Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. Translational science Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroups Simon Rothwell,‍ ‍ 1 Hector Chinoy,‍ ‍ 2,3 Janine A Lamb,4 Frederick W Miller,5 Lisa G Rider,5 Lucy R Wedderburn,6,7 Neil J McHugh,‍ ‍ 8 Andrew L Mammen,‍ ‍ 9,10 Zoe E Betteridge,8 Sarah L Tansley,8,11 John Bowes,‍ ‍ 12 Jiří Vencovský,13 Claire T Deakin,6,7 Katalin Dankó,14 Limaye Vidya,15 Albert Selva-O’Callaghan,16 Lauren M Pachman,17 Ann M Reed,18 Øyvind Molberg,19 Olivier Benveniste,20 Pernille R Mathiesen,21 Timothy R D J Radstake,22 Andrea Doria,‍ ‍ 23 Jan de Bleecker,24 Annette T Lee,25 Michael G Hanna,26 Pedro M Machado,27,28 William E Ollier,4,29 Peter K Gregersen,30 Leonid Padyukov,31 Terrance P O’Hanlon,5 Robert G Cooper,32 Ingrid E Lundberg,31 on behalf of Myositis Genetics Consortium (MYOGEN) Handling editor Josef S Abstract Smolen Key messages Objectives Idiopathic inflammatory myopathies ►► Additional material is (IIM) are a spectrum of rare autoimmune diseases What is already known about this subject? published online only. To view characterised clinically by muscle weakness and ►► The strongest genetic risk among patients please visit the journal online heterogeneous systemic organ involvement. (http://d​ x.​doi.o​ rg/​10.​1136/​ with idiopathic inflammatory myopathies (IIM) The strongest genetic risk is within the major is thought to be within the human leucocyte Protected by copyright. annrheumdis-​2019-​215046) histocompatibility complex (MHC). Since autoantibody antigen (HLA) region in autoantibody defined For numbered affiliations see presence defines specific clinical subgroups of IIM, we subgroups. end of article. aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with What does this study add? Correspondence to IIM autoantibodies. Simon Rothwell, Centre for ►► This study shows for the first time genetic Methods We collected available autoantibody data in Genetics and Genomics, Arthritis differences between adult-onset and juvenile- our cohort of 2582 Caucasian patients with IIM. High Research UK, University of onset patients with anti-TIF1 autoantibodies. Manchester, Manchester M13 resolution human leucocyte antigen (HLA) alleles and ►► Amino acid imputation identifies novel 9PL, UK; corresponding amino acid sequences were imputed using s​ .​rothwell@m ​ anchester.​ac.u​ k associations with autoantibodies that are SNP2HLA from existing genotyping data and tested for stronger than classical HLA associations, association with 12 autoantibody subgroups. Received 9 January 2019 indicating key positions within HLA molecules Revised 14 March 2019 Results We report associations with eight that may confer risk. Accepted 30 March 2019 autoantibodies reaching our study-wide significance Published Online First level of p<2.9×10–5. Associations with the 8.1 ancestral How might this impact on clinical practice or 28 May 2019 haplotype were found with anti-Jo-1 (HLA-B*08:01, future developments? p=2.28×10–53 and HLA-DRB1*03:01, p=3.25×10–9), ►► Differing associations in adult and juvenile anti-PM/Scl (HLA-DQB1*02:01, p=1.47×10–26) and anti- onset disease with the same autoantibody cN1A autoantibodies (HLA-DRB1*03:01, p=1.40×10–11). suggests distinct aetiologies and disease Associations independent of this haplotype were found mechanisms. with anti-Mi-2 (HLA-DRB1*07:01, p=4.92×10–13) ►► As autoantibodies in myositis correlate and anti-HMGCR autoantibodies (HLA-DRB1*11, with specific clinical features of disease, p=5.09×10–6). Amino acid positions may be more understanding genetic risk underlying strongly associated than classical HLA associations; for development of certain autoantibody profiles example with anti-Jo-1 autoantibodies and position 74 will have implications for future research in IIM. of HLA-DRB1 (p=3.47×10–64) and position 9 of HLA-B (p=7.03×10–11). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify © Author(s) (or their Introduction employer(s)) 2019. Re-use haplotypes that may differ between adult-onset and permitted under CC BY-NC. No juvenile-onset patients with these autoantibodies. The idiopathic inflammatory myopathies (IIM) are commercial re-use. See rights Conclusions These findings provide new insights a spectrum of rare autoimmune diseases charac- and permissions. Published regarding the functional consequences of genetic terised clinically by muscle weakness and systemic by BMJ. organ involvement. Clinically, IIM are heteroge- polymorphisms within the MHC. As autoantibodies in To cite: Rothwell S, IIM correlate with specific clinical features of disease, neous and may be subclassified as dermatomyositis Chinoy H, Lamb JA, understanding genetic risk underlying development of (DM), inclusion body myositis (IBM), immune-me- et al. Ann Rheum Dis autoantibody profiles has implications for future research. diated necrotising myopathy, polymyositis (PM) 2019;78:996–1002. and antisynthetase syndrome.1 2 996   Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. Myositis autoantibodies can be detected in approximately Autoantibody testing 60%–70% of children and adults with IIM.3 4 Autoantibodies Myositis relevant autoantibodies were detected using immu- unique to IIM are known as myositis-specific autoantibodies noprecipitation (IP), line blot or ELISA, as detailed in online (MSA), with