Alejandra Sánchez

Fine Mapping of a Major Histocompatibility Complex in Ankylosing Spondylitis Association of the HLA-DPA1 and HLA-DPB1 Regions

OBJECTIVE To investigate the potential association of major histocompatibility complex (MHC) markers other than HLA-B27 with ankylosing spondylitis (AS). METHODS A total of 603 patients with AS and 542 healthy control subjects, all of whom were HLA-B27 positive, were selected for this study based on clinical criteria. First, high-density genotyping across the MHC region (2,360 single-nucleotide polymorphisms [SNPs]) was performed in a cohort of 191 patients and 241 control subjects. After a fine-mapping study, 5 SNPs from the HLA-DPA1/DPB1 region were validated in a second cohort of 412 patients with AS and 301 healthy control subjects. RESULTS Seventeen SNPs located within or near the HLA-DPA1 and HLA-DPB1 loci showed association with AS (P = 1.38 × 10⁻⁵ to 0.05). In addition, multimarker tests, both linkage disequilibrium and sliding windows, showed association of some groups of adjacent SNPs within the HLA-DPA1/DPB1 region with AS (P = 1.0 × 10⁻⁴ to 3.96 × 10⁻⁷). We validated the association by genotyping 5 SNPs from the DPA1/DPB1 region in an additional cohort and obtained P values from 6.42 × 10⁻⁵ to 0.01 in the analysis of the combined cohorts. Subtyping analysis of HLA-DPA1 and HLA-DPB1 showed that HLA-DPA1*01:03, A1*02:01, and B1*13:01 were the subtypes most susceptible to AS. CONCLUSION HLA markers and linkage disequilibrium blocks near HLA-DPA1 and HLA-DPB1 are statistically associated with AS. We identified a region located around the HLA-DPA1 and HLA-DPB1 loci associated with AS, another region within the MHC that is different from HLA-B27.

Genetic polymorphisms, inside and outside the MHC, improve prediction of AS radiographic severity in addition to clinical variables

OBJECTIVE The aim of this study was to analyse if single nucleotide polymorphisms (SNPs) inside and outside the MHC region might improve the prediction of radiographic severity in AS. METHODS A cross-sectional multi-centre study was performed including 473 Spanish AS patients previously diagnosed with AS following the Modified New York Criteria and with at least 10 years of follow-up from the first symptoms of AS. Clinical variables and 384 SNPs were analysed to predict radiographic severity [BASRI-total (BASRI-t) corrected for the duration of AS since first symptoms] using multivariate forward logistic regression. Predictive power was measured by the area under the receiver operating characteristic curve (AUC), specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS The model with the best fit measured radiographic severity as the BASRI-t 60th percentile and combined eight variables: male gender, older age at disease onset and six SNPs at ADRB1 (rs1801253), NELL1 (rs8176785) and MHC (rs1634747, rs9270986, rs7451962 and rs241453) genes. The model predictive power was defined by AUC = 0.76 (95% CI 0.71, 0.80), being significantly better than the model with only clinical variables, AUC = 0.68 (95% CI 0.63, 0.73), P = 0.0004. Internal split-sample analysis proved the validation of the model. Patient genotype for SNPs outside the MHC region, inside the MHC region and clinical variables account for 26, 38 and 36%, respectively, of the explained variability on radiographic severity prediction. CONCLUSION Prediction of radiographic severity in AS based on clinical variables can be significantly improved by including SNPs both inside and outside the MHC region.

