British Journal of Rheumatology 1992;31:701-705
CONFERENCE REPORT
IMMUNOGENETICS OF RHEUMATOID ARTHRITIS AND THE ARTHRITIS AND RHEUMATISM COUNCIL'S NATIONAL REPOSITORY BY M. J. WALPORT, W. E. R. OLLIER* AND A. J. SILMAN* Rheumatology Unit, Department of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN; and *ARC Epidemiology Research Unit, Stopford Building, University of Manchester, Oxford Road, Manchester Ml 3 9PT
conferred by gene products in the major histocompatibility locus and that 200 families should be sufficient to detect almost any significant disease susceptibility locus. HOW STRONG IS THE GENETIC BASIS OF RA? The most powerful method of addressing this question is a comparison of the prevalence of RA in monozygotic and dizygotic twins. Alan Silman, Manchester, presented preliminary results of the ARC's national RA twin study. Monozygotic twin pairs, containing at least one member with RA, were identified during March and April 1989 both from outpatient clinics and by means of an extensive media campaign. The main surprise of the meeting was the low concordance figure amongst the monozygotic twins, subsequently confirmed at 15%, comparable to the Finnish twin registry study but much lower than the frequently quoted figures from the earlier studies by Lawrence (Table I). There was considerable discussion about the meaning of these results which, taken at face value, imply only a small genetic contribution to disease, which could be explained by major histocompatibility complex (MHC) genes alone. Why are these concordance rates so much lower than those of previous studies? Two possibilities, both of which are probably partly correct, are: (i) a tendency to over-ascertain twins concordant for disease during previous studies; and (ii) the inclusion in the present study of twins in whom the diseased proband only suffered from mild disease, in which disease susceptibility genes may play a much smaller part, as discussed below.
PRINCIPLES OF GENETIC ANALYSIS OF DISEASE-AFFECTED SIBLING PAIRS The reason for studying sibling pairs who are both already suffering from disease is that environmental influences become irrelevant, since any such influences have already exerted their effect. The principle of the genetic analysis is to search for regions of the genome showing higher than expected sharing of polymorphic alleles between diseased siblings (Fig. 1). In order to accomplish this task, there are two requirements: (i) a genetic map of the entire human genome; and (ii) characterization of highly polymorphic loci regularly spaced throughout the genome, such that there is a high probability that any individual will be heterozygous at each locus. The required resolution of the genetic map requires identification of a highly polymorphic locus spaced approximately 15 cM (or 15 megabases of DNA) or less apart throughout the genome. Two types of polymorphic locus are proving particularly useful, minisatellite repeats and microsatellites (see glossary). The rapid pace of analysis of the human genome means that these mapping requirements will soon be met. This approach was illustrated by Dr Francois Cornelis, working in Professor John Bell's laboratory in Oxford, who presented estimates which suggested that 50 families with affected sib pairs would be sufficient to detect any locus which conferred disease susceptibility with a similar strength to that
MHC ASSOCIATIONS WITH RA Much of the meeting was spent in discussion of what is already known about disease susceptibility genes for RA. Several speakers described the current state of knowledge of MHC associations with RA. WHAT IS THE MHC ASSOCIATION WITH RA? Numerous studies have demonstrated that DR1 and particular subtypes of DR4 (DRB 1*0401 (Dw4), DRB 1*0404/8 (Dwl4), DRB 1*0405 (Dwl5)) were most strongly associated with RA. Subsequent sequencing of these alleles [3, 4] revealed that they shared a highly conserved DRB1 sequence of amino acids in the third hypervariable region (amino acid positions 70-74), namely QKRAA or QRRAA. It was
Submitted 28 June 1992; accepted 3 July 1992. Correspondence to M. J. Walport.
© 1992 British Society for Rheumatology
0263-7103/92/010710 + 05 $08.00/0 701
Downloaded from http://rheumatology.oxfordjournals.org/ at Carleton University on March 16, 2015
THE Arthritis and Rheumatism Council's (ARC) National Repository of lymphocytes from multicase arthritis families formed the focus for a discussion meeting about the immunogenetics of rheumatoid arthritis (RA) recently held at Holly Royde Conference Centre, Manchester University, organized by Professor Alan Silman and Dr Bill Oilier. The core of the National Repository is a collection of Epstein-Barr virus-transformed lymphocyte cell lines from sibling pairs, in which both members suffer from well-documented RA, and from other non-affected family members. This collection is well underway and it is planned to extend it to include cell lines from sibling pairs with juvenile chronic arthritis, systemic lupus erythematosus (SLE) and other rheumatic diseases. The Repository will form a unique resource for analysing the genetic basis of rheumatic disease.
