A search for genes of predisposition to rheumatoid arthritis

Rheumatoid arthritis belongs to the group of autoimmune multifactor diseases with an essential involvement of genetic components in its genesis. The HLA DRB1* polymorphism was studied in 68 RA patients and in their 75 healthy relatives. 135 blood donors, who were tested at the Institute for Immunology of the Ministry of Health, Russian Federation, Moscow, were in the control group. The carrier-state of the HLA DRB1* 04 gene contributes to a higher probability of RA onset by 8.5 times, while the presence, in genotype, of genes HLADRB1* 02, 05, 06 reduces the risk of RA by 2.1, 2.3 and 7.2 times, respectively. Allele *0401 is encountered reliably more often in RA patients versus healthy controls. Within a sample of patients with familial RA, 43.9% turned out to be the carriers with various combinations of two alleles of genes coding the conservative amine acids of sequences QKRAA or QRRAA, which were named "shared epitope" (SE), versus 5.1% among the controls. The presence of homozygous "SE genotypes" among the RA patients contributed to a higher risk of morbidity by 5.8 times, while the carrier state of haplotypes with "duel SE positivity" enhanced the risk of morbidity by 17.8 times mainly due to the 01/0401 halotype. The RA linkage with two intragenic DNA markers, i.e. with the polymorphous micro-satellite replication of gene TCRA (CA)n and with the point-type changeability (localized in the coding area of the second variable region of V2 TCRD gene), was analyzed. The maximal possible value of the lod-point for (CA)n, i.e. replication of TCRA gene, was equal to +1.30 in males under the condition of zero recombination frequency, and to 16% of frequency recombination in females. The maximal possible value of the lod-point for the point-type changeability of gene TCRD was equal to +0.70 in males under the conditions of zero frequency recombination and to 40% of frequency recombination in females. The maximal lod-point value amounting to +1.20 in males and females in an identical frequency recombination of 5% was found on the basis of a three-point analysis of the linkage between RA and two intragenic markers from gene clusters coding the alpha- and beta-chains of T-cellular receptors. Therefore, our familial data are indicative of the opportunity of localizing the gene predisposed to RA is at a distance of 5 cM, in the direction of the telomere, from the locus of the examined (CA)n, i.e. replication of gene TCRA.     

A complex genetic rearrangement in a t(10;14)(q24;q11) associated with T-cell acute lymphoblastic leukemia.

The t(10;14)(q24;q11) is observed in the leukemia cells of 5-10% of cases of T-cell acute lymphoblastic leukemia (T-ALL). Recently, molecular analyses of a number of these translocations revealed simple reciprocal translocations between the T-cell receptor delta chain gene (TCRD) and a region of 10q24. We have characterized, at the molecular level, a t(10;14)(q24;q11) in a patient with T-ALL. The translocation in this case, in contrast to the previous cases, is part of a complex genetic rearrangement. In addition to a reciprocal translocation between the D delta 3 gene segment of TCRD and a region of 10q24, a local inversion occurred within TCRD, involving the D delta 2 and V delta 2 gene segments. As a consequence, the entire joining and constant regions and most of the diversity regions of TCRD are located on the derivative 14 chromosome, whereas the joining and constant regions of TCRA are positioned on the derivative 10 chromosome. The chromosome 10 breakpoint in our patient, as in other t(10;14), clusters within a 9 kb breakpoint region. The occurrence of seven breakpoints within a localized region of chromosome 10 implies the existence of a nearby gene whose activation may have conferred a selective advantage on the leukemia cells. Moreover, as in the previous cases, the translocation in the present study exhibits recombination signal sequences or signal-like sequences adjacent to the breakpoint junction. The presence of such motifs suggests the involvement of the recombinase enzyme system in the generation of this genetic alteration.     

