EBV immortalized B lymphocyte cell lines have been extensively used as a source of biological material for functional and molecular studies and represent a potentially limitless source of genomic DNA. Current technologies for EBV transformation are costly and use relatively large volumes of peripheral blood. Alternative methods were examined to determine whether smaller volumes of cryo-preserved whole blood could be subsequently transformed and which could provide a more cost-effective strategy for large population-based studies such as UK Biobank. A successful method was established where viable B cells were positively selected from 0.5 ml cryo-preserved whole blood samples. These were EBV transformed in microtitre plates and subsequently expanded in culture. A pilot study within UK Biobank was performed, which confirmed its potential usefulness for this study.

We have identified two novel polymorphisms in the transforming growth factor beta 2 (TGFβ2) gene; an insertion in the 5′-untranslated region (5′UTR) and a single nucleotide polymorphism (SNP) in exon 1. A 895-bp fragment was analysed covering part of the 5′UTR and exon 1. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was performed to detect sequence variations. This was followed by the sequencing of samples demonstrating distinct banding patterns. A 4-bp insertion (ACAA) in the 5′UTR and a SNP (G > A) within exon 1 was identified. The 5′UTR polymorphism was found to be common in three Caucasian populations from Spain, Turkey and the UK. Exon 1 polymorphism is rare and results in an R to H amino acid substitution in codon 91. Both polymorphisms may prove useful for investigating possible associations of TGFβ2 with disease.

Allelic variants of the apolipoprotein E (APOE) gene influence the course of several neurological diseases. In multiple sclerosis the concentration of APOE in cerebrospinal fluid and its intrathecal synthesis is reduced. Specific isoforms of APOE may also be important and it has been suggested that possession of the e4 allele may be associated with a more aggressive disease process. These data prompted us to re-examine, in a large group of patients with multiple sclerosis, the proposal that allelism in the apolipoprotein gene influences disease course. Genotypes were determined in a well-defined group of 370 unrelated Caucasians with clinically definite multiple sclerosis and in 159 healthy controls. Age at onset, sex, disease duration, disease subtype were recorded. Disability was measured using the Kurtzke expanded disability status score in patients with a disease duration of 10 years or greater. There was no significant difference in APOE allele or genotype frequencies between patients and controls, between disease subtypes or between genders. APOE genotype did not significantly influence age of onset, and no significant relationship between genotype, allele frequency and disease severity was found. This study suggests that individual APOE alleles or genotypes do not determine disease susceptibility or the clinical course of multiple sclerosis.

Objective. To examine TNF microsatellite allele frequencies in SLE patients in the Greek population, where disease susceptibility is less associated with HLA-DR3 haplotypes.Methods. A cohort of 46 Greek SLE patients were investigated. Allele frequencies for the TNF microsatellite markers a, b, c and d were determined using a fluorescence based DNA fragment sizing technique. HLA class II typing was performed using a molecular based technique.Results. Associations between SLE and DRB1*1501, *1601 and *0701 were observed and DRB1*0301 was only marginally increased in patients. Linkage disequilibrium was found between DRB1*1501 and TNF a11 and also for DR3 and TNF a2, b3, d2. Stratification of patients suggested that DRB1*1501 and TNF a11 frequencies were higher in SLE patients with renal disease and TNF a2 and b3 frequencies in those without, although these differences did not reach statistical significance.Conclusions. SLE in this Greek population appears to be associated with a number of HLA-DRB1 alleles. The development of renal complications in these patients may be related to the TNF polymorphism encoded on these HLA haplotypes.

Objectives-Polymorphism of the phagocyte IgG receptor Fc gamma RIIa may modulate immune complex mediated inflammation, particularly when immune complexes contain IgG2. Previous studies suggest that this polymorphism may be an important risk factor for lupus nephritis. Fc gamma RIIa is biallelic, the alleles R and H each having a gene frequency of about 50%. Nephritis has been associated with an increased frequency of the R allele. The frequency of common Fc gamma RIIa alleles was examined in white subjects from the United Kingdom and Greek subjects with systemic lupus erythematosus (SLE) and healthy controls.Methods-Fc gamma RIIa genotyping was performed using a single step polymerase chain reaction technique, which differentiates the two major alleles, R and H. Two study populations were examined: (a) white subjects from the United Kingdom : 66 controls and 81 with SLE (19 of whom had renal disease) and (b) Greek: 52 controls and 42 with SLE (19 with renal disease).Results-No significant relation was observed between Fc gamma RIIa genotype and susceptibility to SLE or SLE nephritis.Conclusions-The Fc gamma RIIa R allele does not seem to be associated with SLE (with or without renal disease) in our United Kingdom white or Greek populations.