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
702
ab Q —|I—
1
1 ac
cd
O ad
be
a, b, c and d are alleles at a locus not linked to disease susceptibility. The probability of the diseased siblings (denoted by blackened symbols) sharing allele a is 50%.
cd
O be
bd
be
a, b, c and d are alleles at a locus which is linked to disease susceptibility. The disease susceptibility gene is linked to allele b. The probability of the diseased siblings sharing allele b is greater than 50%. Note that the third sibling without disease also shares allele b—this could be because that individual has not been exposed to the appropriate environmental trigger or because that individual has not inherited other necessary disease susceptibility genes in addition to allele b. Fig. 1.—Principle of studying disease-affected sibling pairs to locate regions of the genome containing disease susceptibility genes. The best loci to use to study allele sharing are those expressing a high degree of polymorphism and heterozygosity. Problems may arise if there is limited polymorphism. For example, if both parents expressed allele b, then the probability of allele b being present in any two siblings by chance is 75%, and it is impossible to discern whether the paternal or maternal b allele is linked to the disease susceptibility gene.
suggested that this may form the basis of a shared DRB1 epitope involved in conferring RA susceptibility [5]. Further population studies have indicated that amino acid position 86 may also be critical for this epitope, and DR4/DR1 variants having a glycine (G) at position 86 are thought to be more associated with RA [6]. The mechanism of the association of Class II molecules with disease is not understood, but presumably follow from the physiological activity of Class II molecules in presentation of peptide antigens to T cell receptors and in the selection of the T cell repertoire within the thymus during ontogeny. It follows from these activities that association of particular Class II molecules with disease could follow from: (i) the selective ability of certain Class II molecules to present as antigens particular peptides leading to autoimmunity; and (ii) thymic selection of populations of potentially pathogenic T cells.
WHAT IS THE PREVALENCE OF RA IN DIFFERENT PARTS OF THE WORLD? A related question to that of the MHC association is to establish the prevalence of disease in different populations. Alan Silman (Manchester) reported that a community-based RA prevalence study in the Yoruba of Nigeria confirmed previous anecdotal reports of low RA prevalence, with no RA cases identified in the 2000 subjects studied. One possible explanation for this low RA prevalence may be the low frequency of DRB1 alleles carrying the shared RA epitope. If DRBl*0102 is excluded from the shared epitope and DRBl*10 included, only 14.5% of those studied possessed the shared epitope. Wendy Thomson (Manchester) also presented RA prevalence and immunogenetic data for Hong Kong Chinese. The prevalence of RA in Hong Kong (0.35%) is lower than that seen in the UK. Of the cases diagnosed as RA 55.6% were typed as DRB1 *04 and were predominantly the DRBl*0405 variant.
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Several papers at the meeting addressed the question of whether HLA-DR molecules bearing the shared epitope really are the relevant disease susceptibility genes for RA located in the MHC. An important method for answering this question is to study different ethnic populations. Results were presented from diverse populations including two studies of Hong Kong Chinese patients (Alex So, London; Wendy Thomson, Manchester), the Yoruba of Nigeria (Alan Silman, Manchester), Zimbabweans (Shirley Cutbush, London), South African blacks (Paul Wordsworth, Oxford) and two studies of Greek caucasoids (Jerry Lanchbury, London; Bill Oilier, Manchester). The results of each of these studies showed consistent associations of disease with the shared epitope, however, this association appears to be much weaker in some populations than others. This is particularly the case in southern Europe. Initial studies of Greek RA patients had failed to show any association with DR4, and a weak association with DR1 [7] and similar results had been reported from Israel [8, 9]. Over 50% of patients with RA were negative for the 'susceptibility haplotype'. In Greeks, a third DR product, DRwlO, with the third AHVR sequence, RRRAA (containing one amino acid substitution distinguishing the sequence from that in DR4 and DR1) appears to be associated with disease susceptibility. Jerry Lanchbury, London, presented a study of 100 consecutive attendees with RA at a clinic in Ioannina—the three sequences comprising the shared epitope, QKRAA, QRRAA and RRRAA, had a relative risk for disease in this population of 3.8 with a prevalence of 43.5% in the patients compared with 17% amongst the controls. Similar results were reported from an Athenian population by Bill Oilier. The significance of the weaker association of disease in these populations with the shared epitope is uncertain—it is possible that disease is milder in these populations or alternatively that the particular cohorts of patients studied had milder disease (see below).
