中国汉族白血病患者及其相关人群罕见的HLA-DR/DQ连锁不平衡研究

 目的 研究中国汉族白血病患者及其相关人群罕见的HLA-DR/DQ连锁不平衡单倍型。方法 对2000－2005年在我院进行异基因造血干细胞移植前HLA配型的白血病患者及与患者有血缘关系的家系供者共1500例的血液标本，采用低分辨序列特异性引物聚合酶链反应（PCR-SSP）方法进行HLA-DR/DQ基因分型，并对两位点间连锁不平衡参数进行统计学分析。1500例中患者650例，平均年龄25岁；家系供者850例，平均年龄42岁。结果 在41例的血液标本中发现13种罕见的连锁不平衡单倍型，主要为HLA-DQ8 或HLA-DQ9与不同DR位点的连锁。其中DR14/DQ4、DR4/DQ5、DR9/DQ6、DR9/DQ7、DR8/DQ8、DR9/DQ8、DR12/DQ8、DR13/DQ8和DR14/DQ9共9种单倍型尚未见报道。650例白血病患者中有20例存在12种罕见的连锁不平衡单倍型，850例家系供者中有21例存在8种罕见的连锁不平衡单倍型。DR8/DQ8单倍型只见于家系供者，而DR14/DQ4、DR12/DQ6、DR11/DQ8、DR13/DQ8和DR14/DQ9单倍型则只见于白血病患者。41例HLA-DR/DQ基因分型结果显示，连锁不平衡单倍型与DR52（DRB3）宽抗原相关联者占58.5%（24/41），与DR53（DRB4）宽抗原相关联者占36.6%（15/41），而与DR51（DRB4）宽抗原相关联者仅占4.9%（2/41）。所发现单倍型频率最高的为DR12/DQ8（0.0023）和DR9/DQ8（0.0023），其次为DR11/DQ9（0.0020）和DR12/DQ9（0.0017）。13种连锁不平衡单倍型的绝对及相对连锁不平衡参数均为负值，说明它们在中国汉族人群中较为罕见，并处于连锁不稳定状态。结论 发现了罕见的DR/DQ连锁不平衡单倍型，对补充中国汉族人群HLA-DR/DQ基因的连锁不平衡类型，提高HLA分型结果的准确性具有一定意义；同时，DR/DQ连锁不平衡单倍型在不同人群中的差异为疾病关联研究提供了思路。     

15个HLA—DR5HTC的RFLP结果分析及与细胞学分型比较结果

本文报告国内外15个DR 5纯合细胞(HTC)的RFLP结果,其中9个HTC还做了MLC棋盘。研究结果表明,DR5是一个高度多态的抗原决定簇。根据DR和DQ的多态性可将DR5HTC分成若干亚型,这些亚型与细胞学检出的D_w特异性相吻合,说明DR和DQ都参与了D_w特异性的表达。15个HTC中有4个是中国人DR5HTC,RFLP和MLC棋盘结果都表明,它们的D_w特异性与现知的D_w5、DB2和DB6都不同,分别属于3种新的D_w特异性。     

CⅡTA基因编码区不同单倍型cDNA的功能研究

目的 研究MHCⅡ类反式激活因子(cⅡTA)基因编码区非同义单核苷酸多态性(SNP)位点C19170G(Leu45Val)和C30799G(Ala500Gly)构成的4种不同单倍型cDNA的功能.方法 将4种不同单倍型的真核表达载体和卒载体分别转染至HeLa细胞.用RT-PCR和间接细胞免疫荧光技术检测未经转染的HeLa细胞、转染4种真核表达载体及卒载体的HeLa细胞cⅡTA mRNA与3种HLAⅡ类分子(HLA-DR、DP、DQ)的表达,并用流式细胞技术对其表达的3种HLAⅡ类蛋白进行定量分析.结果 未经转染和转染空载体的HeLa细胞均尤CⅡTA mRNA和3种HLAⅡ类分子的表达,而转染4种不同单倍型真核表达载体的HeLa细胞均出现CⅡTA mRNA表达,并表达3种HLAⅡ类分子.证实了转染4种不同单倍型真核表达载体后的HeLa细胞3种HLAⅡ类分子表达水平差异无统计学意义(P均>0.05).结论 中国人CⅡTA基因编码区这两个SNP位点的多态性(2个位点氨基酸的改变)不影响CⅡTA反式激活HLAⅡ类基因表达的能力.     

