The role of germline polymorphisms in the T-cell receptor in susceptibility to ankylosing spondylitis

The role of germline polymorphisms of the T-cell receptor A/D and B loci in susceptibility to ankylosing spondylitis was investigated by linkage studies using microsatellite markers in 215 affected sibling pairs. The presence of a significant susceptibility gene (lambda ≤ 1.6) at the TCRA/D locus was excluded (LOD score < -2.0). At the TCRB locus, there was weak evidence of the presence of a susceptibility gene (P = 0.01, LOD score 1.1). Further family studies will be required to determine whether this is a true or false-positive finding. It is unlikely that either the TCRA/D or TCRB loci contain genes responsible for more than a moderate proportion of the non-MHC genetic susceptibility to ankylosing spondylitis.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: Implications for the tertiary structure of the receptor and for an n-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7/1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Suggestive linkage of 2p22-25 and 11q12-13 with low bone mineral density at the lumbar spine in the Irish population

Osteoporosis is a disease characterized by low bone mineral density (BMD) and poor bone quality. Peak bone density is achieved by the third decade of life, after which bone is maintained by a balanced cycle of bone resorption and synthesis. Age-related bone loss occurs as the bone resorption phase outweighs the bone synthesis phase of bone metabolism. Heritability accounts for up to 90% of the variability in BMD. Chromosomal loci including 1p36, 2p22-25, 11q12-13, parathyroid hormone receptor type 1 (PTHR1), interleukin-6 (IL-6), interleukin 1 alpha (IL-1α) and type II collagen A1/vitamin D receptor (COL11A1/VDR) have been linked or shown suggestive linkage with BMD in other populations. To determine whether these loci predispose to low BMD in the Irish population, we investigated 24 microsatellite markers at 7 chromosomal loci by linkage studies in 175 Irish families of probands with primary low BMD (T-score ≤ -1.5). Nonparametric analysis was performed using the maximum likelihood variance estimation and traditional Haseman-Elston tests on the Mapmaker/Sibs program. Suggestive evidence of linkage was observed with lumbar spine BMD at 2p22-25 (maximum LOD score 2.76) and 11q12-13 (MLS 2.55). One region, 1p36, approached suggestive linkage with femoral neck BMD (MLS 2.17). In addition, seven markers achieved LOD scores > 1.0, D2S149, D11S1313, D11S987, D11S1314 including those encompassing the PTHR1 (D3S3559, D3S1289) for lumbar spine BMD and D2S149 for femoral neck BMD. Our data suggest that genes within a these chromosomal regions are contributing to a predisposition to low BMD in the Irish population.

Evidence of genetic association between TNFRSF1A encoding the p55 tumour necrosis factor receptor, and ankylosing spondylitis in UK Caucasians

Objectives: To replicate the possible genetic association between ankylosing spondylitis (AS) and TNFRSF1A. Methods: TNFRSF1A was re-sequenced in 48 individuals with AS to identify novel polymorphisms. Nine single nucleotide polymorphisms (SNPs) in TNFRSF1A and 5 SNPs in the neighbouring gene SCNN1A were genotyped in 1604 UK Caucasian individuals with AS and 1019 matched controls. An extended study was implemented using additional genotype data on 8 of these SNPs from 1400 historical controls from the 1958 British Birth Cohort. A meta-analysis of previously published results was also undertaken. Results: One novel variant in intron 6 was identified but no new coding variants. No definite associations were seen in the initial study but in the extended study there were weak associations with rs4149576 (p=0.04) and rs4149577 (p=0.007). In the metaanalysis consistent, somewhat stronger associations were seen with rs4149577 (p=0.002) and rs4149578 (p=0.006). Conclusions: These studies confirm the weak genetic associations between AS and TNFRSF1A. In view of the previously reported associations of TNFRSF1A with AS, in Caucasians and Chinese, and the biological plausibility of this candidate gene, replication of this finding in well powered studies is clearly indicated.

Genetics of ankylosing spondylitis and rheumatoid arthritis: Where are we at currently, and how do they compare?

Both ankylosing spondylitis (AS) and rheumatoid arthritis (RA) are common, highly heritable conditions, the pathogenesis of which are incompletely understood. Gene-mapping studies in both conditions have over the last couple of years made major breakthroughs in identifying the mechanisms by which these diseases occur. Considering RA, there is an over-representation of genes involved in TNF signalling and the NFκB pathway that have been shown to influence the disease risk. There is also considerable sharing of susceptibility genes between RA and other autoimmune diseases such as systemic lupus erythematosus, type 1 diabetes, autoimmune thyroid disease and celiac disease, with thus far little overlap with AS. In AS, genes involved in response to IL12/IL23, and in endoplasmic reticulum peptide presentation, have been identified, but a full genomewide association study has not yet been reported.

Are we waiting too long for the cardiovascular outcome trials with the glucagon-like peptide-1 receptor agonists?

Several new medicines are in development for the treatment of type 2 diabetes, and cardiovascular outcome trials are the gold standard for these medicines. This editorial demonstrates that despite being available for over 10 years, there are no cardiovascular outcome studies for any of the glucagon-like peptide-1 (GLP-1) receptor agonists, which demonstrate cardiovascular safety or benefit in subjects with high cardiovascular risk. The author argues that the FDA should be ensuring that clinical outcome studies for subjects with type 2 diabetes and high cardiovascular risk be undertaken in a timelier manner.

Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: A Mendelian randomisation analysis

Background To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18–0·25; 12·5%; p=9·3 × 10−33), concentrations of interleukin 6 decreased by 0·02 SD (−0·04 to −0·01; −1·7%; p=3·5 × 10−3), and concentrations of C-reactive protein decreased by 0·03 SD (−0·04 to −0·02; −3·4%; p=7·7 × 10−14). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08–1·22; p=1·8 × 10−6) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02–1·04; p=3·9 × 10−10). Per-allele odds ratios were 0·97 (0·95–0·99; p=9·9 × 10−4) for rheumatoid arthritis, 0·99 (0·97–1·01; p=0·47) for type 2 diabetes, 1·00 (0·98–1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04–1·12; p=1·8 × 10−5) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Funding UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7.

A new regulatory variant in the interleukin-6 receptor gene associates with asthma risk

The main genetic determinant of soluble interleukin 6 receptor (sIL-6R) levels is the missense variant rs2228145 that maps to the cleavage site of IL-6R. For each Ala allele, sIL-6R serum levels increase by ∼20 ng ml -1 and asthma risk by 1.09-fold. However, this variant does not explain the total heritability for sIL-6R levels. Additional independent variants in IL6R may therefore contribute to variation in sIL-6R levels and influence asthma risk. We imputed 471 variants in IL6R and tested these for association with sIL-6R serum levels in 360 individuals. An intronic variant (rs12083537) was associated with sIL-6R levels independently of rs4129267 (P=0.0005), a proxy single-nucleotide polymorphism for rs2228145. A significant and consistent association for rs12083537 was observed in a replication panel of 354 individuals (P=0.033). Each rs12083537:A allele increased sIL-6R serum levels by 2.4 ng ml -1. Analysis of mRNA levels in two cohorts did not identify significant associations between rs12083537 and IL6R transcription levels. On the other hand, results from 16 705 asthmatics and 30 809 controls showed that the rs12083537:A allele increased asthma risk by 1.04-fold (P=0.0419). Genetic risk scores based on IL6R regulatory variants may prove useful in explaining variation in clinical response to tocilizumab, an anti-IL-6R monoclonal antibody.

Common variants in the HLA-DRB1-HLA-DQA1 HLA class II region are associated with susceptibility to visceral leishmaniasis

To identify susceptibility loci for visceral leishmaniasis, we undertook genome-wide association studies in two populations: 989 cases and 1,089 controls from India and 357 cases in 308 Brazilian families (1,970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, and combined analysis across the three cohorts for rs9271858 at this locus showed P combined = 2.76 × 10 -17 and odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.30-1.52. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion, the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species. © 2013 Nature America, Inc. All rights reserved.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Fine mapping and replication of genetic risk Loci in primary sclerosing cholangitis

Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10 -6, OR A vs G = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10 -4, OR A vs G = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10 -4, OR A vs T = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10 -12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10 -8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association. © Informa Healthcare.

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10-8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10-6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Genetic determinants of bone density and fracture risk: State of the art and future directions

Context: Osteoporosis is a common, highly heritable condition that causes substantial morbidity and mortality, the etiopathogenesis of which is poorly understood. Genetic studies are making increasingly rapid progress in identifying the genes involved. Evidence Acquisition and Synthesis: In this review, we will summarize the current understanding of the genetics of osteoporosis based on publications from PubMed from the year 1987 onward. Conclusions: Most genes involved in osteoporosis identified to date encode components of known pathways involved in bone synthesis or resorption, but as the field progresses, new pathways are being identified. Only a small proportion of the total genetic variation involved in osteoporosis has been identified, and new approaches will be required to identify most of the remaining genes.

Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case-control study and meta-analysis of published series

Objectives. It has been shown previously that IL-23R variants are associated with AS. We conducted an extended analysis in the UK population and a meta-analysis with the previously published studies, in order to refine these IL-23R associations with AS. Methods. The UK case-control study included 730 new cases and 1331 healthy controls. In the extended study, the 730 cases were combined with 1088 published cases. Allelic associations were analysed using contingency tables. In the meta-analysis, 3482 cases and 3150 controls from four different published studies and the new UK cases were combined. DerSimonian-Laird test was used to calculate random effects pooled odds ratios (ORs). Results. In the UK case-control study with new cases, four of the eight SNPs showed significant associations, whereas in the extended UK study, seven of the eight IL-23R SNPs showed significant associations (P < 0.05) with AS, maximal with rs11209032 (P < 10-5, OR 1.3), when cases with IBD and/or psoriasis were excluded. The meta-analysis showed significant associations with all eight SNPs; the strongest associations were again seen not only with rs11209032 (P = 4.06 × 10-9, OR ∼1.2) but also with rs11209026 (P < 10-10, OR ∼0.6). Conclusions. IL-23R polymorphisms are clearly associated with AS, but the primary causal association(s) is(are) still not established. These polymorphisms could contribute either increased or decreased susceptibility to AS; functional studies will be required for their full evaluation. Additionally, observed stronger associations with SNPs rs11209026 and rs11465804 upon exclusion of IBD and/or psoriasis cases may represent an independent association with AS. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.