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Mapping of a novel susceptibility gene for rheumatoid arthritis in the telomeric MHC region
www.elsevier.com/locate/issn/10434666 Cytokine 32 (2005) 71e75
Review Article
Mapping of a novel susceptibility gene for rheumatoid arthritis in the telomeric MHC region Rachael Kilding, Anthony G. Wilson* Division of Genomic Medicine, The University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, United Kingdom
Abstract Rheumatoid arthritis (RA) is a complex heterogeneous disease with an estimated genetic contribution to of 30e50%. Approximately one third arises from the major histocompatibility complex (MHC) at 6p21.3. The contribution of specific DRB1 alleles encoding the shared epitope has been well described, however, several recent studies have suggested that additional telomeric genetic influences may exist. This region is difficult to study as a result of the presence of strong linkage disequilibrium (LD) within the MHC and high gene density particularly in the central class III region. In this article we review the current data supporting the existence of a non-DRB1 susceptibility gene for rheumatoid arthritis, in particular within the class III region. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Rheumatoid arthritis; Genetics; Polymorphism; MHC
1. Introduction Rheumatoid arthritis (RA) is a complex heterogeneous disease characterised by persistent joint inflammation resulting in bony erosion and cartilage loss and often systemic tissue. It affects w1% of most Caucasian populations, with a female to male prevalence of 2.5:1 [19]. The aetiology of RA remains unknown, but is multifactorial with a polygenic component influencing both disease susceptibility and severity. 2. The shared epitope The association of RA with DRB1*0401 (DRw1) was initially discovered by Stastny [37] and almost 10 years later a similar association with DRB1*0404 was reported [28]. Subsequent studies led to the description of association of a group of HLA-DR genes with RA susceptibility, leading to the shared epitope (SE) hypothesis [11]. These encode a similar amino acid sequence QRRAA, QKRAA or RRRAA, in position 70e74 on the third hypervariable region of the DR-b chain. * Corresponding author. Tel.: C44 114 271 2232; fax: C44 114 271 1863. E-mail address: [email protected] (A.G. Wilson). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.07.015
Pocket 4 of 9 in the HLA-DR molecule encompasses amino acid positions 13, 70, 71, 74 and 78 of the b chain and position 9 of the a chain. Positions 70, 71 and 74 are part of the RA associated sequence. The amino acid at position 71 plays a vital role in determining which amino acid side chain will be accepted into the peptide binding pocket. Shared epitope alleles associated with RA present negatively, but not positively charged, amino acids at position 4 of the presented peptide, the reverse is true of the protective DRB1*0402 allele [38,42,43]. The alleles of the shared epitope consist of HLADRB1*0101, *0102, *0401, *0404, *0405, *0408, *0409, *0410, *1001, *1402, *1406 and *1409. The contribution of the shared epitope to RA susceptibility is yet to be clarified, whether it directly results in disease susceptibility or influences prognosis is still open for debate. Notably not all patients with RA are SE positive and w30% of the healthy UK Caucasian population are HLA-DRB1*04 positive. An alternative theory suggests that susceptibility is encoded by HLA-DQ, modulated by certain protective HLA-DRB1 alleles [45]. This effect, however, is probably a reflection of linkage disequilibrium with SE alleles [8]. Certain HLA-DRB1 alleles appear to predispose to more aggressive disease [40] with an increased relative risk of developing erosions [9] and may also influence response to
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treatment [31]. The most severe outcome is seen in rheumatoid patients homozygous for *0401 or *0404 or *0401/*0404/ *0408 heterozygotes. Homozygosity of HLA-DRB1*0401 has been shown to be significantly increased in patients with major organ involvement [44]. The compound heterozygote *0404/*0401 has a significantly greater risk of severe erosive RA [25]. 3. Evidence of non-DRB1 genetic contribution within the MHC Recent evidence has suggested that the SE hypothesis does not explain all of the genetic association of the MHC with RA. The TNF gene is located at the telomeric end of the class III region approximately 1 Mb telomeric of DRB1 and, in view of the biological role of this cytokine in RA, this locus has been examined intensively for a role in the genetic background of RA. A Spanish study reported a SE-independent association of TNF microsatellites with erosive RA [12]. Similar effects have also been detected in British and Peruvian studies with evidence in the British study of genetic interaction with the SE in female patients [5,24]. In view of the reported effects on gene expression a number of case control studies have examined TNF promoter SNPs in RA with several reporting evidence of a genetic effect independent of the SE [4,6,39]. Most of these studies have, however, only typed the DRB1 and TNF loci. Recently several case control studies have used a larger set of genetic markers spanning the MHC. A Japanese group typed 5 TNF promoter SNPs and 18 microsatellites spanning the MHC in 248 controls and 120 patients and found evidence of an additional genetic effect in the telomeric class III region close to TNF [33]. A Dutch group examined similar sized cohorts and typed 6 microsatellites around the junction of the class III and I regions and reported evidence of a DRB1-independent genetic effect derived from the A1B8-DR3 (8.1) haplotype [46]. We recently reported results of a family based analysis specifically searching the MHC using the transmission disequilibrium test (TDT) [36]. Thirteen SNPs were genotyped in 164 British rheumatoid arthritis families containing at least one affected offspring. Using unconditional and DRB1-conditioned transmission disequilibrium tests at least one additional nonDRB1 susceptibility locus for RA was identified within an interval encompassing the junction of the class III and I regions. The effect was detected with a SNP in the second intron of the leucocyte specific transcript-1 (LST-1) gene, this is situated almost 10 kb upstream of TNF and w940 kb telomeric of DRB1. This polymorphism appears to have no biological significance, and sequence analysis of the coding and regulatory region of LST-1 did not reveal other polymorphisms in either healthy controls or RA patients. On balance it seems more likely that the observed association with RA is secondary to LD with a functional genetic variant in another gene [17]. Our findings share similarities with the recently reported family study by Jawaheer et al. [16], where two additional genetic effects were described within the MHC, independent of HLA-DRB1. One of these lies in the central MHC, within
a 497-kb region, on a segment of the A1-B8-DRB1*03 ancestral haplotype, this is the same region we identified with LST-1. However, our results suggest that the gene probably lies within a smaller interval between BAT3 and BAT1, although the exact boundaries of the block of LD need to be refined. Further genes were also proposed to exist within a 697-kb interval in the class I region in a subset of DRB1*0404 haplotypes, we found a second smaller effect in this region with a marker in PG8. A recent case control study from Japan has reported the association of the IkBL-62 marker with RA, speculating that this polymorphism may alter a motif for the transcriptional repressor dEF1 [32]. However, using both unconditional and DRB1conditioned TDT we did not find evidence implicating this polymorphism in RA. These contradictory findings may be related to differences in LD in the two populations, we found strong LD between this IkBL marker and *0301, a non-SE allele, however, LD with DRB1 alleles was not examined in the Japanese study and perhaps the association reported is secondary to LD between IkBL-62 and a SE allele in the Japanese population. A further study of over 270 RA patients and controls positive for HLA-DRB1*04, typed for 6 SNPs within the LTAe TNF interval suggested that genes outside this region were acting as modifiers of the effect of DRB1 on RA susceptibility. Significant variation in the LTAeTNF haplotype frequencies were observed depending on the HLA-DRB1*04 subtype. For DRB1*0401 carriers the LTAeTNF2 haplotype was significantly less frequent in cases compared with controls (OR 0.5 95% confidence interval 0.3e0.8, p Z 0.007), however, the opposite was observed in DRB1*0404 carriers (OR 16.7 95% confidence interval 1.8e240, p Z 0.007) [30]. We observed a similar effect in the variation of the high-risk LST-1 allele depending on the HLA-DRB1 haplotype. On the DRB1 1/SEC background allele 2 of LST-1 was overtransmitted (OR 4.4), however, on the DRB1 4/SEC and DRB1 15/SEÿ background allele 1 was overtransmitted (OR 1.48 and 6.79, respectively). This variation in the high-risk allele dependent on the DRB1 background might explain why no effect at LST-1 was detected in the unconditional analysis and only became apparent after conditioning for the effect of DRB1. 4. Candidate genes The telomeric class III and centromeric class I regions of the MHC contain numerous potential immunoregulatory genes including: allograft inhibitory factor 1 (AIF-1), G1, 1C7, LST1, lymphotoxin (LT)b, TNF, LTa, NB6, IkBL and BAT1 [29]. The function of many of these is yet to be determined but several are known to play an active part in modulating the immune response and are therefore exciting potential candidates. 4.1. Tumour necrosis factor The pivotal role of TNF in disease pathogenesis means it has long been considered a potential candidate gene and as
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such has been extensively studied. Studies have, however, failed to consistently show a direct genetic contribution to RA. The SE-independent association of TNF microsatellites with RA in a Spanish population was reported by Hajeer et al. [12]. A similar effect has been reported in British and North American populations [5,24]. Further case control studies have suggested a SE-independent protective effect of TNFC489 and ÿ238 [4,39]. In contrast TNFC489 did not appear to confer a protective effect in a combined Spanish and British case control study [20]. Recently several case control studies have used a larger set of genetic markers spanning the MHC. A Japanese group typed 5 TNF promoter SNPs and 18 microsatellites spanning the MHC in 248 controls and 120 patients and found evidence of an additional genetic effect in the telomeric class III region close to TNF [33]. A Dutch group examined similar sized cohorts and typed 6 microsatellites around the junction of the class III and I regions and reported evidence of a DRB1independent genetic effect derived from the A1-B8-DR3 (8.1) haplotype [46]. Although case control studies have the power to detect weak genetic effects they are also susceptible to the effects of population stratification, in order to avoid this potential bias the transmission disequilibrium test (TDT) can be applied to affected families. Distorted transmission of TNF haplotypes in multiplex RA families has been reported in an Irish population [27] and independently of the SE in Spain [22].