HLA—DR位点的PCR—SSP基因分型

目的：探讨适用于肾移植供体HLA-DR的快速基因分型方法。方法：自行设计合成引物建立顺序特异性引物聚合酶链反应技术,并对110例肾移植供体作HLA-DR基因分型。结果：DNA提取方法可以满足PCR-SSP分型方法对DNA模板的要求。全部操作耗时120min,110例标本均分型成功。基因频率范畴在0.0045-0.1227之间,以DR4、DR15和DR9占多数。结论：PCR-SSP法作HLA-DR分型具有准确,简便,快速等优点。适合在临床进行推广应用。     

应用毛细管参考链介导的构象分析法进行HLA-DR4等位基因分型

目的应用毛细管参考链构象分析法对HLA—DR4进行等位基因分型,并与测序分型结果相比较,评价该方法的准确性及实用性。方法首先应用FAM标记的引物扩增HLA-DRBl等位基因为纯合型的样本DRB1*0803、DRB1*0901的第二外显子作为参考链,与标准样品HLA-DR1第二外显子杂交获得异源双链,借助测序仪通过毛细管电泳建立中国人HLA-DR48个高频率等位基因的标准迁移率,再对30例DR4样本进行分型,并与测序分型法检测结果进行比较。结果30例样本中28例与测序法测得的等位基因结果一致。结论毛细管参考链介导的构象分析法具有准确性高、分辨率高的优势,与测序法相比具有成本低、高通量的特点,值得在移植、相关疾病等领域的HLA分型中推广应用。     

强的松对小儿原发性肾病综合症患者PBMC HLA表达的调节作用

目的: 初步探讨糖皮质激素对外周血单个核细胞(PBMC)人类白细胞抗原(HLA)表达的调控作用.方法: 肾病综合征(NS)患儿PBMC体外经IFN-γ联合PHA 刺激诱导活化,用不同浓度强的松干预,采用流式细胞术检测其PBMC组成性、活化后和强的松处理后HLA-Ⅰ/Ⅱ类分子和HLA-DR7分子的表达量并与正常对照组进行分析比较.结果: (1)体外培养NS患儿及正常人PBMC组成性表达HLA-Ⅰ、Ⅱ类分子和HLA-DR7分子,且NS患儿高于正常人(尤以HLA-Ⅱ类分子突出,为正常人的11.78倍,P＜0.01);(2)体外经 IFN-γ联合PHA活化后HLA-Ⅰ/类分子和DR7分子表达增加(P＜0.01);(3)经不同浓度强的松处理后,HLA-Ⅰ/类分子和DR7分子比活化后表达减少(P＜0.01);(4)经不同浓度强的松预处理后,与活化组比较HLA-Ⅰ、Ⅱ类分子和HLA-DR7表达减少(P＜0.01),对NS患儿的抑制率要高于正常人,并且HLA-Ⅱ类分子表达的抑制作用表现出剂量依赖性(P＜0.01).结论: NS患儿体内存在异常活化的T细胞;糖皮质激素对IFN-γ诱导活化的PBMC HLA表达有抑制作用.     

3个X染色体短串联重复的复合扩增及其多态性

目的　建立 3个 X染色体特异性短串联重复 ( X- chromosome specific shorttandem repeat,X- STR)基因座的复合扩增体系 ,并调查其多态性。方法　复合扩增 DXS6 799、DXS6 80 4和 DXS6 85 4 3个基因座 ,并用聚丙烯酰胺凝胶电泳和银染法显色技术进行基因分型。结果　在广东汉族 2 6 2名无关男性个体及 2 5 5名无关女性个体中 ,3个基因座均发现了 7个等位基因 ,男性个体共检出 73种单倍型 ,单倍型多样性为 0 .96 74。结论　这 3个基因座的复合扩增系统有较高的多态性信息 ,在个体识别和亲权鉴定中有潜在的应用价值     