patients rarely possessing more than one MSA.5 supplementary table 2. Further details are described in online Autoantibodies that present in patients with myositis in associa- supplementary method. tion with another connective tissue disease are known as myosi- tis-associated autoantibodies (MAA). Genotyping Clinical classification of IIM can lead to heterogeneous Genotyping was performed in accordance with UK-based and subgroups, however, some MSA positive subgroups have specific US-based Illumina protocols (Centre for Genetics and Genomics clinical features and may respond differently to treatment. Arthritis Research UK, University of Manchester; Feinstein Therefore, autoantibody status may be a more meaningful way Institute, New York; University College London, London, UK). to characterise patients to understand pathogenesis and predict Standard quality control (QC) was performed as described prognosis.3 For example, patients with anti-histidyl–tRNA-syn- previously.8 thetase (anti-Jo-1) commonly present with myositis, Raynaud’s phenomenon, polyarthritis and a high frequency of interstitial HLA imputation lung disease.6 Patients with autoantibodies directed against anti- Classical HLA alleles and corresponding amino acid sequences Mi-2 present with hallmark cutaneous manifestations of DM, were imputed using SNP2HLA with reference data collected by generally milder myositis and a favourable response to immuno- the Type 1 Diabetes Genetics Consortium.15 Cases and controls suppressive treatment.7 were imputed together. Postimputation QC removed variants IIM are thought to be complex genetic diseases, initiated by with a minor allele frequency <0.01 and variants with an infor- immune activation following specific environmental events in mation score <0.8. genetically predisposed individuals. The major histocompat- ibility complex (MHC), also known as the human leucocyte Statistical analysis antigen (HLA) region, has consistently been identified as the We employed a case-control study design in each autoanti- strongest risk factor for IIM and clinical subgroups.8 Studies body subgroup analysis. Controls were matched for ethnicity have shown that the strongest HLA associations among using principal components analysis (PCA), due to absence of patients with IIM are found when stratifying by autoanti- geographically matched controls for each IIM cohort. Four body status.9–11 In particular, the strongest risk is with the 8.1 healthy controls for each case were matched for ethnicity using Protected by copyright. ancestral haplotype (8.1 AH), a common haplotype of exten- (PCA) coordinates, using a method described previously.16 sive linkage disequilibrium (LD) in Caucasian populations that Analysis was performed in R (V.3.1.0) on the dosage of vari- confers susceptibility to IIM and many other autoimmune or ants (genotype probabilities) which takes into account imputa- immune-mediated diseases.12 Associations with alleles inde- tion uncertainty. Single-test association was performed for HLA pendent of the 8.1 AH have also been reported.9 New MSAs alleles and omnibus tests for multiallelic sites. For multiallelic and MAAs have since been discovered, and small studies have sites, such as amino acids, we identified the most common identified potential HLA associations with these autoantibodies, residue or allele in the control population to be selected as the for example, HLA-DRB1*11:01 in patients with anti-3-hy- reference. A logistic regression assuming an additive model was droxy-3-methylglutaryl-coenzyme A reductase (anti-HMGCR) used to test for association, with gender and the top 10 principal autoantibodies in adult-onset disease.13 components included as covariates. Forward stepwise logistic Here, we aimed to correlate serotype with genotype in patients regression was used to test for independent effects conditional on with IIM, with sufficient statistical power, to identify novel risk the variant of interest. ORs ratios were generated with multivar- variants in the MHC region that confer susceptibility to IIM iate logistic regression. Significance was defined as p<2.9×10–5 autoantibodies and to provide a definitive replication cohort for based on a Bonferroni correction of the pre-QC 1700 variants previously reported associations. imputed by SNP2HLA. Methods Results Study population Autoantibody frequencies in the study population Genetic data were available for 2582 patients with IIM, the Through the Myositis Genetics Consortium (MYOGEN), 2582 majority of whom were tested for at least one autoantibody. Caucasian cases from 14 countries were recruited (online supple- The number of autoantibody positive cases present in the cohort mentary table 1).8 Written informed consent was obtained from is shown in table 1, for groups where n>10. Autoantibody all patients with approval from research ethics committees of frequencies are as a proportion of those in tested individuals and institutional review boards at each participating centre. Patients do not necessarily represent the prevalence of autoantibodies in were enrolled in to MYOGEN if they fulfilled Bohan and Peter an IIM population. criteria for PM and adult and juvenile DM,8 or Griggs, Medical Research Council (MRC) or European Neuromuscular Centre (ENMC) criteria for patients with IBM.14 Shared control geno- Strong