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sequence conservation between 1C7 and the members of the Ig superfamily, at least 2 of the 1C7 transcripts are probably associated with expression of a functional protein. Although further work is needed to determine the function of this gene it is a potential candidate gene for autoimmune disorders [29]. 4.5. Inhibitor of kappa B-like (IkBL) Just 20 kb telomeric of the TNF cluster lies the inhibitor of kappa B-like (IkBL) gene. This gene shows sequence homology with the inhibitor of kappa light chain gene enhancer in B cells alpha (IkBagene) involved in the regulation of nuclear localisation of the transcription factor, nuclear factor kappa B (NFkB), which in turn stimulates the transcription of many genes including proinflammatory cytokines such as TNFa [7]. Suggesting that IkBL could also be involved in regulation of transcription of proinflammatory cytokines, making it an interesting potential candidate gene. Three SNPs have been identified within IkBL, 2 within the promoter and a non-synonomous C / T polymorphism at position C738 results in a cysteine to arginine substitution in a predicted protein kinase C phosphorylation domain [1]. This polymorphism is associated with susceptibility to ulcerative colitis [7] and multiple sclerosis [1]. A recent case control and family association study investigated the potential association of this SNP with rheumatoid arthritis. No significant difference in rare allele frequency was detected between 225 RA patients and 291 controls, similarly the TDT applied to 58 families did not demonstrate any transmission distortion [23].
4.2. Leucocyte specific transcript-1 (LST-1) 4.6. HLA-B-associated transcript 1 (BAT1) The LST-1 gene is situated almost 10 kb upstream of TNF and w940 kb telomeric of DRB1. Although its exact function is yet to be determined, support for an immunoregulatory role is suggested by expression in lymphoid tissues, T cells, B cells, dendritic cells, macrophages and histiocyte cell lines. In addition it is IFNg inducible [14] and has recently been shown to have an inhibitory effect on lymphocyte proliferation [35] indicating that it could be an attractive candidate gene in autoimmune conditions including RA. 4.3. Lymphotoxin alpha (LTa) Lymphotoxin alpha, also known as TNFb, lies telomeric of TNF and shows 35% identity and 50% homology in amino acid sequence with TNF. Lymphotoxin alpha is a soluble protein secreted by activated lymphocytes and believed to act in the immune response. The association of the TNF-308 polymorphism, GeA, reported in females with extensive colitis is even more marked in those carrying an A allele at position 720 in the LTA gene suggesting that it has a modulator role [18].
The HLA-B-associated transcript 1 (BAT1) gene lies w40 kb telomeric of TNFA. It belongs to the DEAD-box family of RNA-binding proteins and is expressed in a wide variety of cells and tissues including lymphocytes and monocytes [2]. Although the function of this gene has yet to be determined it is believed to have an indispensable role. Using anti-sense DNA corresponding to exons 2e5 of BAT1 Allock et al. [3] reported higher levels of proinflammatory cytokines (including TNF and IL-1) in anti-sense transfectants following mitogenic stimulation, suggesting a possible role as a fast acting negative regulator of inflammation. 4.7. HLA-B-associated transcript 3 (BAT3) Also within this region is the HLA-B-associated transcript 3 (BAT3), this encodes a large proline-rich protein, as yet its function is unknown. 4.8. Major histocompatibility complex class I chain related gene A (MICA)
4.4. 1C7 The 1C7 gene lies 15 kb centromeric of TNF, a putative member of the Ig superfamily it is expressed at RNA level in numerous alternatively spliced forms. There is striking
The major histocompatibility complex class I chain related gene A (MICA) is a class-I like molecule and acts as the activating ligand for the NKG2D receptor present in some subsets of natural killer cells and g/d T cells. It is polymorphic
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with a transmembrane microsatellite polymorphism within exon 5. A recent Spanish family based study suggested that one of the MICA alleles might be an independent marker of protection against RA in SE positive patients [21]. Further studies have reported the association of MICA with seronegative arthritis including psoriatic arthritis [10] and Behcet’s syndrome [26], although here, subsequent work suggests the results may be explained by linkage disequilibrium with a known disease associate [41]. There is a growing body of evidence suggesting a second susceptibility locus for RA lies within the central MHC, further work is needed to enable fine mapping and ultimately identification. This will require the combined use of large case control studies, gene expression and animal models of human inflammatory arthritis, such as transgenic and knockout mice. Although the TDT is a robust method for detecting relatively large genetic effects it frequently lacks power to detect weaker ones but can be complemented by large case control studies. This has previously been employed in the fine mapping of the NOD2/CARD15 gene in Crohn’s disease [15]. In vitro gene expression studies enable the examination of the relationship between a genetic variant and the resulting transcript for example examining expression of a candidate gene within diseased synovium using quantitative PCR or measuring the level of a specific cytokine or receptor mRNA, or protein expressed following the in vitro stimulation of cells in culture, or the use of cloning and cell line transfection. In a recent paper Ramensky et al. [34] describe the use of SNP databases to determine which SNPs are likely to have relevant biological significance and predict the effects on protein structure and function. The increasing availability of the new microarray technology means that expression of many different genes can be rapidly, simultaneously evaluated in a wide variety of cell types. Although expensive with limited availability the technique has already been widely employed to investigate a variety of diseases including rheumatoid arthritis [13]. 5. Conclusion It is clear that new susceptibility loci exist for RA and it is likely that these exert smaller, probably additional genetic effects to that of the SE. The number of supportive studies for a secondary effect within the MHC telomeric to DRB1 is increasing. However, disease heterogeneity, the strong dominating effect of the SE, the density of potential candidates in this region and the current ignorance about the exact function of many of these continues to hinder the identification of a new disease susceptibility gene.