Activating killer immunoglobulin-like receptors genes are associated with increased susceptibility to ankylosing spondylitis

The aim of this study was to analyse the association of specific killer cell immunoglobulin‐like receptors (KIR) genes and haplotypes with susceptibility to ankylosing spondylitis (AS) and its different clinical manifestations in a Spanish population. The presence or absence of all KIR genes was studied for their association with AS. A total of 176 patients with AS and 435 healthy control subjects were selected for this study based on clinical criteria. The commercial KIR‐sequence‐specific oligonucleotides (SSO) typing kit was used to investigate KIR typing. Frequencies of KIR2DS1 and KIR3DS1 genes were increased significantly in patients compared with healthy controls [52·8 versus 38·2%, PBonf < 0·01, odds ratio (OR) = 1·81 (1·28–2·59); 51·7 versus 37·5%, PBonf < 0·01, OR = 1·79 (1·25–2·54)]. Moreover, the frequency of activating genotypes in the AS patient group was significantly higher than in the healthy control group (P < 0·05). KIR2DS1 and KIR3DS1, in addition to human leucocyte antigen (HLA)‐B27, may play an important role in the pathogenesis of AS. However, we show that the contribution of the KIR genes to AS susceptibility extends beyond the association with individual KIRs, with an imbalance between activating and inhibitory KIR genes seeming to influence the susceptibility to AS.

A high density SNP genotyping approach within the 19q13 chromosome region identifies an association of a CNOT3 polymorphism with ankylosing spondylitis

Objective To identify genomic variants in the 19q13 chromosome region associated with ankylosing spondylitis (AS) in human leucocyte antigen (HLA)-B27-positive populations. Methods High-throughput genotyping of 1536 haplotype-tag single nucleotide polymorphisms (SNPs) was performed in 249 patients with AS and 302 healthy controls. Some of the identified associations were validated by genotyping four SNPs in two additional cohorts consisting of 412 cases/301 controls and 144 cases/203 controls. All individuals selected (both cases and controls) were HLA-B27-positive. Results Two markers in two different genes (CNOT3 and LAIR2) showed significant association (p<10−3) with AS. In addition, sliding windows analysis showed association of groups of adjacent SNPs in regions located around CNOT3 (Chr19: 59347459-59356564, p=2.43×10−4 to 6.54×10−4). The associations were validated by genotyping four SNPs from regions located near LAIR2 and CNOT3 genes (rs1055234, rs8111398, rs2287828 and rs4591276) in two additional cohorts. The CNOT3 polymorphism (rs1055234) remained associated with AS (combined p=9.73×10−6). One SNP, located downstream of KIR3DL1, was detected which, tested in combination with HLA-Bw4I80, was associated with AS. Conclusion A novel significant association was detected between SNP rs1055234 and AS susceptibility.

Association of the Intergenic Single-Nucleotide Polymorphism rs10865331 (2p15) with Ankylosing Spondylitis in a Spanish Population

Objective. A recent genome-wide association study has identified 2 single-nucleotide polymorphisms (SNP) associated with ankylosing spondylitis (AS), rs10865331 (2p15) and rs2242944 (21q22). We assessed the association of these SNP with AS in a Spanish population. Methods. Four hundred fifty-six patients with AS fulfilling the modified New York Criteria and 300 healthy donors were analyzed. Result. SNP rs10865331 (allele A: p = 0.039; genotype: p = 0.016) was significantly associated with AS, while no association was found for rs2242944. Conclusion. This is the first study that replicates in an independent cohort the association of the intergenic SNP rs10865331 with susceptibility to AS.

A Single Nucleotide Polymorphism in the Il17ra Promoter Is Associated with Functional Severity of Ankylosing Spondylitis