3个X染色体短串联重复的复合扩散及其多态性

目的 建立3个X染色体特异性短串联重复(X-chromosome specific shortt andem repeat，X-STR)基因座的复合扩增体系，并调查其多态性。方法 复合扩增DXS6799、DXS6804和DXS68543个基因座。并用聚丙烯酰胺凝胶电泳和银染法显色技术进行基因分型。结果 在广东汉族262名无关男性个体及255名无关女性个体中，3个基因座均发现了7个等位基因，男性个体共检出73种单倍型，单倍型多样性为0．9674。结论 这3个基因座的复合扩增系统有较高的多态性信息，在个体识别和亲权鉴定中有潜在的应用价值。     

瘢痕疙瘩与HLA-II类基因相关性研究

目的 :探讨免疫遗传学因素在瘢痕疙瘩发病机制中的作用。方法 :用聚合酶链反应 序列特异性引物 (PCR SSP)技术对 5 0例瘢痕疙瘩患者进行HLA Ⅱ类基因分型 ,并以 1 0 0例正常人的HLA Ⅱ类基因为对照。结果 :瘢痕疙瘩组HLA DQ5抗原频率 ( 3 0 % )明显高于对照组 ( 1 4% ) ,相对危险率 (RR) =2 .63 ;HLA DR1 7和HLA DQ8抗原频率 ( 2 % )明显低于对照组 ( 1 4% ,1 5 % ) ,RR =0 .1 3 ,0 .1 2。结论 :瘢痕疙瘩与HLA DQ5基因呈正相关 ,与HLA DR1 7和HLA DQ8基因呈负相关。HLA DQ5、HLA DR1 7和HLA DQ8基因可能是瘢痕疙瘩患者易感的单倍型     

IDDM易感性中HLA遗传因素的异质性

人们已知胰岛素依赖性糖尿病(IDDM)与HLAⅡ类抗原DR3和DR4相关,在DR3/DR4杂合个体中产生IDDM的危险性明显增高。自发现HLA-DQ多态性以来,在IDDM研究中应用了RFLP分析,并发现一些与特定HLA-DR等位基因连锁不平衡     

Analysis of HLA-DRB and -DQB gene RFLPs in DR7 homozygous cell lines: associations with Dw11, Dw17 and DB1

The DR7-associated Dw specificities, Dw11, Dw17 and DB1 were investigated with regard to DRB- and DQB-gene polymorphism, as revealed by RFLP analysis using the restriction enzyme TaqI. In the 22 DR7 homozygous cell lines investigated, each of these Dw specificities was found to correlate to one specific RFLP defined DR-DQ haplotype. In addition, a clear linkage disequilibrium to a specific HLA-B locus allele for each Dw specificity was noted, indicating that the Dw subtypes of DR7 often are associated with a conserved HLA-B-DR-DQ haplotype. Only one genetically homozygous cell line, PLH, deviated from these correlations. This cell line, notably derived from an individual with a deletion of the 21-hydroxylase B-gene (21-OHB), caries the HLA haplotype Bw47, DR7, DQw2, DB1, but displayed a DRB RFLP otherwise found in association with Dw17.     

HLA-DR4 associated Dw types in rheumatoid arthritis

Frequencies of HLA-DR4 and its related Dw types were compared between randomly selected normal controls and the index cases of multiplex rheumatoid arthritis (RA) families. A DR4 frequency of 68.3% was observed in index cases (n = 57) compared to 31.2% in normal controls (n = 96). Cellular typing with homozygous typing cells (HTCs) revealed significant increases of Dw4 (49.1% vs 22.9% RR = 3.2 p less than 0.001) and Dw14 (22.8% vs 2.1% RR = 13.9 p less than 0.001) in the index cases. A non-significant increase was seen for Dw13 (8.8% vs 4.1%). When DR4 positive patients and controls were compared, a significant increase was seen only for Dw14 (34.2% vs 6.6% RR = 7.3 p less than 0.01). Data from HLA genotyped RA and normal families allowed an examination of haplotype combinations of HLA-B antigens and DR4/Dw types to be made. HLA-Dw4 was predominantly found with B44 and Bw62 with nearly all DR4/Bw62 haplotypes being Dw4 positive. HLA-Dw13 was associated with B44 and Dw14 with Bw60, B44 and B27. Based on HTC and normal family data. Dw10 was found to be strongly associated with B38 containing haplotypes. Analysis of 69 C4A, C4B complement typed DR4 haplotypes failed to show any statistically significant association between Dw type and "complotype". However, there was a suggestion of C4A3. BQO being associated with Dw4 (34.2% vs 16.1% X2 = 2.9 p = ns) and C4A3, B1 with Dw14 (45.5% vs 27.6% X2 = 2.1 p = ns).(ABSTRACT TRUNCATED AT 250 WORDS)     

