The effect of plasmapheresis on the concentration of certain plasma proteins: a case identified by an inaccurate LDL-cholesterol estimation.

A 35-year-old man was found to have a negative LDL-cholesterol concentration (-0.05 mmol/L) when estimated on a fasting plasma sample using the Friedewald equation. Plasma urea, electrolytes and liver function tests (LFTs) were normal except for a raised total bilirubin of 74 micromol/L. Haematological results showed both a low haemoglobin and fibrinogen concentration. It transpired that the patient had undergone daily plasmapheresis treatments on the previous four days; plasma had been exchanged with a 5% albumin solution. He had been diagnosed with Evan's syndrome previously (characterized by autoimmune haemolytic anaemia) and had been admitted with severe anaemia, which had proved unresponsive to conventional treatments. The concentration of most plasma substances is reduced by 50-60% after one standard plasmapheresis treatment, with the rate of return to steady state concentrations varying among analytes. The finding of a negative LDL-cholesterol concentration (arising primarily as a result of normal triglyceride concentrations) may reflect the more efficient removal of LDL and HDL lipoproteins during the plasmapheresis procedure (PP) than lipoproteins containing proportionally more triglycerides. Plasma lipids, total protein, immunoglobulins and transferrin had recovered to steady state concentrations by eight days post-plasmapheresis, whereas caeruloplasmin concentrations had not. This case report illustrates the difficulties of obtaining accurate information on the steady state concentrations of plasma analytes, in particular protein bound substances, when analysis is carried out on a sample from a patient that has recently undergone plasmapheresis. The normal plasma albumin in this situation did not flag the possibility of the sample being artefactually diluted.

EBV Immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood.

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.

Two novel polymorphisms in the human transforming growth factor beta 2 gene.

We have identified two novel polymorphisms in the transforming growth factor beta 2 (TGFbeta2) 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 TGFbeta2 with disease.

Polymorphisms of apolipoprotein E; outcome and susceptibility in multiple sclerosis.

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 epsilon4 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.

HLA-TNF haplotype heterogeneity in Greek SLE patients.

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 DRB*1501 and TNF a11 frequencies were higher in SLE patients with renal disease and TNF a2 and b 3 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.

Fc gamma RIIa polymorphism in systemic lupus erythematosus.

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.

Rheumatoid factor isotypes in monozygotic and dizygotic twins discordant for rheumatoid arthritis.

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.

Antinuclear antibodies in early onset pauciarticular juvenile chronic arthritis (JCA) are associated with HLA-DQB1*0603: a possible JCA-associated human leucocyte antigen haplotype.

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.

Absence of TAP 2D in Yoruba Nigerians.

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.

Heterogeneity of disease phenotype in monozygotic twins concordant for rheumatoid arthritis.

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.

HLA-DPB1*0201 is associated with particular clinical features of rheumatoid arthritis.

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 RA group were confirmed using a relative predispositional effect (RPE) analysis. RESULTS: an association between DPB1*0201 and RA was observed (OR 1.8, 95% CI 1.0-3.4). By contrast, negative associations were found with DPB1*0301 (OR 0.5, 95% CI 0.3-1.0) and DPB1*1101 (OR 0.06, 95% CI 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% CI 1.3-8.3), seronegative patients (OR 2.6, 95% CI 0.9-7.3) those with non-erosive disease (OR 2.6, 95% CI 0.9-7.3) or in patients with high titre antinuclear antibodies (OR 2.4, 95% CI 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.

Mannose-binding protein gene polymorphism in systemic lupus erythematosus.

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.

"Homozygosity" for the HLA-DR shared epitope contributes the highest risk for rheumatoid arthritis concordance in identical twins.

OBJECTIVE: To assess the contribution of HLA-DRB1 alleles in determining rheumatoid arthritis (RA) concordance in monozygotic twins. METHODS: Ninety-one monozygotic twins pairs in which at least 1 twin was affected were typed for HLA-DRB1 using both serologic methods and polymerase chain reaction amplification with sequence-specific oligonucleotide hybridization. The role of DR4 and of the shared epitope in disease concordance was investigated. Relative risks (RR) with 95% confidence intervals were determined. RESULTS: Increased concordance for RA was observed in both DR4 positive and shared epitope positive pairs (RR 3.4 and 3.7, respectively). A 5-fold risk for RA concordance was seen in twins who were "homozygous" for the shared epitope, compared with those negative for the shared epitope. CONCLUSION: In the absence of the shared epitope, RA concordance in monozygotic twins is rare. In contrast, "homozygosity" for the shared epitope is the most important factor in determining RA concordance.

Influence of previous exposure to human parvovirus B19 infection in explaining susceptibility to rheumatoid arthritis: an analysis of disease discordant twin pairs.