The aim of this study was to identify new genetic variants associated with the severity of ankylosing spondylitis (AS). We sequenced the exome of eight patients diagnosed with AS, selected on the basis of the severity of their clinical parameters. We identified 27 variants in exons and regulatory regions. The contribution of candidate variants found to AS severity was validated by genotyping two Spanish cohorts consisting of 180 cases/300 controls and 419 cases/656 controls. Relationships of SNPs and clinical variables with the Bath Ankylosing Spondylitis Disease Activity and Functional Indices BASDAI and BASFI were analyzed. BASFI was standardized by adjusting for the duration of the disease since the appearance of the first symptoms. Refining the analysis of SNPs in the two cohorts, we found that the rs4819554 minor allele G in the promoter of the IL17RA gene was associated with AS (p<0.005). This variant was also associated with the BASFI score. Classifying AS patients by the severity of their functional status with respect to BASFI/disease duration of the 60th, 65th, 70th and 75th percentiles, we found the association increased from p60 to p75 (cohort 1: p<0.05 to p<0.01; cohort 2: p<0.01 to p<0.005). Our findings indicate a genetic role for the IL17/ILRA axis in the development of severe forms of AS.

AB0857 Single nucleotide polymorphisms associated with non response to anti-TNF-alpha treatment in ankylosing spondylitis patients. Data from regisponser

Background Taking into account that almost 40% of AS patients remain disease active even on long term biological treatment1, genetic biomarkers that differentiate between responders and non-responders would be useful in selecting the apropiate treatment. Objectives The aim of the study was to investigate candidate single nucleotide polymorphisms (SNPs) responsible for non response to TNF-alpha blockers in Spanish AS patients at the beginning of the first anti- TNF-alpha agent. Methods We undertook an association study on AS patients enrolled in the REGISPONSER. The patients were classified as “non-responders” if they failed to achieve BASDAI50 improvement criteria at the visit that followed treatment initiation. We selected candidate SNPs which were previously described as associated with susceptibility or pathogenesis of AS and/or other spondylartropathies. SNP genotyping was performed using the Illumina Golden gate genotyping platform. Statistical analysis was performed with the SPSS v19.0 and the SVS v7.3.1 softwares.P-values of <0,05 were considered statistically significant. Results Among 121 patients who fulfilled the inclusion criteria, 68 (56.2%) were responders and 53(43.8%) were non-responders.The mean age of studied patients was 47.7 years (±9.5), with mean disease duration since first symptoms of 21.1 years (±8.9). There were no significant differences in baseline clinical and demographic characteristics in the two treatment response groups. From the 345 SNPs tested for association with treatment response status, 10 polymorphisms remained associated after adjustment for multiple testing. Risk alleles of SNPs associated with non-response to anti-TNF-alpha treatment by their OR with 95% confidence intervals (CI) are listed in table 1. Table 1. Allelic variants associated with non response to anti-TNF-alpha treatment according to BASDAI50 response criteria Snp Gene Risk Allele OR non-responder P-adjusted* (95% CI)a rs755622 MIF G 2.92 (1.41–6.03) 0.003 rs917997 IL18RAP A 2.4 (1.32–4.35) 0.005 rs1061622 TNFRSF1B C 2.12 (1.15–3.91) 0.009 rs4343 ACE G 1.82 (1.05–3.13) 0.016 rs4355801 TNFRSF11B A 1.79 (1.04–3.08) 0.051 rs6060369 UQCC A 1.77 (1.03–3.02) 0.032 rs3740691 ARFGAP2 A 1.76 (1.03–3.02) 0.031 rs331377 ASPN G 1.7 (1.01–2.84) 0.043 rs3213718 CALM1 A 1.72 (1.01–2.92) 0.044 rs1800896 IL10 A 1.69 (1.01–2.84) 0.029 *p adjusted using single value permutation testing; a)P values and Odds Ratios (95% CI) were obtained from Chi-squared test of 2×2 tables of allele frequencies in non-responders versus responders. Conclusions Our study identified 10 different polymorphisms in 10 different genes associated with non response to TNF-alpha blockers. Validation of these markers in further cohorts would be of great interest to confirm the robustness of our results. References Baraliakos X, Listing J, Fritz C, et al. Persistent clinical efficacy and safety of infliximab in ankylosing spondylitis after 8 years – early clinical response predicts long-term outcome. J. Rheumatology (Oxford). 2011 Sep;50(9):1690-9. Disclosure of Interest None Declared