classical HLA associations are observed in IIM types were drawn from a pool of 15 651 individuals from 12 autoantibody subgroups countries as described previously.8 Genotypes from an additional Data quality was evaluated after imputation by determining the 19 UK juvenile-onset patients were included from the Juvenile concordance between imputed alleles and existing HLA typing Dermatomyositis Cohort Biomarker Study and Repository. from next generation sequencing technology (HistoGenetics, Patients were classified as having juvenile IIM if age of onset New York, USA) for 162 individuals. There was a high level was <16 years old, except in the USA, where 18 years old was of concordance across all loci for both 2-digit (96.6%–100%) considered the cut-off. and 4-digit (94.8%–100%) alleles (online supplementary table Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 997 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. Table 1 Number of patients with myositis with myositis-specific or associated autoantibodies Adult-onset IIM Juvenile-onset IIM Total number positive (Adult+juvenile- Antibody Number tested Number antibody positive Number tested Number antibody positive onset) Jo-1 1937 325 (16.8%) 447 7 (1.6%) 332 TIF1 1697 91 (5.4%) 356 106 (29.8%) 197 PM/Scl 1883 120 (6.4%) 457 16 (3.5%) 136 Mi-2 1884 87 (4.6%) 456 17 (3.7%) 104 NXP2 1502 28 (1.9%) 360 65 (18.1%) 93 cN1A 232 46 (19.8%) 81 18 (22.2%) 64 SRP 1822 45 (2.5%) 457 0 45 HMGCR 1264 51 (4.0%) 130 1 (0.8%) 52 MDA5 1674 18 (1.1%) 402 17 (4.2%) 35 SAE 1518 31 (2.0%) 397 0 31 PL7 1927 23 (1.2%) 457 0 23 PL12 1927 11 (0.6%) 457 0 11 Myositis-specific/associated autoantibodies n>10 in cohort, stratified by adult-onset and juvenile-onset disease. Frequency is of those patients tested for each autoantibody within the cohort, where testing was not necessarily random. IIM, idiopathic inflammatory myopathies. 3). After stringent QC removing variants with low frequency antisynthetase autoantibodies are included in online supplemen- (<0.01) and poor imputation accuracy (r2<0.8), our anal- tary tables 5-10. ysis included 509 classical HLA alleles and amino acids. In A strong HLA association was observed in patients the primary analysis, adult-onset and juvenile-onset cases were with anti-TIF1 autoantibodies with the HLA-allele group combined and analysed together against matched healthy DQB1*02 (p=2.34×10–10, OR=2.49, 95% CI 1.88 to Protected by copyright. controls. Autoantibody associations reaching study-wide signifi- 3.31). Within this allele group, there were associations with cance of p<2.9×10–5 are reported in table 2. HLA-DQB1*02:01 (p=1.69×10–5) and HLA-DQB1*02:02 The strongest HLA association was found with anti-Jo-1 auto- (p=1.04×10–4). antibodies and HLA-B*08:01 (p=2.28×10–53, OR=3.37, 95% Associations with class II alleles of the 8.1 AH were observed CI 2.37 to 4.83). Conditioning on HLA-B*08:01 in a stepwise for patients with anti-Jo-1, anti-PM/Scl and anti-cN1A. Anti- logistic regression model revealed an independent association HMGCR and anti-Mi-2 were the only autoantibodies tested that with HLA-DRB1*03:01 (p=3.25×10–9, OR=3.09, 95% CI were associated with HLA alleles not forming part of the 8.1 2.08 to 4.30). We analysed antisynthetase autoantibody posi- AH. Although the strong association with anti-HMGCR auto- tive patients as a group (n=381), and the strength of association antibodies and HLA-DRB1*11 was confirmed in adult-onset became stronger (HLA-B*08:01, p=1.79×10–56, OR=3.14, 95% CI 2.27 to 4.38 and HLA-DRB1*03:01, p=3.31×10–9, patients, a stronger association was observed with the 4-digit OR=2.73, 95% CI 1.96 to 3.80). We did not find associations allele HLA-DRB1*11:01 (p=3.22×10–7, OR=11.90, 95% CI with rarer aminoacyl-tRNA-synthetases individually, likely due 4.72 to 31.85) before the allele was removed from the initial to small sample sizes. However, in a small sample size of 23 analysis due to low imputation accuracy (r2=0.75). patients, only 30.4% of patients with anti-PL7 antibodies carried Using a more conservative threshold correcting for the HLA-DRB1*03:01, compared with 79.2% of patients with anti- number of antibodies analysed (p<2.4×10–6), the associa- Jo-1 autoantibodies, and 23% of healthy controls (online supple- tion with HLA-DRB*11 and anti-HMGCR autoantibodies did mentary table 4). This may suggest that not all antisynthetase not reach our significance threshold. There were no signifi- autoantibodies have the same associations with 8.1 AH. The cant associations with classical HLA alleles and anti-NXP2, ‘haplotype phased’ raw HLA genotypes for patients with rarer anti-SRP, anti-MDA5 or anti-SAE autoantibodies. Table 2 Classical HLA allele associations SNP2HLA classical HLA association Autoantibody Number of cases Gene Allele P value OR (95% CI) Jo-1 332 HLA-B 08:01 2.28×10–53 3.37 (2.37 to 4.83) HLA-DRB1 03:01 3.25×10–9 3.09 (2.08 to 4.30) TIF1 197 HLA-DQB1 02 2.34×10–10 2.49 (1.88 to 3.31) PM/Scl 136 HLA-DQB1 02:01 1.47×10–26 17.50 (10.63 to 30.59) Mi-2 104 HLA-DRB1 07:01 4.92×10–13 5.47 (3.48 to 8.77) cN1A 64 HLA-DRB1 03:01 1.40×10–11 9.23 (4.95 to 18.01) HMGCR 52 HLA-DRB1 11 5.09×10–6 4.92 (2.52 to 9.97) 4-digit alleles are preferentially reported unless a 2-digit allele is significantly more associated. P values (uncorrected), OR and 95% CI were calculated in a regression including the most significantly associated independent variants. Associations reaching study-wide significance are reported (p<2.9×10–5). HLA, human leucocyte antigen. 