References [1] Allcock RJ, Christiansen FT, Price P. The central MHC gene IKBL carries a structural polymorphism that is associated with HLA-A3,B7, DR15. Immunogenetics 1999;49:660e5.
[2] Allcock RJ, Price P, Gaudieri S, Leelayuwat C, Witt CS, Dawkins RL. Characterisation of the human central MHC gene, BAT1: genomic structure and expression. Exp Clin Immunogenet 1999;16:98e106. [3] Allcock RJ, Williams JH, Price P. The central MHC gene, BAT1, may encode a protein that down-regulates cytokine production. Genes Cells 2001;6:487e94. [4] Brinkman BM, Huizinga TW, Kurban SS, van der Velde EA, Schreuder GM, Hazes JM, et al. Tumour necrosis factor alpha gene polymorphisms in rheumatoid arthritis: association with susceptibility to, or severity of, disease? Br J Rheumatol 1997;36:516e21. [5] Castro F, Acevedo E, Ciusani E, Angulo JA, Wollheim FA, SandbergWollheim M. Tumour necrosis factor microsatellites and HLA-DRB1), HLA-DQA1) and HLA-DQB1) alleles in Peruvian patients with rheumatoid arthritis. Ann Rheum Dis 2001;60:791e5. [6] Date Y, Seki N, Kamizono S, Higuchi T, Hirata T, Miyata K, et al. Identification of a genetic risk factor for systemic juvenile rheumatoid arthritis in the 5#-flanking region of the TNFalpha gene and HLA genes. Arthritis Rheum 1999;42:2577e82. [7] de la Concha EG, Fernandez-Arquero M, Lopez-Nava G, Martin E, Allcock RJ, Conejero L, et al. Susceptibility to severe ulcerative colitis is associated with polymorphism in the central MHC gene IKBL. Gastroenterology 2000;119:1491e5. [8] de Vries N, van Elderen C, Tijssen H, van Riel PL, van de Putte LB. No support for HLA-DQ encoded susceptibility in rheumatoid arthritis. Arthritis Rheum 1999;42:1621e7. [9] Emery P, Salmon M, Bradley H, Wordsworth P, Tunn E, Bacon PA, et al. Genetically determined factors as predictors of radiological change in patients with early symmetrical arthritis. Bmj 1992;305:1387e9. [10] Gonzalez S, Martinez-Borra J, Torre-Alonso JC, Gonzalez-Roces S, Sanchez del Rio J, Rodriguez Perez A, et al. The MICA-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to the development of psoriatic arthritis and is independent of the association of Cw)0602 in psoriasis. Arthritis Rheum 1999;42: 1010e6. [11] Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205e13. [12] Hajeer AH, Dababneh A, Makki RF, Thomson W, Poulton K, GonzalezGay MA, et al. Different gene loci within the HLA-DR and TNF regions are independently associated with susceptibility and severity in Spanish rheumatoid arthritis patients. Tissue Antigens 2000;55:319e25. [13] Heller R, Schena M, Chai A, Shalon D, Bedilion T, Gilmore J, et al. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc Natl Acad Sci U S A 1997;94:2150e5. [14] Holzinger I, de Baey A, Messer G, Kick G, Zwierzina H, Weiss EH. Cloning and genomic characterization of LST1: a new gene in the human TNF region. Immunogenetics 1995;42:315e22. [15] Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599e603. [16] Jawaheer D, Seldin MF, Amos CI, Chen WV, Shigeta R, Monteiro J, et al. A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases. Am J Hum Genet 2001;68:927e36. [17] Kilding R, Iles MM, Timms JM, Worthington J, Wilson AG. Additional genetic susceptibility for rheumatoid arthritis telomeric of the DRB1 locus. Arthritis Rheum 2004;50:763e9. [18] Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP. Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes Immun 2000;1:185e90. [19] Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini, et al. Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum 1998;41: 778e99. [20] Low AS, Gonzalez-Gay MA, Akil M, Amos RS, Bax DE, Cannings C, et al. TNF C489 polymorphism does not contribute to susceptibility to rheumatoid arthritis. Clin Exp Rheumatol 2002;20:829e32.
