James I. Robinson

Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake.

Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

In situ macromolecular crystallography using microbeams

A sample environment for mounting crystallization trays has been developed on the microfocus beamline I24 at Diamond Light Source. The technical developments and several case studies are described.

Algal growth control by a barley straw extract

In recent years, there has been an apparent increase in the occurrence of harmful algal blooms occurring in potable waters. The potential of a simple barley straw extract to inhibit algal growth was assessed. Algal growth in lakewater was inhibited by the addition of barley straw (1% w/v), with the chlorophyll a concentration remaining below the original level (40 micrograms l-1) throughout the experiment. In contrast, in the presence of wheat straw, algal biomass increased, reaching a final chlorophyll a concentration of 1160 micrograms l-1 after 28 days. Analysis of the remaining particulate straw at the end of the experiment showed that the lignin content of barley straw had increased significantly from 10-33% (w/w). Further, a preparation of a simple aqueous extract from the decomposed-barley straw was found to inhibit the cyanobacteria Microcystis sp. and the algal species Scenedesmus, with chlorophyll a levels some 10-fold lower than in untreated flasks. This study shows that a decomposed-barley extract, even in a very dilute concentration (0.005%) was capable of inhibiting the growth of Microcystis sp., a commonly occurring cyanobacterium which produces the toxin microcystin and has been responsible for some of the most serious pernicious algal blooms in the UK.

Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment

Background The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). Methods Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. Results At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. Conclusions This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

Genetic variants within the MAP kinase signalling network and anti-TNF treatment response in rheumatoid arthritis patients

Background Rheumatoid arthritis (RA) does not always respond to available treatments, including tumour necrosis factor (TNF) antagonists. A study was undertaken to investigate whether genetic variation within genes, encoding proteins in the p38 signalling network, contributes to the variable response to TNF antagonists. Methods 1102 UK Caucasian patients with RA receiving anti-TNF therapy (infliximab, adalimumab and etanercept) were genotyped for 38 pairwise-tagging single nucleotide polymorphisms (SNPs) spanning 12 candidate genes from the p38 network. Regression analyses were performed to test association between genotype and treatment response at 6 months using both absolute change in DAS28 (Disease Activity Score across 28 joints) and European League Against Rheumatism (EULAR) improvement criteria. Stratified analyses were performed to investigate association with individual therapies. Results Seven SNPs, in five genes, were associated with improvement in DAS28 at 6 months at a nominal 0.1 significance level, jointly explaining 3% of variance in outcome in a model adjusting for other predictors. These encoded proteins both upstream (MKK6) and downstream (MAPKAPK2, MSK1, MSK2) of p38, and MAPK14, the p38-α isoform of p38 MAPK. One SNP (rs2716191 in MAP2K6) was associated with EULAR response at the 0.1 level. SNPs generally showed greater correlation with response to infliximab and adalimumab, but not to etanercept. Conclusions More SNPs than would be expected by chance, mapping to the p38 signalling network, showed association with the anti-TNF response as a whole, and particularly with the response to infliximab and adalimumab. Validation of these findings in independent cohorts is warranted.

Analysis of Fcγ receptor haplotypes in rheumatoid arthritis: FCGR3A remains a major susceptibility gene at this locus, with an additional contribution from FCGR3B

The Fcγ receptors play important roles in the initiation and regulation of many immunological and inflammatory processes, and genetic variants (FCGR) have been associated with numerous autoimmune and infectious diseases. The data in rheumatoid arthritis (RA) are conflicting and we previously demonstrated an association between FCGR3A and RA. In view of the close molecular proximity with FCGR2A, FCGR2B and FCGR3B, additional polymorphisms within these genes and FCGR haplotypes were examined to refine the extent of association with RA. Biallelic polymorphisms in FCGR2A, FCGR2B and FCGR3B were examined for association with RA in two well characterized UK Caucasian and North Indian/Pakistani cohorts, in which FCGR3A genotyping had previously been undertaken. Haplotype frequencies and linkage disequilibrium were estimated across the FCGR locus and a model-free analysis was performed to determine association with RA. This was followed by regression analysis, allowing for phase uncertainty, to identify the particular haplotype(s) that influences disease risk. Our results reveal that FCGR2A, FCGR2B and FCGR3B were not associated with RA. The haplotype with the strongest association with RA susceptibility was the FCGR3A–FCGR3B 158V-NA2 haplotype (odds ratio 3.18, 95% confidence interval 1.13–8.92 [P = 0.03] for homozygotes compared with all genotypes). The association was stronger in the presence of nodules (odds ratio 5.03, 95% confidence interval 1.44–17.56; P = 0.01). This haplotype was also more common in North Indian/Pakistani RA patients than in control individuals, but not significantly so. Logistic regression analyses suggested that FCGR3A remained the most significant gene at this locus. The increased association with an FCGR3A–FCGR3B haplotype suggests that other polymorphic variants within FCGR3A or FCGR3B, or in linkage disequilibrium with this haplotype, may additionally contribute to disease pathogenesis.

Outrunning free radicals in room-temperature macromolecular crystallography

A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector.

Inferring relative proportions of DNA variants from sequencing electropherograms

MOTIVATION Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. RESULTS We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. AVAILABILITY AND IMPLEMENTATION QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. CONTACT msjimc@leeds.ac.uk SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

Objective The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy. Methods FcγRIIIa/CD16 expression on CD14low and CD14++ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined. Results Increased FcγRIIIa/CD16 expression on CD14++ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14++ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14++ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers. Conclusion Increased FcγRIIIa/CD16 expression on CD14++ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.

Confirmation of association of FCGR3B but not FCGR3A copy number with susceptibility to autoantibody positive rheumatoid arthritis

The FCGR locus encoding the low‐affinity Fcγ receptors (FcγR) for immunoglobulin G has largely been missed by genome‐wide association studies due to complications with structural variation and segmental duplication. Recently identified copy number variants (CNVs) affecting FCGR3A and FCGR3B have been linked to a number of autoimmune disorders. We have developed and validated a novel quantitative sequence variant assay in combination with an adapted paralogue ratio test to examine independent CNVs carrying FCGR3A and FCGR3B in rheumatoid arthritis (RA) compared with healthy volunteers (n = 1,115 and 654, respectively). Implementation of a robust statistical analysis framework (CNVtools) allowed for systematic batch effects and for the inherent uncertainty of copy number assignment, thus avoiding two major sources of false positive results. Evidence for association with neither duplications nor deletions of FCGR3A was found; however, in line with previous studies, there was evidence of overrepresentation of FCGR3B deletions in RA (odds ratio [OR] 1.50, P = 0.028), which was more apparent in rheumatoid factor positive disease (OR 1.61, P = 0.011). The level of FcγRIIIb, encoded by FCGR3B, expression on neutrophils was shown to correlate with gene copy number. Thus, our results may highlight an important role for neutrophils in the pathogenesis of RA, potentially through reduced FcγRIIIb‐mediated immune complex clearance. Hum Mutat 33:741–749, 2012.