OBJECTIVES: To assess the association between exposure to parvovirus B19 and susceptibility to rheumatoid arthritis (RA). METHODS: One hundred and fifty five twin pairs (76 monozygotic (MZ) and 79 dizygotic (DZ)), discordant for RA, were tested for the presence of IgG antiparvovirus antibodies using ELISA. The data obtained were analysed using conditional logistic regression, from which odds ratios and 95% confidence intervals were calculated. RESULTS: Overall, there was no association between exposure to parvovirus and RA (OR = 1.2, 95% CI: 0.7-1.7). However, in two subgroups there was a suggestion of an association. These were: (1) pairs where the affected twin was rheumatoid factor (RF) seronegative (OR = 2.0, 95% CI: 0.9-12.4) and (2) in opposite-sexed twin pairs where the affected twin was female (OR = 3.0, 95% CI: 0.9-11.6). CONCLUSION: Previous exposure to parvovirus infection did not explain disease susceptibility in both MZ and DZ discordant pairs with rheumatoid arthritis. This infection, however, might be relevant in some subgroups.

Twin concordance rates for rheumatoid arthritis: results from a nationwide study.

We report the concordance rate for RA in a nationwide study of 91 monozygotic (MZ) and 112 dizygotic (DZ) pairs. Twin pairs were recruited from both a national media campaign and a 2-month prospective inquiry of all UK rheumatologists. Disease status was established following a structured clinical and serological appraisal, together with radiological assessment where necessary. Zygosity was confirmed using DNA fingerprinting. In all, 14 (15.4%) of the MZ and four (3.6%) of the DZ pairs were disease concordant (risk ratio: 4.3 95% CI 1.5 to 12.6). There was no difference in the concordance between the media and clinical derived twins. Further the excess MZ concordance persisted after adjusting for age, age at disease onset, sex and rheumatoid factor status. Analysing the data in relation to the timing of disease onset in the first affected twin showed that subsequent disease risk in the initially unaffected co-twins of the MZ affected probands increased with increasing duration of follow-up. We conclude that the overall MZ concordance at 15% is lower than the 30% figure normally quoted from a study some 30 years ago and sets a ceiling at the potential genetic contribution to disease susceptibility.

A shared HLA-DRB1 sequence confers RA susceptibility in Greeks.

Previous serological studies of Greek rheumatoid arthritis (RA) patients have failed to demonstrate an association with DR4. Using sequence specific oligonucleotide typing we have identified the DRB1 alleles in panels of Greek RA patients and controls. When patient and control HLA-DRB1 frequencies were compared, significantly higher frequencies of DRB1*0101 (23.3% vs. 7.0%, odds ratio [OR] 4.0, 95% confidence intervals [CI] 1.4-12.0) and DRB1*1001 (20.9% vs. 5.8%, OR 4.3, 95% CI 1.3-13.7) were found in RA patients compared with controls. No association of DRB1*04 with RA was observed (20.9% vs. 14.0% in controls) confirming earlier reports. However DRB1*04 subtyping demonstrated a small but significant increase of DRB1*0405 in patients (14.0% vs. 3.5%, OR 4.5, 95% CI 1.1-18.9). When the frequency of individuals carrying the shared RA susceptibility epitope was compared between patients and controls it was found to be significantly higher in RA patients (60.5% vs. 22.1%, OR 5.4, 95% CI 2.4-12.0). We conclude that the shared epitope is significantly associated with RA in this population, but that it is predominantly accounted for by DRB1*0101 and DRB1*1001. Previous studies of UK RA patients have demonstrated a negative association of DR2 with disease and articular erosions. HLA-DR2 variants, DRB1*1501 and *1502 are not at reduced frequency in Greek RA patients (DRB1*1501, 14.0% in patients vs. 7.0% in controls; DRB1*1502, 7.0% in patients vs. 7.0% in controls). Genes conferring RA resistance may be in linkage disequilibrium with DR2 in UK patients. This does not appear to be the case in Greek RA patients. No association was seen between RA and HLA-DPB1 type.

Absence of an association between HLA-DRB1*04 and rheumatoid arthritis in newly diagnosed cases from the community.

OBJECTIVES: To determine HLA-DR4 and DR1 allele frequencies in a series of patients with newly diagnosed early inflammatory arthritis. METHODS: HLA-DR1 and DR4 frequencies were determined by oligonucleotide typing of 208 patients classified as having either rheumatoid arthritis (RA) or undifferentiated inflammatory polyarthritis. RESULTS: The frequency of occurrence of DR4 in these patients with RA did not differ significantly from that in controls in the United Kingdom (42 v 37%). HLA-DR1 was increased in the group with inflammatory polyarthritis (25 v 18%). CONCLUSIONS: The frequency of DR4 is not increased in newly diagnosed community based patients with RA. This supports the hypothesis that DR4 is less important as a marker for susceptibility to RA than it is for disease persistence or severity.

Lack of association of increased antibody levels to mycobacterial hsp65 with rheumatoid arthritis: results from a study of disease discordant twin pairs.

OBJECTIVES: To investigate the role of humoral immunity to mycobacterial hsp65 in the aetiology of rheumatoid arthritis. METHODS: Levels of IgG antibodies to recombinant mycobacterial hsp65 were measured by enzyme linked immunosorbent assay (ELISA) in serum samples of 152 twin pairs discordant for RA and in serum samples from 62 normal blood donors. RESULTS: No significant differences between antibody levels in the subjects with RA compared either with their unaffected twins or with a group of normal blood donors was observed. In the monozygotic twins there was a strong but negative association between levels of antibody to hsp65 and disease status. Zygosity, sex, and HLA status did not significantly affect levels of antibody to hsp65. CONCLUSION: Previous reports of an association between hsp65 and RA were not confirmed.