R. Kilding, A.G. Wilson / Cytokine 32 (2005) 71e75 [21] Martinez A, Fernandez-Arquero M, Balsa A, Rubio A, Alves H, PascualSalcedo D, et al. Primary association of a MICA allele with protection against rheumatoid arthritis. Arthritis Rheum 2001;44:1261e5. [22] Martinez A, Fernandez-Arquero M, Pascual-Salcedo D, Conejero L, Alves H, Balsa A, et al. Primary association of tumor necrosis factorregion genetic markers with susceptibility to rheumatoid arthritis. Arthritis Rheum 2000;43:1366e70. [23] Martinez A, Pascual M, Pascual-Salcedo D, Balsa A, Martin J, de la Concha EG. Genetic polymorphisms in Spanish rheumatoid arthritis patients: an association and linkage study. Genes Immun 2003;4: 117e21. [24] Mattey DL, Hassell AB, Dawes PT, Ollier WE, Hajeer A. Interaction between tumor necrosis factor microsatellite polymorphisms and the HLA-DRB1 shared epitope in rheumatoid arthritis: influence on disease outcome. Arthritis Rheum 1999;42:2698e704. [25] McDonagh JE, Dunn A, Ollier WE, Walker DJ. Compound heterozygosity for the shared epitope and the risk and severity of rheumatoid arthritis in extended pedigrees. Br J Rheumatol 1997;36:322e7. [26] Mizuki N, Ota M, Kimura M, Ohno S, Ando H, Katsuyama Y, et al. Triplet repeat polymorphism in the transmembrane region of the MICA gene: a strong association of six GCT repetitions with Behcet disease. Proc Natl Acad Sci U S A 1997;94:1298e303. [27] Mulcahy B, Waldron-Lynch F, McDermott MF, Adams C, Amos CI, Zhu DK, et al. Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis. Am J Hum Genet 1996;59:676e83. [28] Nepom GT, Seyfried CE, Holbeck SL, Wilske KR, Nepom BS. Identification of HLA-Dw14 genes in DR4C rheumatoid arthritis. Lancet 1986;2:1002e5. [29] Neville MJ, Campbell RD. A new member of the Ig superfamily and a V-ATPase G subunit are among the predicted products of novel genes close to the TNF locus in the human MHC. J Immunol 1999;162: 4745e54. [30] Newton J, Brown MA, Milicic A, Ackerman H, Darke C, Wilson JN, et al. The effect of HLA-DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin alpha-tumor necrosis factor haplotype. Arthritis Rheum 2003;48:90e6. [31] O’Dell JR, Nepom BS, Haire C, Gersuk VH, Gaur L, Moore GF, et al. HLA-DRB1 typing in rheumatoid arthritis: predicting response to specific treatments. Ann Rheum Dis 1998;57:209e13. [32] Okamoto K, Makino S, Yoshikawa Y, Takaki A, Nagatsuka Y, Ota M, et al. Identification of I kappa BL as the second major histocompatibility
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Review Article
Mapping of a novel susceptibility gene for rheumatoid arthritis in the telomeric MHC region Rachael Kilding, Anthony G. Wilson* Division of Genomic Medicine, The University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, United Kingdom
Abstract Rheumatoid arthritis (RA) is a complex heterogeneous disease with an estimated genetic contribution to of 30e50%. Approximately one third arises from the major histocompatibility complex (MHC) at 6p21.3. The contribution of specific DRB1 alleles encoding the shared epitope has been well described, however, several recent studies have suggested that additional telomeric genetic influences may exist. This region is difficult to study as a result of the presence of strong linkage disequilibrium (LD) within the MHC and high gene density particularly in the central class III region. In this article we review the current data supporting the existence of a non-DRB1 susceptibility gene for rheumatoid arthritis, in particular within the class III region. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Rheumatoid arthritis; Genetics; Polymorphism; MHC
1. Introduction Rheumatoid arthritis (RA) is a complex heterogeneous disease characterised by persistent joint inflammation resulting in bony erosion and cartilage loss and often systemic tissue. It affects w1% of most Caucasian populations, with a female to male prevalence of 2.5:1 [19]. The aetiology of RA remains unknown, but is multifactorial with a polygenic component influencing both disease susceptibility and severity. 2. The shared epitope The association of RA with DRB1*0401 (DRw1) was initially discovered by Stastny [37] and almost 10 years later a similar association with DRB1*0404 was reported [28]. Subsequent studies led to the description of association of a group of HLA-DR genes with RA susceptibility, leading to the shared epitope (SE) hypothesis [11]. These encode a similar amino acid sequence QRRAA, QKRAA or RRRAA, in position 70e74 on the third hypervariable region of the DR-b chain. * Corresponding author. Tel.: C44 114 271 2232; fax: C44 114 271 1863. E-mail address: [email protected] (A.G. Wilson). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.07.015
