Conserved 33-kb haplotype in the MHC class III region regulates chronic arthritis Anthony C. Y. Yaua, Jonatan Tuncela, Sabrina Haaga, Ulrika Norina, Miranda Houtmanb, Leonid Padyukovb, and Rikard Holmdahla,c,1 aMedical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden; bRheumatology Unit, Department of Medicine, Karolinska Institutet and Karolinska University Hospital, SE-171 76, Stockholm, Sweden; and cSouthern Medical University, Guangzhou 510515, China Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved May 16, 2016 (received for review January 12, 2016) Genome-wide association studies have revealed many genetic loci and lymphotoxin-β (LTB) (10). However, so far there is no evi- associated with complex autoimmune diseases. In rheumatoid ar- dence that polymorphism in the MHC-III region really contributes thritis (RA), the MHC gene HLA-DRB1 is the strongest candidate to the pathogenesis in RA or in its associated animal models. predicting disease development. It has been suggested that other MHC-III is challenging to study because of its high gene density immune-regulating genes in the MHC contribute to the disease and extensive linkage disequilibrium (LD) (9). Animal models for risk, but this contribution has been difficult to show because of RA are attractive alternatives to human association studies for the strong linkage disequilibrium within the MHC. We isolated gene identifications because the use of these models not only genomic regions in the form of congenic fragments in rats to test overcomes genetic heterogeneity and reduces environmental whether there are additional susceptibility loci in the MHC. By effects but also allows the isolation of specific genetic regions to both congenic mapping in inbred strains and SNP typing in wild study their roles in RA in vivo (14–16). Rat arthritis models such rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in as pristane-induced arthritis (PIA) (17, 18), which meet many of the MHC-III region, which regulates the onset, severity, and chro- the criteria for the diagnosis of RA (19), have been used to nicity of arthritis. The Ltab-Ncr3 haplotype consists of five poly- reveal highly significant linkages to unique chromosomal re- morphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb gions, often a much smaller interval with far fewer genes than (lymphotoxin-β), Lst1 (leukocyte-specific transcript 1), and Ncr3 previously postulated for quantitative traits (16, 20, 21). (natural cytotoxicity-triggering receptor 3). Significant correlation We recently established a panel of recombinant strains in the in the expression of the Ltab-Ncr3 genes suggests that interaction MHC region to study the MHC association with T-cell selection of these genes may be important in keeping these genes clustered (22) and showed that RT1-B (the human ortholog is HLA-DQ)in together as a conserved haplotype. We studied the arthritis asso- MHC-II determines the onset and severity of PIA (21). Here, we ciation and the spliceo-transcriptome of four different Ltab-Ncr3 constructed a panel of recombinant strains in MHC-III to study haplotypes and showed that higher Ltb and Ncr3 expression, the association of MHC-III with arthritis on the DA (RT1av1) lower Lst1 expression, and the expression of a shorter splice var- background. By dissecting this gene-dense region, gradually nar- iant of Lst1 correlate with reduced arthritis severity in rats. Inter- rowing down the size of the arthritis-regulating quantitative trait estingly, patients with mild RA also showed higher NCR3 expression locus (QTL), we identified a conserved 33-kb MHC-III haplo- and lower LST1 expression than patients with severe RA. These data type comprising highly polymorphic, differentially expressed, and demonstrate the importance of a conserved haplotype in the regu- spliced genes that regulates the onset, severity, and chronicity of lation of complex diseases such as arthritis. autoimmune arthritis. arthritis | major histocompatibility complex | congenic mapping | Significance haplotype | inflammation The role of the MHC region has been a long-standing issue in he MHC, known as the HLA complex in humans, is the most chronic inflammatory diseases, such as rheumatoid arthritis, and Tgene-dense and polymorphic region in the human genome it has not been possible to identify the underlying specific poly- with strong associations to many autoimmune diseases (1, 2). morphism. Here, we provide evidence that some of the MHC One such example is rheumatoid arthritis (RA). RA is a chronic association must be explained by how closely linked genes – autoimmune inflammatory joint disorder affecting 0.5 1% of the operate together as haplotype blocks. We identified a conserved general population. The genetic association of RA is mainly with haplotype, Ltab-Ncr3, comprising five genes (lymphotoxin α and MHC polymorphism; non-MHC loci [more than 100 RA-risk β,Tnf, leukocyte-specific transcript 1,andnatural