998 Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. Table 3 The strongest amino acid associations in autoantibody subgroups of IIM Amino acid association Autoantibody Number of cases Gene Variant P value OR (95% CI) –64 Jo-1 332 HLA-DRB1 Position 74 Omnibus 3.47×10 Alanine (ref) Arginine 1.24×10--8 2.94 (2.03 to 4.26) Glutamine 0.02 0.59 (0.38 to 0.89) Leucine 6.21×10–5 2.78 (1.67 to 4.55) Glutamic acid 0.5 1.20 (0.70 to 1.96) HLA-B Position 9 Omnibus 7.03×10–11 Tyrosine (ref) Aspartic acid 2.10×10–10 3.33 (2.30 to 4.84) Histidine 0.77 0.96 (0.73 to 1.26) TIF1 197 HLA-DQB1 Position 37 Omnibus 1.49×10–10 Tyrosine (ref) Isoleucine 2.34×10–10 2.49 (1.88 to 3.31) PM/Scl 136 HLA-DRB1 Position 74 Omnibus 2.48×10–40 Alanine (ref) Arginine 5.06×10–25 18.37 (10.88 to 32.98) Glutamine 0.43 1.26 (0.70 to 2.21) Leucine 0.58 0.70 (0.17 to 2.32) Glutamic acid 0.58 1.34 (0.44 to 3.56) Mi-2 104 HLA-DRB1 Position 4 Omnibus 3.69×10–13 Arginine (ref) Glutamine 3.26×10–12 4.93 (3.17 to 7.79) cN1A 64 HLA-DRB1 Position 74 Omnibus 6.15×10–14 Protected by copyright. Alanine (ref) Arginine 1.37×10–9 8.68 (4.43 to 18.1) Glutamine 0.02 0.17 (0.03 to 0.61) Leucine 0.05 3.31 (0.93 to 11.02) Glutamic acid 0.43 0.45 (0.03 to 2.30) SRP 45 HLA-DRB1 Position 74 Omnibus 1.91×10–5 Alanine (ref) Arginine 9.47×10–4 3.51 (1.69 to 7.57) Glutamine 0.13 0.38 (0.08 to 1.18) Leucine 0.63 1.36 (0.32 to 4.34) Glutamic Acid 5.69×10–4 5.07 (2.05 to 13.17) HMGCR 42 HLA-DRB1 Position 58 Omnibus 2.42×10–6 Alanine (ref) Glutamic acid 5.13×10–6 4.91 (2.52 to 9.97) SAE 31 HLA-DQB1 Position 57 Omnibus 2.66×10–6 Aspartic acid (ref) Alanine 1.71×10–5 8.52 (3.42 to 24.47) Valine 0.41 1.61 (0.49 to 4.92) Serine 0.94 0.92 (0.04 to 7.66) OR and 95% CI were calculated in a regression model including significant independent variants, using the most common amino acid in the population as the reference. Associations reaching study-wide significance are reported (p<2.9×10–5). IIM, idiopathic inflammatory myopathies. Amino acid positions may be more strongly associated than autoantibodies, such as anti-Jo-1, anti-PM/Scl and anti-cN1A, classical HLA alleles amino acid associations were markedly stronger than the clas- SNP2HLA was used to impute amino acid positions. We sical HLA associations reported in table 2. Regional association employed a case-control study design in each autoantibody plots for autoantibodies with significant associations are shown subgroup analysis, and autoantibodies with significant amino in online supplementary figure 2A-H. Using a more conserva- acid associations are reported in table 3. tive threshold correcting for the number of antibodies analysed The omnibus p value reports the significance of the amino (p<2.4×10–6), associations with amino acids and anti-SRP, anti- acid position, and the effect sizes of the potential amino acids HMGCR and anti-SAE autoantibodies did not reach our signif- at this location were calculated in a regression model. The loca- icance threshold. tion of associated amino acids within the 3D structure of HLA For anti-Jo-1, anti-PM/Scl, anti-cN1A and anti-SRP auto- molecules are shown in online supplementary figure 1. For some antibodies, the strongest association was with position 74 of Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 999 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. trends were also seen when restricting the analysis to US juve- Table 4 Differences in association at the HLA-DQB1*02 locus in nile-onset patients with anti-TIF1 autoantibodies (n=70); the adult-onset and juvenile-onset patients with anti-TIF1 autoantibodies association with HLA-DQB1*02:01 (p=0.01, OR=2.05, 95% DQB1*02:01 DQB1*02:02 CI 1.18 to 3.54) was stronger than with HLA-DQB1*02:02 Adult-onset TIF1, n=91 P=0.06 P=2.96×10–5 (p=0.76, OR=0.89, 95% CI 0.40 to 1.81). OR=1.54 (0.97 to 2.44) OR=3.31 (1.89–5.84) There were no significant classical HLA or amino acid asso- Juvenile-onset TIF1, n=106 P=3.70×10–5 P=0.13 ciations in either adult-onset or juvenile-onset patients with OR=2.47 (1.61 to 3.80) OR=1.50 (0.87 to 2.51) anti-NXP2 or anti-MDA5 autoantibodies. For anti-Jo-1, OR with 95% CI. anti-PM/Scl, anti-Mi-2 and anti-cN1A, the numbers in the . juvenile-onset cohorts were too small to reach our study-wide significance. However, for alleles associated in the total cohort, the trends for association were in the same direction in juve- HLA-DRB1 (table 3). For anti-Jo-1, anti-PM/Scl and anti- nile-onset IIM (online supplementary table 11). cN1A autoantibodies, an arginine at position 74 of HLA-DRB1 conferred the strongest risk. In patients with anti-PM/Scl and anti-cN1A autoantibodies, an arginine conferred all of the risk at Discussion this position, whereas in patients with anti-Jo-1 autoantibodies We used HLA imputation to find associations with IIM autoanti- there was also evidence of risk attributable to other amino acids. bodies and both classical HLA alleles and amino acid positions that Stepwise conditional analysis showed evidence of multiple may confer risk. By applying this novel approach, we report for independent effects in the HLA region with anti-Jo-1; posi- the first genetic