WALPORT ETAL.: IMMUNOGENETICS OF RA AND THE ARC'S NATIONAL REPOSITORY
703
TABLE I PREVIOUS STUDIES OF THE PREVALENCE OF RA IN MONOZYGOTIC VERSUS DIZYGOTIS TWINS
Monozygotic twins
Dizygotic twins
No. of pairs
% Concordance
No. of pairs
% Concordance
Notes
Reference
97 20 73
32 30 12.3
188 73 173
9 5 3.5
Literature summary ARC survey Finnish twin registry
[1] [1] [2]
These data provide strong support for the idea that the presence of the shared epitope in the third AHVR of the HLA-DRBl*0101 and 0401 alleles is associated with the presence of severe RA. However, this does not answer the question of whether the association is with disease progression or susceptibility. On the one hand it is possible that there is a single aetiology for many cases of early polyarthritis and the presence of the shared epitope in the third AHVR predisposes to disease progression to severe RA. The alternative hypothesis is that there are many aetiologies to polyarthritis, one of which is marked by the possession of the shared epitope, and whose clinical expression is severe RA. DO BOTH MHC HAPLOTYPES MATTER? A further complexity to the association of Class II products with RA came from observations that both alleles at DR may matter. Felty's syndrome is associated with a very high prevalence of DR4, consistently over 90% in the majority of studies. Jerry Lanchbury (London) found that 40 of 43 patients with Felty's were DR4 positive, and 13 of these were DR4homozygotes. Amongst these DR4 homozygotes there was a marked excess of Dw4/Dwl4 heterozygotes. Dwl4 haplotypes only appeared to play a role in combination with Dw4 suggesting that independent synergistic mechanisms may be involved in development of Felty's. Very similar results were presented by Paul Wordsworth, Oxford, who found that DR4 homozygosity was increased both in severe RA and also in Felty's syndrome, and again there was an excess of Dw4/Dwl4or Dw4/Dwl5 heterozygosity. There was also an increase in DR4/DR1 heterozygosity amongst patients with severe RA. What is the explanation for heterozygous associations with DR molecules each bearing the shared epitope, but differing in other polymorphic regions of the molecule? Two explanations that were discussed were: (i) the possibility that the range of pathogenic T cells selected within the thymus is greater; and (ii) that the range of 'pathogenic' peptides presented is increased. THE T CELL RECEPTOR IN RA Now that the main MHC associations with RA appear to have been defined, the focus of attention is switching to analysis of the T cell receptors to which Class II molecules may be presenting peptide antigen, or possibly superantigen (see glossary). How may the T cell receptors influence disease susceptibility? At germ-line level there may be differences in the inher-
Downloaded from http://rheumatology.oxfordjournals.org/ at Carleton University on March 16, 2015
IS THE DRB1 ASSOCIATION WITH PRESENCE OR SEVERITY OF RA? This question has been addressed in three ways by study of: (i) the prevalence of MHC gene products in patients with early disease; (ii) measurement of MHC haplotype sharing in affected sibling pairs with disease of differing severity; and (iii) correlation of MHC gene products with disease progression. Initial studies suggested that HLA-DR4 was mainly associated with severe disease, for example the prevalence of HLADR4 in RA patients studied in different settings showed a progressive increase as follows: general practice, hospital clinic, extra-articular features, Felty's syndrome. Three presentations (Paul Emery, Birmingham; Wendy Thompson, Manchester; Chris Deighton, Newcastle) focused on the characterization of populations of patients with early inflammatory polyarthritis and reached broadly similar conclusions. There was no particular excess of HLA-DR4 or 1 amongst patients presenting within a few months of onset of an inflammatory polyarthritis. Features which correlated with disease progression and with the presence of HLADR4 and 1 were the early onset of erosions and the presence of rheumatoid factor. Chris Deighton, Newcastle, presented the results of a study examining the frequency of HLA haplotype sharing in sib-pairs with RA, stratified according to the severity of the disease. The expected frequency of sharing of haplotypes amongst sibling pairs is two haplotypes 25%, one haplotype 50% and no haplotypes 25%. Amongst sibling pairs with mild RA there was no significant change from the expected frequency of haplotype sharing. In contrast, amongst a cohort of 15 sibling pairs with severe disease 50% shared both MHC haplotypes. Nick de Vries (Nijmegen) and Bernhard Lang (Regensburg) presented data on the association of HLA-DR4 with disease progression and severity. The prevalence of HLA-DR4 rose in parallel with the number of ACR criteria for the presence of RA, an indirect measure of disease severity. De Vries presented data showing that presence of the shared sequence in the third allelic hypervariable region was associated during 3 years of follow-up with greater increase in numbers of erosions and increase in X-ray score than in a matched cohort of rheumatoid patients not possessing this DRB sequence. Similarly, Lang found that HLA-DR4 was associated with greater progression in erosions, more extra-articular features and the development of rheumatoid nodules.