cytotoxicity- loci have been identified in genome-wide association studies triggering receptor 3) within MHC class III, regulating arthritis. We (GWAS)] contribute only to a minor extent (3, 4). found significant coexpression of the Ltab-Ncr3 genes, indicating Recently it has been suggested that six different amino acids in how these genes may work together as a haplotype. Further- HLA-DRB1, HLA-A, HLA-B, and HLA-DPB1 explain most of more, haplotype-specific differences in Ltab-Ncr3 gene expres- the MHC association with RA in seropositive patients (5, 6). In sion and alternative splicing correlate remarkably to susceptibility addition, independent genetic susceptibility for RA has been to arthritis. Our data show that a conserved haplotype within identified in the MHC class III (MHC-III) (7–10). MHC-III is a MHC class III regulates arthritis development. region sandwiched between MHC class I (MHC-I) and MHC class II (MHC-II) in humans. This region consists of many genes Author contributions: A.C.Y.Y., J.T., and R.H. designed research; A.C.Y.Y., J.T., S.H., and U.N. performed research; M.H. and L.P. provided and prepared human samples; A.C.Y.Y. with important immune functions encoding for complement and J.T. analyzed data; and A.C.Y.Y., J.T., S.H., M.H., L.P., and R.H. wrote the paper. proteins, cytokines, and heat-shock proteins. Many of these The authors declare no conflict of interest. MHC-III genes have been described as candidate genes in RA, This article is a PNAS Direct Submission. including a gene encoding for TNF (11), which now is a thera- 1To whom correspondence should be addressed. Email: [email protected]. peutic target in RA and other autoimmune diseases (12, 13). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Other candidate genes include allograft-inflammatory factor-1 (8) 1073/pnas.1600567113/-/DCSupplemental. E3716–E3724 | PNAS | Published online June 14, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1600567113 Downloaded by guest on September 29, 2021 Results intervals in this 2.8-Mb region (Fig. 1). We mapped all the MHC PNAS PLUS Identification of a 33-kb Locus in MHC-III That Regulates the Onset congenic and subcongenic strains on the 2004 (Baylor 3.4/rn4) and Severity of Arthritis. We previously identified RT1-B in the assembly which, based on more than 70 widely dispersed SNP MHC-II region as a susceptibility locus for acute PIA and ex- and short tandem repeat (STR) markers as described in the cur- cluded a contribution of the classical MHC-Ia region (21). Al- rent and a previous study (22), offers more accurate annotation though rats of both the MHC congenic strain DA.1HR2, which of the region than the 2012 (RGSC 5.0/rn5) and 2014 (RGSC spans the entire MHC, and the MHC-II congenic strain DA.1HR61 6.0/rn6) assemblies. We assessed acute arthritis in these strains (Fig. 1) were less susceptible to PIA, we observed a small but re- and found that rats of the subcongenic strains DA.1HR7N, producible difference in arthritis protection between these two strains DA.1HR56, DA.1HR56T, DA.1HR56A, and DA.1HR56D de- from day 14 to day 25 (Fig. 2A) (21). This observation suggested veloped significantly milder acute arthritis than DA rats, having a putative second QTL in the nonclassical MHC-Ib and/or the MHC- lower disease score, reduced weight loss, and a delayed disease III region, which was confirmed with the recombinant strain onset, whereas rats of the subcongenic strains DA.1HR4, DA.1HR7, which carries a fragment from DA.1HR2 in the DA.1HR52C, DA.1HR56C, and DA.1HR56E were not pro- MHC-Ib/MHC-III region (Fig. 1 and Table 1). tected (Fig. 1 and Table 1). These results demonstrate the ex- To identify the underlying genetic regulation in DA.1HR7 istence of a 32.7-kb arthritis-regulating QTL in the MHC-III rats, we generated ten subcongenic strains spanning different region (3.655–3.713 Mb) comprising five genes, lymphotoxin-α GENETICS if1 Bat2 Lst1Ncr3 A RT1-CE1 Bat1 Atp6v1g2 Nfkbil1 Abhd16a Ly6g5c Ly6g6f Ng23 G7c G7a Skiv2l Dxo Stk19 C4a C4b Cyp21a1 Tnxa-ps1 Atf6b fkbpl Prrt1 RT1-Db2 RT1-CE10 Ddah2 Clic1 RT1-CE4 RT1-CE5 RT1-CE2 RT1-CE3 Tnf Ltb Lta Bat3 Apom G4 Gpank1 Csnk2b Ly6g5b Ly6g6c Ly6g6d Ly6g6e G6b Ddah2 Msh5 Hspa1b Hspa1a Neu1 Slc44a4 Ehmt2 Zbtb12 C2 Cfb Nelfe Gpsm3 RT1-Ba RT1-Bb RT1-DOb Tap2 Psmb8 Tap1 Psmb9 Agpat1 Rnf5 Ager Pbx2 Notch4 Btnl5 Btnl3a2 Btnl8 Btnl7 Tesb RT1-DMb RT1-DMa RT1-Ha RT1-DOa Rxrb Col11a2 Slc39a7 Hsd17b8 Ring1 RT1-A3 RT1-A2 RT1-A1 Ppt2 Egfl8 Btnl2 RT1-Da RT1-Db1 Brd2 Btnl3 MHC Ib MHC III MHC II MHCIa telomere 3.50 3.75 4.00 4.25 4.50 4.75 5.00 centromere 1.62 5.447 5.976 HR2* 4.315 4.403 4.815 4.834 HR7* HR83 4.047 4.103 4.274 4.301 4.815 4.830 HR7N* HR61* 3.655 3.658 4.583 4.629 HR4 HR62 3.845 3.864 HR52C 3.430 3.452 epytolpah HR56* 3.850 3.863 HR56T* RT1h 3.801 3.806 HR56A* HR56B 3.711 3.713 HR56D* HR56C 3.635 3.649 HR56E UR2 3.801 3.806 RT1u RT1u UR2A haplotype FR2 2.79 4.815 4.834 RT1f RT1f FR9 haplotype 3.736 3.957 FR5 Ltab-Ncr3 RT1-B Fig.