analyzed using polymerase chain reaction sequence based typing. Thirty HLA-A, 48 HLA-B, 24 HLA-C, 34 HLA-DRB1 and 18 HLA-DQB1 alleles were detected at the 2nd-field level in the Kazak population. Frequencies of HLA alleles, genotypes, and haplotypes were calculated, and some exhibited significantly different distributions among different populations. A neighbor-joining (NJ) tree, heatmap, multidimensional scaling (MDS) and principal component analysis (PCA) were used to explore the genetic relationships between the Kazak population and 32 reference populations distributed in Asia, Africa, America and Europe using frequency data of HLA-A, -B, -C and -DRB1 loci. The NJ tree, heatmap, and MDS of the 33 populations were constructed based on pairwise D_A values of populations obtained by the HLA-A, -B, -C and -DRB1 allele frequencies. Different PCA plots were constructed based on the allele frequencies of HLA-A, -B, -C and -DRB1 or estimated haplotypic frequencies of HLA-A, -B, -C loci. The data obtained in the present research can be used for research on HLA-related diseases or paternity relationships, and aid to finding the best matched donors in stem cell transplantation for Kazak individuals.     

HLA polymorphisms in Sindhi community in Mumbai,India

Indian population is an amalgamation of various ethnicities, cultural and linguistic diversities, primarily due to marriages within a community. HLA-A, B and DRB1 alleles and haplotype frequencies were investigated in the Sindhi and compared with Marathi, Gujarati and North Indian population from Mumbai. This work is a part of a larger effort aimed at analysis of the HLA profile of diverse Indian ethnics to establish an umbilical cord stem cell panel in India. HLA polymorphisms at the HLA-A, B and DRB1 loci were determined in 413 cord blood samples by the molecular method of polymerase chain reaction using sequence-specific primer amplification. The most frequent alleles included A*01, A*02, A*11 and A*24 at A locus, B*35 and B*40 at B locus and DRB1*07 and DRB1*15 in all the four groups, although the frequency fluctuated in individual communities. HLA-DRB1*03 was significantly high (P < 0.05) in the Sindhi. Phylogenetic association using neighbour-joining tree, based on DA genetic distances for HLA-A and HLA-B alleles, indicated that the Sindhis cluster with North Indian and Pakistan Sindhi. The three locus haplotype analysis revealed that A*02-B*40-DRB1*15 and A*33-B*44-DRB1*07 were common haplotypes in all the groups. The three locus haplotypes found suggest an influence from Caucasian and Oriental populations. The data will be useful in developing an umbilical cord stem cell panel in India. The results will have clinical implications in unrelated umbilical cord stem cell for transplantation in India.     

造血干细胞移植受-供者HLA抗原识别部位的核苷酸匹配研究

目的 研究中国人群等待造血干细胞移植受-供者人类白细胞抗原(human leukocyte antigens,HLA)-A、-B、-Cw、-DRBl、-DQB1 5个位点的等位基因及核苷酸匹配情况,从单核苷酸水平探讨最佳供选择方案.方法 采用聚合酶链反应测序分型法(polymerase chain reaction-sequence-based typing,PCR-SBT),对537对中国人群等待造血干细胞移植受-供者HLA-A、-B、-Cw、-DRB1、-DQB1位点的等位基因进行序列分型,应用BLAST工具分析受-供者HLA核苷酸差异.结果 37对受-供者中HLA-A、-B、-Cw、-DRB1、-DQB1五位点核苷酸完全匹配占16.20%,单个等位基因错配的受-供者对分别占8.38%,0.74%,12.29%,2.42%和2.79%,两个或两个以上等位基因错配比率占42.65%.检出A*02:01-A*02:06,A*02:06-A*02:07,Cw*03:04-Cw*15:02,Cw*03:03-Cw*04:01,Cw*03:04-Cw*14:02,Cw *03:03-Cw*08:01,DRB1*04:03:01-DRB1*04:05不容许错配等位基因对.两对受-供者B*07:05:01-B*07:06,Cw*07:01:01-Cw*07:06抗原识别区外核苷酸错配.结论 在造血干细胞移植选择HLA错配的无关供者时注意受-供核苷酸匹配差异,对HLA抗原识别区内的核苷酸匹配差异和抗原识别区外的核苷酸匹配差异应当加以区别.本研究结果为优化供者选择顺序提供科学参考数据.     

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in the Kinh population in Vietnam

Allele and haplotype frequencies of the human leukocyte antigens (HLA) were studied in the Kinh Vietnamese population. We analyzed 170 unrelated healthy individuals. DNA-based HLA typing was performed using a microsphere-based array genotyping platform with sequence-specific oligonucleotide probes to distinguish HLA-A, -B, -C, -DRB1 and -DQB1 alleles. A total of 21 HLA-A, 37 HLA-B, 18 HLA-C, 25 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. HLA-A*1101, A*2402, A*3303, B*1502, B*4601, Cw*0102, Cw*0702, Cw*0801, DRB1*1202, DQB1*0301, DQB1*0303, and DQB1*0501 were found with frequencies higher than 10%. Two representative haplotypes bearing two to five HLA loci were A*1101-B*1502 and A*3303-B*5801 for HLA-A-B; Cw*0801-B*1502 and Cw*0102-B*4601 for HLA-C-B; B*1502-DRB1*1202 and B*4601-DRB1*0901 for HLA-B-DRB1; DRB1*1202-DQB1*0301 and DRB1*0901-DQB1*0303 for HLA-DRB1-DQB1; A*1101-Cw*0801-B*1502 and A*3303-Cw*0302-B*5801 for HLA-A-C-B; A*1101-B*1502-DRB1*1202 and A*2901-B*0705-DRB1*1001 for HLA-A-B-DRB1, A*1101-Cw*0801-B*1502-DRB1*1202-DQB1*0301 and A*2901-Cw*1505-B*0705-DRB1*1001-DQB1*0501 for HLA-A-C-B-DRB1-DQB1. Allele distribution and haplotype analysis demonstrated that the Vietnamese population shares HLA patterns with southern Chinese, Thai, Javanese and Micronesians, while it also retains unique characteristics.     

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.