New HLA-D Alleles Associated with DRI and DR2

The present study describes two new HLA-D specificities : LD 13, associated with DR1, and LD14 associated with DR2. LD13 is defined by an HTC who is the bc offspring of an a: A25, B18, DR7, Dw7/b: A33, B14, DR1, Dx father, and of a c: A24, B14, Dr1, Dx/d: A26, B41, DR5, Dw5 mother. This HTC was included both as a responder and as a stimulator in our cross-reference studies of 8W HTCs. While failing to cluster with any other 8W HTC, it typed 2 of 64 panel members carrying a "blank" HLA-D, linked to DR1. To exclude the possibility that HTC-LD13 might be a split of Dwl, the entire family was tested with the Family Set of 8W HTCs. No typing responses to any 8W Dw1 HTCs were observed. Furthermore, checkerboard experiments between HTC-LD13 and 8WDw1 HTCs showed strong reciprocal stimulation. The LD13 specificity was only found in Ashkenazi Jews and may be in linkage disequilibrium with HLA-B14. LD14 is defined by three, SD different, HTCs deriving from the same family of Sicilian descent. The family was included in the 8th Workshop and each HTC was shown to have inherited DR2, MT1 from both parents. When tested as stimulators, on our HLA-D reference panel, these cells were clustered in a distinct group, LD14, associated with DR2. None of the 8W HTCs appeared to belong to this cluster. The antigen frequency of LD14 is 0.03.     

Cell membrane polymorphisms coded for in the HLA-D/DR region I. Relation between D and DR

The relation of HLA-D and -DR determinants was studied in Dutch Caucasoids. The recognition of subgroups of DR4, DR5, and DR7, and the specificities LB12 and LB13 are described. Phenotype and gene frequencies and a Hardy-Weinberg analysis of DR and local (LB) B-cell groups are given. Excellent correlation between D and DR typing was obtained when HTCs were studied by selected B-cell antisera. When the same sera were used to type a panel of D typed cells, the correlation was decreased (with the exception of DR3 and Dw3). In the case of discrepancies the DR specificity, but not the corresponding D specificity, always could be found and not the other way around.

The data fit best the assumption that HLA-D and -DR are carried by the same molecule, although they might be different determinants on this molecule. A number of possible explanations for the observed discrepancies has been given.     

Ninth International Histocompatibility pre-workshop testing of Dw6 HTCs. Two subtypes of Dw6

In the scope of the cellular part of the 9th International Histocompatibility Workshop, the offered homozygous typing cells (HTCs) of several specificities have been screened in a pre-Workshop. In Leiden and Seattle all HTCs typing for "HLA-Dw6" have been tested. This implied that in both laboratories mixed lymphocyte culture (MLC) matrices were performed between the Dw6 HTCs and that all HTCs were tested as stimulator cells against a panel of heterozygous responder cells. The results clearly demonstrated that "HLA-Dw6" as defined by the different participating laboratories can be split into two major groups, Dw6.a and Dw6.b. This observation confirms and extends previous reports. None of the 9th Workshop B-cell sera could discriminate between the two presently described subgroups of HLA-Dw6.     

DNA restriction fragment length polymorphisms characteristic for Dw subtypes of DR2

DNA restriction fragment length polymorphisms (RFLP) can be easily demonstrated in DNA of cells expressing different DR specificities when class II cDNA probes are used for hybridization. Previous studies of DR4+ homozygous typing cells (HTCs) carrying different Dw subtypes, however, detected no RFLP correlating with subtypes. In contrast, we report here Southern blotting studies of DR2+ HTCs carrying different subtypes which showed RFLP patterns characteristic for each subtype, using both DR beta and DQ beta probes and several restriction enzymes. The RFLP between subtypes of DR2 was, however, appreciably lower than that found between DR specificities.     