1. Doria A, Ghirardello A, Zambiasi P, Ru€atti A, Gambari PF. Japanese diagnostic criteria for mixed connective tissue disease in Caucasian patients. J Rheumatol 1992;19:259±64. 2. Asano S, Akaike Y, Jinnouchi H, Muramatsu T, Wakasa H. Necrotizing lymphadenitis: a review of clinicopathological, immunohistochemical and ultrastructural studies. Hematol Oncol 1990;8:251±60. 3. Dorfman RF, Berry GJ. Kikuchi's histiocytic necrotizing lymphadenitis: an analysis of 108 cases with emphasis on di€erential diagnosis. Semin Diagn Pathol 1988;5:329±45. 4. Imamura M, Ueno H, Matsumura A et al. An ultrastructural study of subacute necrotizing lymphadenitis. Am J Pathol 1982;107:292±9. 5. Tumiati B, Bellelli A, Portioli I, Prandi S. Kikuchi's disease in systemic lupus erythematosus: an independent or dependent event? Clin Rheumatol 1991;10:90±3. 6. Litwin MD, Kirkham B, Henderson DRF, Milazzo SC. Histiocytic necrotising lymphadenitis in systemic lupus erythematosus. Ann Rheum Dis 1992;51:805±7. 7. Ohta A, Matsumoto Y, Ohta T, Kaneoka H, Yamaguchi M. Still's disease associated with necrotizing lymphadenitis (Kikuchi's disease): report of three cases. J Rheumatol 1988;15:981±3. 8. Lin S, Ko W, Lee H, Hwang W. Kikuchi's disease associated with lupus-like syndromeÐa fatal case. J Rheumatol 1992;19:1995±6. 9. Aqel NM, Henry K, Woodrow D. Skin involvement in Kikuchi's disease: an immunocytochemical and immunōuorescence study. Virchows Arch (A) 1997;430:349±52. 10. Aqel NM, Henry K. Kikuchi's disease: an immunocytochemical study. J Pathol 1994;173:200A.

We have characterized TAP allele frequencies in a panel of 71 Yoruba Nigerians using ARMS-PCR. With the exception that TAP 2D was absent in Nigerians, TAP 2 allele frequencies in this population were found to be similar to those in a UK white population. HLA-DR4 also was found to be at a low frequency in Yoruba Nigerians (1.4%). This may reflect the absence of TAP 2D in Nigerians as DR4 and TAP 2D are in linkage disequilibrium in UK Caucasoids. The most frequent TAP 1 allele in Yoruba Nigerians was TAP 1A (49%). However, this value will be an underestimate as TAP1 alleles could not be unequivocally assigned in 41% of subjects using the ARMS-PCR methodology.

A panel of 43 early onset pauciarticular (EOPA) juvenile chronic arthritis (JCA) patients have been typed for human leucocyte antigens (HLA) DRB1, DPB1, DQA1 alleles, and DQB1*0603 status using molecular-based methods. Increased frequencies of DRB1*08 [odds ratio (OR) 7.7, 95% confidence interval (CI) 2.6-22.3], DRB1*11 (OR 3.1, 95% CI 1.2-8.1), DRB1*1301 (OR 7.7, 95% CI 2.6-22.3), DPB1*0201 (OR 3.5, 95% CI 1.6-8.0), DQA1*0103 (OR 4.4, 95% CI 1.5-13.3), DQA1*0501 (OR 2.9, 95% CI 1.3-6.6), DQA1*0601 (OR 30, 95% CI 3.6-241) and DQB1*0603 (OR 7.3, 95% CI 3.0-17.6) were found in the EOPA-JCA group compared with Caucasoid controls. Stratification of the EOPA-JCA group into antinuclear antibody (ANA) positive (n = 18) and ANA negative (n = 25) individuals revealed that ANA positivity was only associated with DRB1*1301 (OR 4.2, 95% CI 1.0-17.3), DPB1*0201 (OR 4.0, 95% CI 1.0-15.7) and DQB1*0603 (OR 11.5, 95% CI 2.5-53.4). Further analysis of the relative contributions of HLA antigens to ANA status revealed that DQB1*0603 determined the primary HLA effect. No apparent interaction between DQB1*0603 and DRB1*1301 or between DQB1*0603 and DPB1*0201 was found to contribute to the association with ANA. We suggest that those ANA positive individuals with a restricted HLA background, (DQB1*0603 positive), defines a group of EOPA-JCA patients which will be especially valuable in the characterization of the ANA associated with EOPA-JCA.