Pocket 4 of 9 in the HLA-DR molecule encompasses amino acid positions 13, 70, 71, 74 and 78 of the b chain and position 9 of the a chain. Positions 70, 71 and 74 are part of the RA associated sequence. The amino acid at position 71 plays a vital role in determining which amino acid side chain will be accepted into the peptide binding pocket. Shared epitope alleles associated with RA present negatively, but not positively charged, amino acids at position 4 of the presented peptide, the reverse is true of the protective DRB1*0402 allele [38,42,43]. The alleles of the shared epitope consist of HLADRB1*0101, *0102, *0401, *0404, *0405, *0408, *0409, *0410, *1001, *1402, *1406 and *1409. The contribution of the shared epitope to RA susceptibility is yet to be clarified, whether it directly results in disease susceptibility or influences prognosis is still open for debate. Notably not all patients with RA are SE positive and w30% of the healthy UK Caucasian population are HLA-DRB1*04 positive. An alternative theory suggests that susceptibility is encoded by HLA-DQ, modulated by certain protective HLA-DRB1 alleles [45]. This effect, however, is probably a reflection of linkage disequilibrium with SE alleles [8]. Certain HLA-DRB1 alleles appear to predispose to more aggressive disease [40] with an increased relative risk of developing erosions [9] and may also influence response to
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treatment [31]. The most severe outcome is seen in rheumatoid patients homozygous for *0401 or *0404 or *0401/*0404/ *0408 heterozygotes. Homozygosity of HLA-DRB1*0401 has been shown to be significantly increased in patients with major organ involvement [44]. The compound heterozygote *0404/*0401 has a significantly greater risk of severe erosive RA [25]. 3. Evidence of non-DRB1 genetic contribution within the MHC Recent evidence has suggested that the SE hypothesis does not explain all of the genetic association of the MHC with RA. The TNF gene is located at the telomeric end of the class III region approximately 1 Mb telomeric of DRB1 and, in view of the biological role of this cytokine in RA, this locus has been examined intensively for a role in the genetic background of RA. A Spanish study reported a SE-independent association of TNF microsatellites with erosive RA [12]. Similar effects have also been detected in British and Peruvian studies with evidence in the British study of genetic interaction with the SE in female patients [5,24]. In view of the reported effects on gene expression a number of case control studies have examined TNF promoter SNPs in RA with several reporting evidence of a genetic effect independent of the SE [4,6,39]. Most of these studies have, however, only typed the DRB1 and TNF loci. Recently several case control studies have used a larger set of genetic markers spanning the MHC. A Japanese group typed 5 TNF promoter SNPs and 18 microsatellites spanning the MHC in 248 controls and 120 patients and found evidence of an additional genetic effect in the telomeric class III region close to TNF [33]. A Dutch group examined similar sized cohorts and typed 6 microsatellites around the junction of the class III and I regions and reported evidence of a DRB1-independent genetic effect derived from the A1B8-DR3 (8.1) haplotype [46]. We recently reported results of a family based analysis specifically searching the MHC using the transmission disequilibrium test (TDT) [36]. Thirteen SNPs were genotyped in 164 British rheumatoid arthritis families containing at least one affected offspring. Using unconditional and DRB1-conditioned transmission disequilibrium tests at least one additional nonDRB1 susceptibility locus for RA was identified within an interval encompassing the junction of the class III and I regions. The effect was detected with a SNP in the second intron of the leucocyte specific transcript-1 (LST-1) gene, this is situated almost 10 kb upstream of TNF and w940 kb telomeric of DRB1. This polymorphism appears to have no biological significance, and sequence analysis of the coding and regulatory region of LST-1 did not reveal other polymorphisms in either healthy controls or RA patients. On balance it seems more likely that the observed association with RA is secondary to LD with a functional genetic variant in another gene [17]. Our findings share similarities with the recently reported family study by Jawaheer et al. [16], where two additional genetic effects were described within the MHC, independent of HLA-DRB1. One of these lies in the central MHC, within
a 497-kb region, on a segment of the A1-B8-DRB1*03 ancestral haplotype, this is the same region we identified with LST-1. However, our results suggest that the gene probably lies within a smaller interval between BAT3 and BAT1, although the exact boundaries of the block of LD need to be refined. Further genes were also proposed to exist within a 697-kb interval in the class I region in a subset of DRB1*0404 haplotypes, we found a second smaller effect in this region with a marker in PG8. A recent case control study from Japan has reported the association of the IkBL-62 marker with RA, speculating that this polymorphism may alter a motif for the transcriptional repressor dEF1 [32]. However, using both unconditional and DRB1conditioned TDT we did not find evidence implicating this polymorphism in RA. These contradictory findings may be related to differences in LD in the two populations, we found strong LD between this IkBL marker and *0301, a non-SE