differences between adult-onset and juvenile-onset tion 74 of HLA-DRB1 and position 9 of HLA-B. These were anti-TIF1-positive patients. There are independent associations of associated at a similar level of significance (p=3.47×10–64 vs HLA-DRB1 and HLA-B for the development of anti-Jo-1 autoanti- p=5.25×10–64) and remained significant after conditioning on bodies, implicating multiple genetic features of the 8.1 AH. Amino either position. Analysing antisynthetase autoantibody positive acid imputation identified novel associations with autoantibodies patients as a group increased the strength of association with that are stronger than the classical HLA allelic association, indi- these independent amino acid positions (HLA-DRB1 Position cating key positions within HLA molecules that may confer risk 74, p=5.69×10–67 and HLA-B Position 9, p=8.63×10–11). for various antibodies. For certain autoantibodies, the strongest amino acid associations Protected by copyright. HLA alleles may differentiate adult-onset and juvenile-onset were at a similar level of significance as the strongest specific HLA patients with anti-TIF1 autoantibodies association (online supplementary figure 2A-H). These amino For autoantibodies that occur at high frequencies in both adult- acids may act as a proxy for the risk allele due to carriage on onset and juvenile-onset patients with IIM, the cohort was strat- risk haplotypes. In other instances, amino acid associations were ified to investigate potential differences in HLA associations markedly more significant than any HLA allele. For example, in between these populations. Patients with anti-TIF1 autoanti- patients with anti-PM/Scl autoantibodies, amino acid position 74 bodies were stratified into adult-onset (n=91) and juvenile-onset of HLA-DRB1 had a stronger association (p=2.48×10–40) than (n=106) disease. Of these, all but six adult-onset patients were any allelic association (p=1.47×10–26). For patients with anti-Jo-1 recorded as having DM. The association at the HLA-DQB1 autoantibodies, forward stepwise conditional analysis suggested locus differed between adult-onset and juvenile-onset patients independent associations within the 8.1 AH of HLA-B*08:01 and (table 4). In adult-onset IIM, the strongest 4-digit HLA asso- HLA-DRB1*03:01. Other MSAs may have multiple independent ciation was with HLA-DQB1*02:02 (p=2.96×10–5, OR=3.31, associations within the HLA region that we were underpowered 95% CI 1.89 to 5.84). In contrast, in juvenile-onset patients, a to detect, in line with other seropositive autoimmune diseases.17 strong association with HLA-DQB1*02:01 was observed, but the Imputation of classical HLA alleles confirmed strong associ- strongest association was with HLA-DRB1*03:01 (p=6.21×10– ations between alleles of the 8.1 AH and anti-Jo-1, anti-PM/Scl 6 and anti-cN1A autoantibodies. Specific HLA alleles are thought to , OR=2.69, 95% CI 1.75 to 4.15), which is on the same haplo- type as HLA-DQB1*02:01. target particular autoantigens, resulting in a breakdown in immu- To ensure that this difference was not due to the effects of nological tolerance to self-antigens. However, studies have shown different methods of autoantibody detection, analysis was that there are additional genetic features of the 8.1 AH that predis- restricted to UK patients serotyped using IP in the same centre pose individuals to immune-mediated diseases, such as NF-kappaB at the University of Bath. Nineteen additional juvenile-onset and TNF-alpha polymorphisms,18 19 and gene copy number vari- patients were included, recently genotyped through the Juvenile ants of complement genes.20 The association of alleles independent Dermatomyositis Cohort Biomarker Study and Repository. This of the 8.1 AH with anti-Mi-2 and anti-HMGCR, as well as finding subanalysis included UK adult-onset (n=40) and juvenile-onset that some autoantibodies do not have strong HLA associations, patients (n=48) with anti-TIF1 autoantibodies. The strongest suggests that not all patients with IIM share a common genetic associations for juvenile-onset patients were with alleles of the risk. The 8.1 AH may be more critical for generation of a particular 8.1 AH, which were not significant in the adult-onset cohort. autoantibody repertoire rather than for the development of IIM. For example, HLA-DQB1*02:01 had a stronger association in Environmental triggers including viruses, infections, UV radiation, the juvenile-onset cohort (p=0.004, OR=2.48, 95% CI 1.33 drugs and additional genetic risk factors may contribute to disease to 4.61) in comparison to the adult-onset cohort (p=0.11, heterogeneity. OR=1.69, 95% CI 0.87 to 3.20). Conversely, HLA-DQB1*02:02 The position of amino acids may give insight into whether these was more strongly associated in adult-onset (p=0.01, OR=2.38, associations are functionally important. For example, amino acid 95% CI 1.16 to 4.76) than in juvenile-onset disease (p=0.09, position 74 faces inwards of the peptide binding groove in HLA OR=1.85, 95% CI 0.89 to 3.64), thus replicating trends from DR molecules and may change the structure to accommodate the original cohort. However, due to the reduced sample size, autoantigenic peptides, whereas amino acid position 4 lies outside these associations did not reach study-wide significance. These of the peptide binding groove (online supplementary figure 1). 