Aims: to determine the effect of HLA-DPB1 status on rheumatoid arthritis (RA) susceptibility and disease expression.Methods: HLA-DPB1 alleles were identified in 158 RA patients and 106 controls using PCR-sequence specific oligonucleotide probing. HLA-DPB1 allele frequencies were compared between patient and control groups and the strength of associations assessed using odds ratios and with 95% confidence intervals (CI). Associations observed in the total Ri group were confirmed using a relative predispositional effect (RPE) analysis.Results: an association between DPB1*0201 and RA was observed (OR 1.8, 95% Cl 1.0 - 3.4). By contrast, negative associations were found with DPB1*0301 (OR 0.5, 95% Cl 0.3- 1.0) and DPB1*1101 (OR 0.06, 95% Cl 0.001 - 0.5). These associations were confirmed using RPE analysis. On further analysis the increase in DPB1*0201 and decrease in DPB1*0301 frequencies in RA was found to be independent of DR4 status. The association of DPB1*0201 with RA appears to be most pronounced in male patients (OR 3.3, 95% Cl 1.3-8.3), seronegative patients (OR 2.6, 95% Cl 0.9-7.3) those with non-erosive disease (OR 2.6, 95% Cl 0.9-7.3) or in patients with high titre antinuclear antibodies (OR 2.4, 95% Cl 0.8-7.1).Conclusions: HLA-DPB1 alleles may be associated with the pattern of disease expression in certain RA patients and in some cases confer protection against disease.

OBJECTIVE:To determine whether an allelic form of mannose-binding protein (MBP) incapable of activating complement is associated with susceptibility to systemic lupus erythematosus (SLE).METHODS:MBP allele frequencies were determined by amplification refractory mutation system-polymerase chain reaction in 102 white SLE patients and 136 controls.RESULTS:The MBP allele that is unable to activate complement was present in 42 SLE patients (41%) and in 41 controls (30%) (P = 0.08, odds ratio [OR] = 1.6, 95% confidence interval [95% CI] 1.0-2.8). The gene frequency of this allele was 0.25 in SLE patients and 0.19 in controls (P = 0.08, OR = 1.5, 95% CI 1.0-2.3).CONCLUSION:Our results suggest that this allele of the MBP gene represents a minor risk factor for SLE.

OBJECTIVE:To examine the influence of genetic factors in determining the occurrence of rheumatoid factor (RF) isotypes. We investigated the hypothesis that, in twin pairs discordant for rheumatoid arthritis (RA), a genetic influence would be indicated by a higher rate of occurrence of RF among the unaffected monozygotic (MZ) when compared with the unaffected dizygotic (DZ) co-twins of seropositive affected twins.METHODS:IgM, IgA, and IgG RF were measured by ELISA in 70 MZ and 84 DZ disease discordant pairs using a cutoff for seropositivity defined using a normal control population. The risk of seropositivity in the unaffected twins of MZ when compared with DZ seropositive index twins was examined using odds ratios (OR).RESULTS:For all 3 RF isotypes, levels in the unaffected twins of seropositive index twins were higher than in the control population. MZ unaffected twins showed an increased risk for seropositivity for IgM and IgG RF when compared with DZ unaffected twins: IgM OR = 2.2 (95% CI 0.9-5.4), IgG OR = 2.4 (95% CI 0.9-6.6). The greatest excess risk for seropositivity occurred for IgM RF amongst the unaffected twin of an index twin with past or current documented evidence of RF seropositivity, OR = 3.4 (95% CI 1.4-8.5). For IgA RF, seropositivity risk in MZ unaffected twins was not increased, OR = 1.0 (0.3-3.1). The seropositivity risk for all 3 isotypes was independent of the age of the pair, the age of disease onset in the index twin, and the sex, HLA-DRB1*01 and DRB1*04 status of the unaffected twin.CONCLUSION:Genetic factors are important in determining the level of IgM and IgG RF. A genetic contribution to RA seropositivity exists that is independent of HLA-DR.