Role of T cell receptor delta gene in susceptibility to celiac disease

There is a strong genetic influence on the susceptibility to celiac disease. Although in the vast majority of patients with celiac disease, the HLA-DQ(l ^* 0501, 1 ^* 0201) heterodimer encoded by the alleles HLADQA1 ^* 0501 and HLA-DQB1 ^* 0201 seems to confer the primary disease susceptibility, it cannot be excluded that other genes contribute to disease susceptibility, as indicated by the difference in concordance rates between monozygotic twins and HLA identical siblings (70% vs. 30%). Obviously other genes involved in the genetic control of T cell mediated immune response could potentially influence susceptibility to celiac disease. The density of T cells using the T cell receptor (TCR) is considerably increased in the jejunal epithelium of patients with celiac disease, an abnormality considered to be specific for celiac disease. This suggests an involvement of T cells in the pathogenesis of the disease. To ascertain whether the TCR delta (TCRD) gene contributes to celiac disease susceptibility we carried out an association study and genetic linkage analysis using a highly polymorphic microsatellite marker at the TCRD locus on chromosome 14q11.2. The association study demonstrated no significant difference in allele frequencies of the TCRD gene marker between celiac disease patients and controls; accordingly, the relative risk estimates did not reach the level of statistical significance. In the linkage analysis, performed in 23 families, the logarithm of the odds (LOD) scores calculated for celiac disease versus the TCRD gene marker excluded linkage, suggesting that there is no determinant contributing to celiac disease status at or 5 cM distant to the analyzed TCRD gene marker. In conclusion, the results of the present study provide no evidence that the analyzed TCRD gene contributes substantially to celiac disease susceptibility.Abbreviations AGA Antigliadin antibodies - EMA Antiendomysium antibodies - LOD score Logarithmic odds ratio - PCR Polymerase chain reaction - RR Relative risk - TCR T cell receptor     

T cell receptor excision circle assessment of thymopoiesis in aging mice

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in na?ve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of na?ve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 na?ve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.     

Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction

Abstract: Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.     

Identification of a new cluster of T-cell receptor delta recombining elements

Within the human T-cell receptor delta (TCRD) gene we have identified a new cluster of seven delta recombining elements (deltaRec2.1-2.7), located 2.6-5.2 kilobases downstream of the Vdelta2 gene segment. The deltaRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Ddelta3 and Jdelta1 segments of the TCRD gene as well as with the psiJalpha of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical deltaRec rearrangements. The total frequency of deltaRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, we assume that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-psiJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-psiJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. We hypothesize, that in a similar manner to the classical deltaRec rearrangements, the deltaRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-alphabeta lineage.     

Association of MICA polymorphism with rheumatoid arthritis patients in Koreans

To investigate whether genetic variations of MICA are associated with susceptibility to rheumatoid arthritis (RA), the (GCT)n microsatellite polymorphism of the transmembrane domain was analyzed in 144 Korean patients with RA and in 297 unrelated healthy controls. The allele frequency of MICA*A9 significantly decreased in RA patients compared with controls (9.0% vs. 15.3%, odds ratio [OR] = 0.55, p = 0.0098, p_c = 0.049), whereas the frequency of the MICA*A4 and MICA*A5.1 alleles tended to increase in RA patients (21.2% vs. 14.8%, OR = 1.55, p = 0.018, p_c > 0.05; 20.5% vs. 15.0%, OR = 1.46, p = 0.0403, p_c > 0.05). By subgroup analysis, the MICA*A4 allele significantly increased in seropositive RA patients versus controls (23.0% vs. 14.8%, OR = 1.69, p = 0.0082, p_c = 0.041). HLA-DRB1*0405 was strongly associated with RA (p_c = 0.0000008), and strong linkage disequilibrium was observed between HLA-DRB1*0405 and MICA*A4 alleles in controls (p_c = 0.000004) as well as in RA patients (p_c = 0.0012). In Korean patients, HLA-DRB1*0405 was primarily associated with RA and the weak association of RA with MICA*A4 was secondary to that with HLA-DRB1*0405. Additionally, MICA*A9 might have a weak protective effect on the susceptibility to RA in Koreans.     

IkappaBalpha promoter polymorphisms in patients with rheumatoid arthritis

To investigate the role of inhibitor of kappaBalpha promoter polymorphisms in the pathogenesis of rheumatoid arthritis (RA), 140 patients with RA and 115 healthy controls were enrolled in this study. The IkappaBalpha promoter polymorphisms were determined using the polymerase chain reaction/restriction fragment length polymorphisms method. In comparison with IkappaBalpha-826 C/C, the genotype frequency of IkappaBalpha-826 C/T was significantly higher in the patients with RA than that of the controls (P = 0.009, OR = 2.0, 95% CI = 1.2-3.4). The allele frequency of IkappaBalpha-826 T was also significantly increased in patients with RA when compared with that of the controls (P = 0.027, OR = 1.6, 95% CI = 1.1-2.4). In comparison with IkappaBalpha-550 A/A, the genotype frequency of IkappaBalpha-550 A/T was significantly decreased in patients with RA when compared with that of the controls (P = 0.02, OR = 0.2, 95% CI = 0.06-0.8). The allele frequency of IkappaBalpha-550 A was significantly increased in patients with RA (P = 0.007, OR = 5.1, 95% = 1.4-18.2). This study also revealed that the IkappaBalpha-826 T -550 A -519 C haplotype was significantly increased in patients with RA in comparison to that of controls (P = 0.01, OR = 1.8, 95% CI = 1.1-2.8). The IkappaBalpha-826 T and -550 A alleles are associated with susceptibility to RA. Moreover, the IkappaBalpha-826 T -550 A -519 C haplotype is associated with susceptibility to RA in Taiwan.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