1000 Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. An arginine at position 74 of HLA-DRB1 was the most strongly Author affiliations 1 associated amino acid position in patients with anti-Jo-1, anti-PM/ Centre for Genetics and Genomics, Arthritis Research UK, University of Manchester, Manchester, UK Scl and anti-cN1A autoantibodies and has been implicated in 2 National Institute for Health Research Manchester Biomedical Research Centre, other autoimmune diseases in Caucasian individuals.21 Arg-74 Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK 3 almost exclusively resides on HLA-DRB1*03 alleles, which may Manchester Academic Health Science Centre, Salford Royal NHS Foundation Trust, explain the strong association with many IIM autoantibodies and Salford, UK 4 Centre for Epidemiology, The University of Manchester, Manchester, UK HLA-DRB1*03:01. It will be interesting to examine whether risk 5 Environmental Autoimmunity Group, National Institute of Environmental Health alleles in other ethnic populations share the same amino acids as Sciences, Bethesda, Maryland, USA 6 in Caucasian populations.22 Our method of analysis was unable to NIHR Great Ormond Street Biomedical Research Centre, University College London, distinguish whether risk was attributable to amino acid motifs or to London, UK 7 Arthritis Research UK Centre for Adolescent Rheumatology, University College individual amino acid positions due to the high level of LD within London, London, UK these molecules. Previous studies have identified HLA-DRB1 motif 8 Pharmacy and Pharmacology, University of Bath, Bath, UK 9 9 EYSTS13 as risk for IIM,23 and the HLA-DRB1 motif 70QKXXR74 Muscle Disease Unit, Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Musculoskeletal and Skin Diseases, National Institutes of Health, for patients with anti-Jo-1 and anti-PM/Scl autoantibodies.9 In Bethesda, Maryland, USA both of these cases, however, the motifs are no stronger than the 10 Departments of Neurology and Medicine, Johns Hopkins University School of classical association with HLA-DRB1*03:01, or indeed an arginine Medicine, Baltimore, Maryland, USA 11 at position 74 of HLA-DRB1. Royal National Hospital for Rheumatic Diseases, Royal United Hospitals Bath NHS Foundation Trust, Bath, UK We found evidence of different haplotypic associations for 12 Arthritis Research UK Centre for Genetics and Genomics, The University of adult-onset and juvenile-onset patients with anti-TIF1 autoanti- Manchester, Manchester, UK bodies. After stratifying by age, there was an association between 13 Institute of Rheumatology and Department of Rheumatology, Charles University, anti-TIF1 autoantibodies and the 8.1 AH in juvenile-onset patients Prague, Czech Republic 14 Internal Medicine, University of Debrecen, Debrecen, Hungary that was absent in adult-onset patients. Differences in association 15 Rheumatology Unit, Royal Adelaide Hospital, University of Adelaide, Adelaide, could be due to distinct aetiologies or to epitope spreading. An South Australia, Australia 16 association of anti-TIF1 autoantibodies with cancer in adult-onset Internal Medicine Department, Vall d’Hebron General Hospital, Universitat disease is not present in juvenile-onset disease.24 25 Cancer-associ- Autonoma de Barcelona, Barcelona, Spain 17 Ann & Robert H. Lurie Children’s Hospital of Chicago, Northwestern University, ated myositis may develop as a paraneoplastic process, with neoan- Feinberg School of Medicine, Chicago, Illinois, USA tigens in the cancer triggering autoimmunity in adult disease.26 In 18 Pediatrics, Duke University, Durham, North Carolina, USA Protected by copyright. 19 juvenile-onset patients with anti-TIF1 autoantibodies, however, Department of Rheumatology, University of Oslo, Oslo, Norway 20 an association with the 8.1 AH may suggest a different aetiology, Department of Internal Medicine and Clinical Immunology, Pitié-Salpêtrière University Hospital, France, France where environmental risk factors such as infections,27 28 UV expo- 21 Department of Paediatrics and Adolescent Medicine, Rigshospitalet, Copenhagen, sure29 or others,30 may initiate disease. Differences in association Denmark 22 with anti-HMGCR autoantibodies in adult-onset patients (HLA- Department of Rheumatology and Clinical Immunology, Utrecht Medical Center, DRB1*11:01) and juvenile onset-patients (HLA*DRB1*07:01) Utrecht, The Netherlands 23 Division of Rheumatology, University of Padova, Padova, Italy have recently been described.13 24 Department of Neurology, Ghent University, Ghent, Belgium There are some limitations with our study. Due to the nature 25 Robert S. Boas Center for Genomics and Human Genetics, The Feinstein Institute of this large multicentre collection, not all patients were tested for Medical Research, Manhasset, New York, USA 26 using gold standard IP. Alternatively, patients were included if they MRC Centre for Neuromuscular Diseases, Department of Neuromuscular Diseases, University College London Institute of Neurology, London, UK tested positive for an autoantibody by a commercially available 27 Department of Rheumatology, University College London Hospital NHS Foundation line blot. Although concordance between these different methods Trust, London, UK 28 has not been comprehensively studied, there is