Five HLA-D clusters associated with HLA-DR4

In order to investigate the HLA-D clusters associated with DR4, 54 DR4-positive, Dw4- and Dw10-negative responders, together with selected Dw4- or Dw10-positive responders, were tested with 22 HTCs that define DR4-associated D specificities. The results are consistent with previous data defining four distinct D clusters--Dw4, Dw10, DB3, and DYT--and have identified a new cluster provisionally termed LD40. In addition, the DB3 cluster is complex and appears to give typing response patterns overlapping those of the KT2 cluster originally defined as being associated with DR4 in Japanese populations. Of 116 DR4-positive haplotypes tested, 44% typed as Dw4, 18% were LD40, 16% were Dw10, 9% were DB3, 3% were DYT, and 10% gave no typing response to the HTCs defining any of these clusters. These studies are informative not only in defining the DR4-associated D clusters and in supporting the concept that D and DR cannot be considered identical but also in emphasizing the complexity of the D region.     

Extended HLA haplotypes in families with insulin-dependent diabetes mellitus in Northern Finland

Haplotypes including HLA, Bf and C4 loci were analyzed in a material comprising 55 families with diabetic children. One hundred and ten haplotypes found in IDDM patients were compared with 101 haplotypes present only in healthy family members. Two complotypes, BfSC4A3B3 and SC4A0B1 . were significantly more common (P <0.05) in the diabetic haplotypes, and these were in most cases found in haplotypic combinations with HLA-B15,Dw4,DR4 and HLA-B8.Dw3,DR3 genes, respectively. The B8/DR3 haplotype was better conserved, as 72% included the BfSC4A0B1 complotype as compared with only 35% of the B15/DR4 haplotypes with "high risk" C4A3B3 complement alleles (p <0.05). DR3 was found in 26% of the diabetic haplotypes and DR4 in 43%. DR4 associated with the Dw4 in 69% of cases and with D w14 in 26% of the diabetic haplotypes. Our results confirm that the two phenotypes found earlier to be associated with IDDM in Northern Finland, e.g. "B15, BfS, C4A3B3, Dw4, DR4" and "B8, BfS, C4A0B1, Dw3, DR3" are inherited as haplotypes.     

B27-bearing HLA haplotypes in rheumatoid arthritis: characterization in Finnish patients

The frequency of human leukocyte antigen (HLA)-B27 has been found to be increased in rheumatoid arthritis (RA) in Finland and marginally also in some other populations. In the present study HLA-B27-bearing haplotypes in RA patients were found to carry DR1 and DR4 genes more often than do B27 haplotypes in control population. B27;DR4,DW14;DQw7 was a typical B27-DR4 haplotype whereas DR4 in the majority of other haplotypes occurs with Dw4 and DQw8 genes. The result indicates that the B27 association with RA is not independent of DR1 and DR4, but whether the B27;DR4;DQw7 haplotype subjects a person to a higher disease risk than do other DR4 haplotypes, or is associated with a more severe course of the disease, remains to be investigated.     

DR2+ haplotypes in insulin-dependent diabetes: analysis of DNA restriction fragment length polymorphisms

Insulin-dependent diabetes (IDD) is strongly associated with certain HLA class II (Ia) antigens. The frequency of DR2 is significantly reduced in IDD; among DR2+ patients, the frequency of the subtype specificity Dw2 defined with homozygous typing cells (HTCs) is significantly reduced compared to DR2+ controls, and the specificity LD-MN2, which we have defined using primed lymphocyte typing reagents, is significantly increased. We have studied DNA restriction fragment length polymorphisms (RFLP) of DR2-LD-MN2+ individuals and homozygous typing cells carrying specificities antigenically related to LD-MN2. Using a number of different restriction enzymes, a characteristic pattern of fragments could be defined for DR2-LD-MN2 using both DQ beta and DR beta cDNA probes. This pattern was shared with some but not all of the antigenically related HTCs, and was distinct from that of DR2-Dw2. The RFLP pattern of DR2-LD-MN2 obtained with the DQ beta probe is identical, except for one band, to that of DR1-Dw1, suggesting that at least some part of the DQ region is identical in these two haplotypes. These results indicate that analysis of RFLP patterns can be used to help identify the genetic regions and, eventually, genes most important in the association of HLA and IDD.