allele, however, LD with DRB1 alleles was not examined in the Japanese study and perhaps the association reported is secondary to LD between IkBL-62 and a SE allele in the Japanese population. A further study of over 270 RA patients and controls positive for HLA-DRB1*04, typed for 6 SNPs within the LTAe TNF interval suggested that genes outside this region were acting as modifiers of the effect of DRB1 on RA susceptibility. Significant variation in the LTAeTNF haplotype frequencies were observed depending on the HLA-DRB1*04 subtype. For DRB1*0401 carriers the LTAeTNF2 haplotype was significantly less frequent in cases compared with controls (OR 0.5 95% confidence interval 0.3e0.8, p Z 0.007), however, the opposite was observed in DRB1*0404 carriers (OR 16.7 95% confidence interval 1.8e240, p Z 0.007) [30]. We observed a similar effect in the variation of the high-risk LST-1 allele depending on the HLA-DRB1 haplotype. On the DRB1 1/SEC background allele 2 of LST-1 was overtransmitted (OR 4.4), however, on the DRB1 4/SEC and DRB1 15/SEÿ background allele 1 was overtransmitted (OR 1.48 and 6.79, respectively). This variation in the high-risk allele dependent on the DRB1 background might explain why no effect at LST-1 was detected in the unconditional analysis and only became apparent after conditioning for the effect of DRB1. 4. Candidate genes The telomeric class III and centromeric class I regions of the MHC contain numerous potential immunoregulatory genes including: allograft inhibitory factor 1 (AIF-1), G1, 1C7, LST1, lymphotoxin (LT)b, TNF, LTa, NB6, IkBL and BAT1 [29]. The function of many of these is yet to be determined but several are known to play an active part in modulating the immune response and are therefore exciting potential candidates. 4.1. Tumour necrosis factor The pivotal role of TNF in disease pathogenesis means it has long been considered a potential candidate gene and as
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such has been extensively studied. Studies have, however, failed to consistently show a direct genetic contribution to RA. The SE-independent association of TNF microsatellites with RA in a Spanish population was reported by Hajeer et al. [12]. A similar effect has been reported in British and North American populations [5,24]. Further case control studies have suggested a SE-independent protective effect of TNFC489 and ÿ238 [4,39]. In contrast TNFC489 did not appear to confer a protective effect in a combined Spanish and British case control study [20]. Recently several case control studies have used a larger set of genetic markers spanning the MHC. A Japanese group typed 5 TNF promoter SNPs and 18 microsatellites spanning the MHC in 248 controls and 120 patients and found evidence of an additional genetic effect in the telomeric class III region close to TNF [33]. A Dutch group examined similar sized cohorts and typed 6 microsatellites around the junction of the class III and I regions and reported evidence of a DRB1independent genetic effect derived from the A1-B8-DR3 (8.1) haplotype [46]. Although case control studies have the power to detect weak genetic effects they are also susceptible to the effects of population stratification, in order to avoid this potential bias the transmission disequilibrium test (TDT) can be applied to affected families. Distorted transmission of TNF haplotypes in multiplex RA families has been reported in an Irish population [27] and independently of the SE in Spain [22].
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sequence conservation between 1C7 and the members of the Ig superfamily, at least 2 of the 1C7 transcripts are probably associated with expression of a functional protein. Although further work is needed to determine the function of this gene it is a potential candidate gene for autoimmune disorders [29]. 4.5. Inhibitor of kappa B-like (IkBL) Just 20 kb telomeric of the TNF cluster lies the inhibitor of kappa B-like (IkBL) gene. This gene shows sequence homology with the inhibitor of kappa light chain gene enhancer in B cells alpha (IkBagene) involved in the regulation of nuclear localisation of the transcription factor, nuclear factor kappa B (NFkB), which in turn stimulates the transcription of many genes including proinflammatory cytokines such as TNFa [7]. Suggesting that IkBL could also be involved in regulation of transcription of proinflammatory cytokines, making it an interesting potential candidate gene. Three SNPs have been identified within IkBL, 2 within the promoter and a non-synonomous C / T polymorphism at position C738 results in a cysteine to arginine substitution in a predicted protein kinase C phosphorylation domain [1]. This polymorphism is associated with susceptibility to ulcerative colitis [7] and multiple sclerosis [1]. A recent case control and family association study investigated the potential association of this SNP with rheumatoid arthritis. No significant difference in rare allele frequency was detected between 225 RA patients and 291 controls, similarly the TDT applied to 58 families did not demonstrate any transmission distortion [23].