evidence that these Department of Rheumatology, Northwick Park Hospital, London North West assays are robust.31 32 A subanalysis of patients with anti-Jo-1 and University Healthcare NHS Trust, London, UK 29 School of Healthcare Sciences, Manchester Metropolitan University, Manchester, , anti-PM/Scl autoantibodies serotyped using EUROIMMUN line UK blots resulted in the same associations as those serotyped by IP 30 Center for Genomics and Human Genetics, The Feinstein Institute for Medical (data not shown). Another limitation is the potential mischarac- Research, Manhasset, New York, USA 31 Division of Rheumatology, Department of Medicine, Karolinska University Hospital, terisation of patients as antibody negative. Patients may have rare Stockholm, Sweden autoantibodies not currently included on commercially available 32 MRC/ARUK Centre for Integrated Research into Musculoskeletal Ageing, University line-blots, undiscovered antibodies not detectable in cell lines used of Liverpool, Liverpool, , UK for IP or low/negative titres of antibody during disease remission. Acknowledgements We thank Mr. Paul New (Salford Royal Foundation Trust) for Mischaracterising patients as antibody negative would result in a ethical and technical support. We thank all of the patients and their families who type-2 error and should not affect our primary findings. For these contributed to this study. Thanks to Douglas Bell (NIH/NIEHS) and Elaine Remmers (NIH/NHGRI) for critical review of the manuscript. The authors would like to reasons, we did not compare antibody positive against antibody acknowledge the assistance given by IT Services at The University of Manchester. A negative-patient groups. full list of acknowledgments is included in the supplementary materials. In conclusion, these results confirm strong associations Collaborators The following members of the US Childhood Myositis Heterogeneity between HLA alleles and autoantibody specificities present in Study Group contributed to this study: Drs Barbara S Adams (University of Michigan, Caucasian patients with IIM. As well as strong associations with Ann Arbor, MI), Catherine A Bingham (Hershey Medical Center, Hershey, PA), Gail the 8.1 AH, we show that there are associations independent D Cawkwell (All Children’s Hospital, St. Petersburg, FL), Terri H Finkel (Children’s Hospital of Philadelphia, Philadelphia, PA), Steven W George (Ellicott City, MD), of this haplotype and that risk within this region may differ Harry L Gewanter (Richmond, VA), Ellen A. Goldmuntz (Children’s National between adult-onset and juvenile-onset patients with anti-TIF1 Medical Center, Washington, DC), Donald P Goldsmith (St Christopher’s Hospital autoantibodies. As autoantibodies in myositis correlate with for Children, Philadelphia, PA), Michael Henrickson (Children’s Hospital, Madera, CA), Lisa Imundo (Columbia University, New York, NY), Ildy M Katona (Uniformed specific clinical features of disease, understanding the mecha- Services University, Bethesda, MD), Carol B Lindsley (University of Kansas, Kansas nisms behind development of different autoantibodies should be City), Chester P Oddis (University of Pittsburgh, Pittsburgh, PA), Judyann C Olson a focus of ongoing research. (Medical College of Wisconsin, Milwaukee), David Sherry (Children’s Hospital of Rothwell S, et al. Ann Rheum Dis 2019;78:996–1002. doi:10.1136/annrheumdis-2019-215046 1001 Myositis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215046 on 28 May 2019. Downloaded from http://ard.bmj.com/ on February 7, 2022 at Univesity Autonomous of Barcelona. Philadelphia, Philadelphia, PA), Scott A Vogelgesang (Walter Reed Army Medical 7 Love LA, Leff RL, Fraser DD, et al. A new approach to the classification of idiopathic Center, Washington, DC), Carol A Wallace (Children’s Medical Center, Seattle, WA), inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous Patience H White (George Washington University, Washington, DC) and Lawrence S patient groups. Medicine 1991;70:360–74. Zemel (Connecticut Children’s Hospital, Hartford). 8 Rothwell S, Cooper RG, Lundberg IE, et al. Dense genotyping of immune-related loci in idiopathic inflammatory myopathies confirms HLA alleles as the strongest genetic Contributors JAL, RGC, IEL, FWM, LGR, NJMcH and HC devised the study concept risk factor and suggests different genetic background for major clinical subgroups. and design and obtained funding. SR, HC and JAL wrote the manuscript. SR Ann Rheum Dis 2016;75:1558–66. performed the statistical analysis. JB contributed statistical support. JAL, RGC, IEL, 9 O’Hanlon TP, Carrick DM, Targoff IN, et al. Immunogenetic risk and protective factors FWM, NJMcH, HC, LGR, LP and LRW contributed to the interpretations of findings. for the idiopathic inflammatory myopathies: distinct HLA-A, -B, -Cw, -DRB1, and Other authors contributed samples and/or data and all authors contributed to and -DQA1 allelic profiles distinguish European American patients with different myositis approved the manuscript. autoantibodies. Medicine 2006;85:111–27. Funding This study was supported in part by: Association Francaise Contre Les 10 Chinoy H, Salway F, Fertig N, et al. In adult onset myositis, the presence of interstitial Myopathies (AFM), The European Union Sixth Framework Programme (project lung disease and myositis