异质性胞核核糖核蛋白A2基因的克隆、表达、纯化及其临床应用

目的：构建含异质性胞核核糖核蛋白A2（hnRNP A2) cDNA片段的基因克隆，制备并纯化重组蛋白hnRNP A2，用以检测抗hnRNP A2/RA33的抗体，方法，从人外周血单个核细胞提取细胞总RNA，应用RT－PCR方法扩增hnRNP A2 cDNA，将其克隆于pUC-T1质粒中，测定其序列。构建高效表达载体pET28a-hnRNP A2，转化大肠杆菌BL21（DE3）plys S并进行诱导表达，将含目的蛋白的组分经金属螯合树脂亲和层析柱进行蛋白纯化。以该纯化蛋白为包被抗原，ELISA方法检测类风湿关节炎（RA）97例，系统性红斑狼疮（SLE）50例，混合性结缔组织病（ECTD）8例，其他关节炎29例，其他弥漫性结缔组织病（CTD）99例，结果：构建hnRNP A2重组表达载体并在大肠杆菌中获得hnRNP A2高效表达；在RA，SLE，MCTD，其他关节炎，其他CTD患者中抗hnRNP A2/RA33抗体阳性率分别为36.8%,24%,75%,3.75%,10.10%，在RA中特异性为86．67％，结论：以纯化的基因重组蛋白hnRNP A2为抗原检测hnRNP A2/RA33抗体是早期诊断RA的可靠方法。     

Manganese superoxide dismutase and cytochrome P450 1A1 genes polymorphisms in rheumatoid arthritis in Taiwan

To investigate the role of manganese superoxide dismutase (MnSOD) and cytochrome P450 1A1 (CYP1A1) gene polymorphisms in the pathogenesis of rheumatoid arthritis (RA) in Taiwan, MnSOD and CYP1A1 genes polymorphisms were determined by he polymerase chain reaction/restriction fragment length polymorphism method in 112 patients with RA and 96 controls. There were no significant differences in the genotype, allele, and phenotype frequencies of MnSOD Ala-9Val (C1183T) polymorphisms between patients with RA and controls. The polymorphism of MnSOD 5777T, threonine at the 58th amino acid, cannot be found in RA patients and controls in Taiwan. The allele and phenotype frequencies of CYP1A1 4887A and genotype frequency of CYP1A1 4887C/A were lower in RA patients than in controls, whereas the significant difference was lost after correction. MnSOD C1183T polymorphisms were not associated with the clinical manifestations of RA. However, RA patients with CYP1A1 4889G/G have significantly higher frequency of Sj?gren's syndrome, especially in the presence of MnSOD 1183T/T. Patients with CYP1A1 4887C/A also have a trend to develop Sj?gren's syndrome in the presence of MnSOD 1183T/T. The linkage disequilibrium between CYP1A1 4889G and CYP1A1 6235C can be found in this study. MnSOD gene polymorphisms are not related to susceptibility to RA in Taiwan, whereas individuals with CYP1A1 4887A tend to avoid the development of RA. Moreover, CYP1A1 4889G/G and 4887C/A may play a role in the development of Sj?gren's syndrome, especially in the presence of MnSOD 1183T/T. These findings are preliminary. A further confirmation study is necessary.     

The Presence of Anti-Protein A Antibodies in Patients with Rheumatoid Arthritis

Sixteen patients with rheumatoid arthritis (RA) were examined for the presence of anti-protein A antibodies. The F(ab')2 preparations from five RA patients showed significant binding to IgG-free protein A on ELISA. The protein A binding was further examined by immunoblotting. The F(ab')2 preparations of high protein A-binding protein gave a specific reaction with IgG-free protein A on nitrocellulose paper. This demonstrates the presence of anti-protein A antibodies in patients with RA. Those RA patients with anti-protein A antibodies had more active disease as judged by the Lansbury's activity index. The level of serum rheumatoid factor (RAHA) was significantly higher in patients with anti-protein A antibodies than in those without anti-protein A antibodies.     