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
DOES GENETIC VARIATION WITHIN THE TcR LOCI DETERMINE DISEASE SUSCEPTIBILITY? This question has been addressed at the population level by study of polymorphisms located within the TcR loci identified by Southern blotting and no clear answer has emerged. This is not surprising considering the large size of the TcR loci (approximately 600 kb for TcR and 1000 kb for TcR-a) and our lack of knowledge of the extent of linkage disequilibrium between genes encoded in these regions. However, this is one of the first questions that can be answered by use of material from the affected sib pairs in the ARC repository, and suitable polymorphic marker loci within the TcR loci are available to undertake this work. One surprising finding that was presented by Nick de Vries (Nijmegen) at the meeting, based on a popula-
CONCLUSIONS AND A PLEA TO THE READER The major contribution of molecular genetics to medicine to date has been the characterization of the molecular basis of monogenic disorders. The majority of common chronic inflammatory diseases are probably mediated by a combination of disease susceptibility genes, coupled with environmental factors. Methods are now available to tackle polygenic disorders and a vital part of this work is the collection of material from informative families. The ARC National Repository is an important step in this direction. The search for sibling pairs with RA juvenile arthritis or SLE is a major task and will require international collaboration. If you are looking after sibling pairs affected by one of these diseases, please contact: Irene Smith, Family Studies Coordinator, ARC Epidemi-
Downloaded from http://rheumatology.oxfordjournals.org/ at Carleton University on March 16, 2015
GENE INTERACTIONS IN ANIMAL MODELS The complexity of the problems to be addressed was illustrated by Peter Gregersen (New York) who reviewed the complex interactions between the MHC and the repertoire of T cell receptors in the induction of the collagen-induced arthritis in mice. This disease can be induced by immunization of susceptible animals with native collagen in Freund's adjuvant and its pathogenesis is dependent on a cellular and T cell-dependent humoral response. Disease can be transferred from mouse to mouse using T cell clones positive for T cell receptors of the V(38 family. However susceptibility to disease is also linked to the MHC (I-Aq). It is of great interest that the SWR strain of mice has I-Aq and yet is not susceptible to collagen arthritis and the explanation for this lack of susceptibility is almost certainly that the SWR has deletion of a block of T cell receptor variable genes including V(38 [10, 11]. In mice, the expressed T cell receptor repertoire is influenced by the expression of genes encoding endogenous 'superantigens' (see glossary). One such is called Mlsa (minor lymphocyte stimulating system of the mouse) and is associated with deletion of VP8 TcR. Expression of Mlsa in susceptible strains of mice is associated with marked protection against induction of collageninduced arthritis [12].
tion study, was that a DNA restriction fragment length polymorphism, demonstrated using a Vp8 probe, appeared to correlate with disease severity in a cohort studied at the time of presentation and 3 years later. WHAT IS THE TcR REPERTOIRE IN RA? If there was limited heterogeneity of expression of T cell receptors at sites of inflammation in a disease mediated by T cells, this would be an attractive target for therapy. The results of initial experiments in animals with models of autoimmune diseases looked extremely promising in this respect and candidate TcR were identified in experimental autoallergic encephalomyelitis [13, 14], collagen arthritis [11, 15] and murine diabetes [16]. The results in outbred humans appear different and less encouraging. An initial report from Paliard and colleagues [ 17] showed an excess of TcR bearing receptors expressing |3-chains encoded by the Vpi4 family, but this has not been confirmed by others. Alex So and Claudio Lunardi (Hammersmith Hospital) and Jerry Lanchbury (Guy's Hospital) presented their own findings and reviewed the current state of knowledge of the repertoire of TcR expressed in RA. The following conclusions seemed to be generally accepted: (i) that in certain patients the expressed TcR repertoire in synovium is different from that of peripheral blood; (ii) in some patients there is expression of dominant clonotypes within synovium but that these differ from one subject to another. There are many important caveats in the interpretation of all such data. The stage at which disease is studied may be extremely important—it may be that restricted TcR expression may only be a feature of early disease. Indeed, it was shown some years ago by Pelton and colleagues [18] that in advanced RA, synovial tissue looks and behaves like a lymph node. They demonstrated an anamnestic response to a number of common infectious and immunizing stimuli occurring within joints, demonstrating that the specificity of recruitment of lymphocytes to synovial tissue may have been lost (if it ever existed). Other important factors that will require careful control experiments are MHC typing and disease severity.