specific/associated antibodies are governed by HLA class II AutoCure; LSH-018661), European Science Foundation (ESF) in the framework of haplotype, rather than by myositis subtype. Arthritis Res Ther 2006;8. the Research Networking Programme European Myositis Network (EUMYONET), 11 Miller FW, Chen W, O’Hanlon TP, et al. Genome-wide association study identifies HLA The Swedish Research Council and grants provided by the Stockholm County 8.1 ancestral haplotype alleles as major genetic risk factors for myositis phenotypes. Council (ALF project), the Swedish Rheumatism Association, King Gustaf V 80 Genes Immun 2015;16:470–80. year Foundation, the intramural research programs of the National Institute of 12 Price P, Witt C, Allcock R, et al. The genetic basis for the association of the 8.1 Environmental Health Sciences (NIEHS) and the National Institute of Arthritis and ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Musculoskeletal and Skin Diseases (NIAMS), the National Institutes of Health (NIH), Immunol Rev 1999;167:257–74. European Community’s FP6, AutoCure LSHB CT-2006-018661, The UK Myositis 13 Mammen AL, Gaudet D, Brisson D, et al. Increased Frequency of DRB1 * 11 : 01 in Support Group, Arthritis Research UK (18474 and 20380), Medical Research Anti – Hydroxymethylglutaryl-Coenzyme A Reductase – Associated Autoimmune Council (MR/N003322/1), The Cure JM Foundation, the Wellcome Trust, the Henry Myopathy. Arthritis Care Res. 2012;64:1233–7. Smith Charity UK, Action Medical UK. The Czech cohort was supported by Project 14 Rothwell S, Cooper RG, Lundberg IE, et al. Immune-Array analysis in sporadic for Conceptual Development of Research Organization 00023728 from Ministry of inclusion body myositis reveals HLA-DRB1 amino acid heterogeneity across the Health in the Czech Republic. Antibody testing in the UK JDRG cohort was supported myositis spectrum. Arthritis Rheumatol 2017;69:1090–9. by a BMA Dorris Hillier Grant 2012 and the Bath Institute of Rheumatic Diseases. 15 Jia X, Han B, Onengut-Gumuscu S, et al. Imputing amino acid polymorphisms in Thank you to the Research Fund of Region Zeeland, Denmark. Pedro M. Machado human leukocyte antigens. PLoS One 2013;8:e64683. is supported by the National Institute for Health Research (NIHR) University College 16 Gregersen PK, Kosoy R, Lee AT, et al. Risk for myasthenia gravis maps to a 151 London Hospitals (UCL) Biomedical Research Centre (BRC). LRW is supported by Pro→Ala change in TNIP1 and to human leukocyte antigen-B*08. Ann Neurol. the National Institute for Health Research (NIHR) Great Ormond Street Hospital 2012;72:927–35. (GOSH) Biomedical Research Centre (BRC). This report includes independent research 17 Betteridge ZE, Chinoy H, Cooper RG, et al. 175 myositis-specific autoantibodies supported by the NIHR Biomedical Research Centre Funding Scheme. rarely coexist with each other: an analysis of the Ukmyonet and Eumyonet cohorts. Rheumatology 2016;55. Disclaimer The views expressed in this publication are those of the authors and Protected by copyright. 18 Chinoy H, Li CK-C, Platt H, et al. Genetic association study of NF-κB genes in UK not necessarily those of the NHS, the National Institute for Health Research or the Caucasian adult and juvenile onset idiopathic inflammatory myopathy. Rheumatology Department of Health. 2012;51:794–9. Competing interests JV reports grants from Ministry of Health in the Czech 19 Chinoy H, Salway F, John S, et al. Tumour necrosis factor-alpha single nucleotide Republic, grants from European Community’s FP6, AutoCure LSHB CT-2006-01866, polymorphisms are not independent of HLA class I in UK Caucasians with adult onset grants from European Science Foundation, during the conduct of the study. IEL idiopathic inflammatory myopathies. Rheumatology 2007;46:1411–6. reports grants from Swedish Research Council, grants from European Science 20 Lintner KE, Patwardhan A, Rider LG, et al. Gene copy-number variations (CNVs) of Foundation, grants from Association Francaise Contre Les Myopathies (AFM), grants complement C4 and C4A deficiency in genetic risk and pathogenesis of juvenile from Stockholm County Council, grants from The European Union Sixth Framework dermatomyositis. Ann Rheum Dis 2016;75:1599–606. Programme, during the conduct of the study. 21 Menconi F, Osman R, Monti MC, et al. Shared molecular amino acid signature in the HLA-DR peptide binding pocket predisposes to both autoimmune diabetes and Patient consent for publication Not required. thyroiditis. Proceedings of the National Academy of Sciences 2010;107:16899–903. Ethics approval Written informed consent was obtained from all cases with 22 Okada Y, Kim K, Han B, et al. Risk for ACPA-positive rheumatoid arthritis is driven by approval from research ethics boards at each participating centre. shared HLA amino acid polymorphisms in Asian and European populations. Hum Mol Genet 2014;23:6916–26. Provenance and peer review Not commissioned; externally peer reviewed. 23 Rider LG, Shamim E, Okada S, et al. Genetic risk and protective factors for idiopathic Data sharing statement No data are available. inflammatory myopathy in Koreans and American whites: a tale of two loci. Arthritis Open access This is an open access article distributed in accordance with the Rheum 1999;42:1285–90. 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