S100A4在类风湿关节炎滑膜中的表达及其对成纤维样滑膜细胞分泌VEGF促进血管生成的影响

目的:研究S100钙结合蛋白A4(S100A4)在类风湿关节炎(rheumatoid arthritis,RA)患者和正常人膝关节滑膜中的表达水平,以及S100A4对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis fibroblast-like synoviocytes,RAFLSs)促进血管生成的影响。方法:滑膜分别取自RA患者(RA组)及正常人(control组)膝关节,免疫组化法观察S100A4和VEGF蛋白在2组滑膜中的表达情况。RAFLSs分离自活动性RA滑膜;ELISA法检测S100A4刺激RAFLSs分泌血管内皮生长因子(vascular endothelial growth factor,VEGF)的作用;用rh S100A4孵育RAFLSs的条件培养基作用于人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),检测S100A4体外血管生成能力。结果:S100A4及VEGF蛋白在RA组滑膜中高表达(P0.05),rh S100A4显著刺激RAFLSs分泌VEGF,呈时间和剂量依赖性(P0.05);rh S100A4孵育RAFLSs的条件培养基可促进HUVECs在体外形成管腔。结论:S100A4蛋白在RA患者膝关节滑膜中高表达,S100A4可通过促进RAFLSs分泌VEGF来刺激滑膜血管生成。这些结果提示S100A4可作为治疗RA的潜在靶点。     

Gamma/delta T cells and their subpopulations in blood and synovial fluid from rheumatoid arthritis and spondyloarthritis.

T cell receptor (TCR) gamma/delta bearing lymphocytes in peripheral blood and synovial fluid from rheumatoid arthritis (RA) and seronegative spondyloarthritides (SSA) were evaluated by means of double label immunofluorescence and cytofluorographic analysis. Three monoclonal antibodies (MAb) were used. TCR delta-1 against a common delta chain epitope, BB3 directed against the T cell subset whose TCR is encoded by the V delta 2 gene, and A13 directed against the V delta 1 subset. Peripheral blood T gamma delta cells were significantly reduced compared to normal control blood in RA patients who had joint effusion but not in other RA patients. The decrease of T gamma delta in the RA PB was mainly confined to the A13+ subset. In RA synovial fluid (RA SF) T gamma delta cells were significantly increased compared to paired but not normal peripheral blood, the most significant being the increase of A13+ cells. In contrast, in patients with SSA, no change in T gamma delta cells was observed on PB or SF. These data suggest that in RA, but not SSA, T gamma delta cells migrate from PB to inflamed synovium and thus may be involved in the pathogenesis of RA.     

骨保护素基因单核苷酸多态性与类风湿关节炎发病的相关性研究

 目的：探讨骨保护素（OPG）基因163A/G及245T/G单核苷酸多态性（SNPs）与我国汉族人群类风湿关节炎（RA）发病的相关性。方法：采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术检测我国南方汉族正常人群及RA患者的OPG 163A/G 和245T/G 2个SNP位点;进行Hardy-Weinberg平衡检验;计算基因型和等位基因频率,及这2个位点的连锁关系,并分析这2个SNP位点与RA的关系。结果：所研究基因分布符合Hardy-Weinberg平衡,163A/G 位点基因型AA、AG、GG分布频率在2组比较有显著差异（P<0.05）;等位基因A、G分布比较在2组有显著差异（P<0.05）,携带163GG基因型者发生RA的危险性是非携带者的1.219倍（OR=1219, 95％CI：1066~2.339, P<0.05)。但245T/G位点各基因型及等位基因频率在2组中均未见差异（P>005）。结论：OPG 基因 163A/G SNP可能与我国汉族人群RA发病相关,携带G等位基因可能是发病的危险因素。     

Association of FCRL3 Genotypes with Susceptibility of Iranian Patients to Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a complex disease, the hallmark of which is synovial joint inflammation. The substantial contribution from genetic factors in susceptibility to RA has been well-defined. The Fc receptor-like3 (FCRL3) gene is one of the genes that have recently shown a significant association with RA. To determine the possible role of FCRL3-169 C/T and FCRL3-110 A/G gene polymorphisms in the development of RA in Iranian patients, 320 RA patients and 302 healthy subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism. No significant difference was found in genotype and allele frequencies of FCRL3-169 C/T between patients and controls. In contrast, at position ‐110 A/G, the frequency of the AA genotype and A allele was significantly decreased in RA patients compared to controls (p = 0.005). After Bonferroni correction for multiple testing, no significant correlations between FCRL3-169 C/T and ‐110 A/G polymorphism and laboratory and clinical features of the patients was observed. In conclusion, the results of this study showed a significant association between FCRL3-110 A/G polymorphism and susceptibility to RA.