A panel of 43 early onset pauciarticular (EOPA) juvenile chronic arthritis (JCA) patients have been typed for human leucocyte antigens (HLA) DRB1, DPB1, DQA1 alleles, and DQB1*0603 status using molecular-based methods. Increased frequencies of DRB1*08 [odds ratio (OR) 7.7, 95% confidence interval (CI) 2.6-22.3], DRB1*11 (OR 3.1, 95% CI 1.2-8.1), DRB1*1301 (OR 7.7, 95% CI 2.6-22.3), DPB1*0201 (OR 3.5, 95% CI 1.6-8.0), DQA1*0103 (OR 4.4, 95% CI 1.5-13.3), DQA1*0501 (OR 2.9, 95% CI 1.3-6.6), DQA1*0601 (OR 30, 95% CI 3.6-241) and DQB1*0603 (OR 7.3, 95% CI 3.0-17.6) were found in the EOPA-JCA group compared with Caucasoid controls. Stratification of the EOPA-JCA group into antinuclear antibody (ANA) positive (n = 18) and ANA negative (n = 25) individuals revealed that ANA positivity was only associated with DRB1*1301 (OR 4.2, 95% CI 1.0-17.3), DPB1*0201 (OR 4.0, 95% CI 1.0-15.7) and DQB1*0603 (OR 11.5, 95% CI 2.5-53.4). Further analysis of the relative contributions of HLA antigens to ANA status revealed that DQB1*0603 determined the primary HLA effect. No apparent interaction between DQB1*0603 and DRB1*1301 or between DQB1*0603 and DPB1*0201 was found to contribute to the association with ANA. We suggest that those ANA positive individuals with a restricted HLA background, (DQB1*0603 positive), defines a group of EOPA-JCA patients which will be especially valuable in the characterization of the ANA associated with EOPA-JCA.

Objectifs. Etudier l'influence de l'allele HLA-DPB1 sur le risque de polyarthrite rhumatoide et l'expression clinique de la maladie. Methodes. Les alleles HLA-DPBI ont ete types par amplification genomique, en utilisant la reaction en chaine de la polymerase avec des sondes oligonucleotidiques specifiques, chez 158 malades souffrant de polyarthrite rhumatoide et chez 106 temoins. Les frequences des alleles HLA-DPB1 ont ete comparees chez les malades et les temoins, et les associations ont ete evaluees en calculant les odds ratios avec leurs intervalles de confiance a 95%. Les associations mises en evidence dans l'ensemble du groupe de malades ont ete confirmees grâce a une analyse de l'effet relatif sur la predisposition. Resultats. Nous avons mis en evidence une association positive entre DPB1*0201 et la polyarthrite rhumatoide (odds ratio, 1,8; intervalle de confiance a 95%, 1,0-3,4). A l'inverse, il y avait une association negative avec DPB1*0301 (0,5 et 0,3-1,0) et DPB1*1101 (0,06 et 0,001-0,5). Ces associations ont ete confirmees par l'analyse de l'effet relatif sur la predisposition. L'augmentation de la frequence de DPB1*0201 et la diminution de DPB1* 0301 dans la polyarthrite rhumatoide etaient independantes de la presence ou de l'absence de DR4. L'association de DPB1*0201 avec la polyarthrite rhumatoide semblait plus nette chez les hommes (odds ratio 3,3; intervalle de confiance a 95%, 1,3-8,3), les sujets seronegatifs (2,6 et 0,9-7,3), ceux qui n'avaient pas d'erosions (2,6 et 0,9-7,3) et ceux qui avaient un titre eleve d'anticorps antinucleaires (2,4 et 0,8-7,1). Conclusions. Les alleles HLA-DPB1 pourraient etre lies a l'expression clinique de la polyarthrite rhumatoide, et certains d'entre eux pourraient diminuer le risque de developper la maladie

The objective of the study was to investigate the genetic contribution to the clinical expression of rheumatoid arthritis (RA) by comparison of disease features in RA-concordant monozygotic (MZ) twin pairs. Fourteen RA-concordant MZ twin pairs recruited from a nation-wide study were examined to determine the degree of similarity in: (a) age of disease onset; (b) pattern of joint involvement; (c) pattern of extra-articular disease; (d) toxic reactions to drugs; (e) disease course; and (f) serology for rheumatoid factor (RF) and antinuclear antibody. There was considerable within-pair diversity in the variables studied. Some similarity within twin pairs was observed for the ages at disease onset (R = 0.63), presence of erosive changes (kappa = 0.61) and the presence of IgM RF (R = 0.87). No important similarity was seen, however, in the pattern of joint involvement, the occurrence of extra-articular disease, adverse drugs reactions, clinical disease course and reported disability level. There is heterogeneity in the genetic contribution to the clinical expression of RA. The overall lack of similarity for the majority of clinical variables indicates the importance of non-genetic factors on the expression of disease.