4.2. Leucocyte specific transcript-1 (LST-1) 4.6. HLA-B-associated transcript 1 (BAT1) The LST-1 gene is situated almost 10 kb upstream of TNF and w940 kb telomeric of DRB1. Although its exact function is yet to be determined, support for an immunoregulatory role is suggested by expression in lymphoid tissues, T cells, B cells, dendritic cells, macrophages and histiocyte cell lines. In addition it is IFNg inducible [14] and has recently been shown to have an inhibitory effect on lymphocyte proliferation [35] indicating that it could be an attractive candidate gene in autoimmune conditions including RA. 4.3. Lymphotoxin alpha (LTa) Lymphotoxin alpha, also known as TNFb, lies telomeric of TNF and shows 35% identity and 50% homology in amino acid sequence with TNF. Lymphotoxin alpha is a soluble protein secreted by activated lymphocytes and believed to act in the immune response. The association of the TNF-308 polymorphism, GeA, reported in females with extensive colitis is even more marked in those carrying an A allele at position 720 in the LTA gene suggesting that it has a modulator role [18].
The HLA-B-associated transcript 1 (BAT1) gene lies w40 kb telomeric of TNFA. It belongs to the DEAD-box family of RNA-binding proteins and is expressed in a wide variety of cells and tissues including lymphocytes and monocytes [2]. Although the function of this gene has yet to be determined it is believed to have an indispensable role. Using anti-sense DNA corresponding to exons 2e5 of BAT1 Allock et al. [3] reported higher levels of proinflammatory cytokines (including TNF and IL-1) in anti-sense transfectants following mitogenic stimulation, suggesting a possible role as a fast acting negative regulator of inflammation. 4.7. HLA-B-associated transcript 3 (BAT3) Also within this region is the HLA-B-associated transcript 3 (BAT3), this encodes a large proline-rich protein, as yet its function is unknown. 4.8. Major histocompatibility complex class I chain related gene A (MICA)
4.4. 1C7 The 1C7 gene lies 15 kb centromeric of TNF, a putative member of the Ig superfamily it is expressed at RNA level in numerous alternatively spliced forms. There is striking
The major histocompatibility complex class I chain related gene A (MICA) is a class-I like molecule and acts as the activating ligand for the NKG2D receptor present in some subsets of natural killer cells and g/d T cells. It is polymorphic
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with a transmembrane microsatellite polymorphism within exon 5. A recent Spanish family based study suggested that one of the MICA alleles might be an independent marker of protection against RA in SE positive patients [21]. Further studies have reported the association of MICA with seronegative arthritis including psoriatic arthritis [10] and Behcet’s syndrome [26], although here, subsequent work suggests the results may be explained by linkage disequilibrium with a known disease associate [41]. There is a growing body of evidence suggesting a second susceptibility locus for RA lies within the central MHC, further work is needed to enable fine mapping and ultimately identification. This will require the combined use of large case control studies, gene expression and animal models of human inflammatory arthritis, such as transgenic and knockout mice. Although the TDT is a robust method for detecting relatively large genetic effects it frequently lacks power to detect weaker ones but can be complemented by large case control studies. This has previously been employed in the fine mapping of the NOD2/CARD15 gene in Crohn’s disease [15]. In vitro gene expression studies enable the examination of the relationship between a genetic variant and the resulting transcript for example examining expression of a candidate gene within diseased synovium using quantitative PCR or measuring the level of a specific cytokine or receptor mRNA, or protein expressed following the in vitro stimulation of cells in culture, or the use of cloning and cell line transfection. In a recent paper Ramensky et al. [34] describe the use of SNP databases to determine which SNPs are likely to have relevant biological significance and predict the effects on protein structure and function. The increasing availability of the new microarray technology means that expression of many different genes can be rapidly, simultaneously evaluated in a wide variety of cell types. Although expensive with limited availability the technique has already been widely employed to investigate a variety of diseases including rheumatoid arthritis [13]. 5. Conclusion It is clear that new susceptibility loci exist for RA and it is likely that these exert smaller, probably additional genetic effects to that of the SE. The number of supportive studies for a secondary effect within the MHC telomeric to DRB1 is increasing. However, disease heterogeneity, the strong dominating effect of the SE, the density of potential candidates in this region and the current ignorance about the exact function of many of these continues to hinder the identification of a new disease susceptibility gene.
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