ited repertoire between different individuals. At the somatic level, factors which could be important are: (i) the T cell receptor repertoire selected within the thymus by interactions with Class I and II molecules associated with self-peptides; (ii) selective usage of T cells bearing particular sets of receptors in particular diseases; and (iii) stimulation by superantigens of T cells expressing receptors expressing (3-chains derived from particular Vp gene families. It has become possible to address each of these questions following a great expansion in knowledge of the molecular structure of the TcR genes. The development of novel techniques for sequencing large numbers of T cell receptors using modifications of the polymerase chain reaction has allowed much of this work to take place.
WALPORT ETAL.:
IMMUNOGENETICS OF RA AND THE ARC'S NATIONAL REPOSITORY
ology Research Unit, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT. Tel: 061-275 5622, Fax 061-275 5043
REFERENCES
1. Aho K, Koskenvuo M, Tuominen J, Kaprio J. Occurrence of rheumatoid arthritis in a nationwide series of twins. J Rheumatol 1986;13:899-902. 2. Lawrence JS. Rheumatoid arthritis—nature or nurture. Ann Rheum Dis 1970;29:357-79. 3. Gregersen PK, Shen M, Song Q-L et al. Molecular diversity of HLA-DR4 haplotypes. Proc Natl Acad Sci USA 1986;83:2642-6. 4. Cairns JS, Curtsinger JM, Dahl CA, Freeman S, Alter BJ, Bach FH. Sequence polymorphism of HLA
DRbetal alleles relating to T-cell-recognized determinants. Nature 1985;317:166-8. 5. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. Arthritis Rheum 1987; 30:1205-13. 6. Gao XJ, Olsen NJ, Pincus T, Stastny P. HLA-DR alleles with naturally occurring amino acid substitutions and risk for development of rheumatoid arthritis. Arthritis Rheum 1990;33:939^6. 7. Papasteriades CA, Kappou ID, Skopouli FN, Barla MN, Fostiropoulos GA, Moutsopoulos HM. Lack of HLA-antigen association in Greek rheumatoid arthritis patients. Rheumatol Int 1985;5:201-3. 8. Schiff B, Mizachi Y, Orgad S, Yaron M, Gazit E. Association of HLA-Aw31 and HLA-DR1 with adult rheumatoid arthritis. Ann Rheum Dis 1982;41:403-4. 9. Brautbar C, Naparstek Y, Yaron M et al. Immunogenetics of rheumatoid arthritis in Israel. Tissue Antigens 1986;28:8-14. 10. Behlke MA, Chou HS, Huppi K, Loh DY. Murine T cell receptor mutants with deletions of b-chain variable region genes. Proc Natl Acad Sci USA 1986;83:767-71. 11. HaqqiTM, Banerjee S, Jones WLef a/. Identification of T-cell receptor V beta deletion mutant mouse strain AU/ssJ (H-2q) which is resistant to collageninduced arthritis. Immunogenetics 1989;29:180—5. 12. Anderson GD, Banerjee S, Luthra HS, David CS. Role of Mls-1 locus and clonal deletion of T cells in susceptibility to collagen-induced arthritis in mice. J Immunol 1991;147:1189-93. 13. Acha-Orbea H, Mitcell DJ, Timmermann L et al. Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell 1988;54:263-73. 14. Urban JL, Kumar V, Kono DH etal. Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cell 1988;54:577-92. 15. Haqqi TM, Anderson GD, Banerjee S, David CS. Restricted heterogeneity in T-cell antigen receptor V beta gene usage in the lymph nodes and arthritic joints of mice. Proc Natl Acad Sci USA 1992;89:1253-5. 16. Reich E-P, Sherwin RS, Kanagawa O, Janeway Jr CA. An explanation for the protective effect of the MHC Class II I-E molecule in murine diabetes. Nature 1989;231:326-8. 17. Paliard X, West SG, Lafferty JA et al. Evidence for the effects of a superantigen in rheumatoid arthritis. Science 1991 ;253:325-9. 18. Pelton BK, Harvey AR, Denman AM. The rheumatoid synovial membrane participates in systemic anti-viral immune responses. Clin Exp Immunol 1985;62:657-61.
Downloaded from http://rheumatology.oxfordjournals.org/ at Carleton University on March 16, 2015
GLOSSARY Genetic polymorphism: inherited variation in the sequence of a particular locus, present at a frequency in a population of greater than 1%. Microsatellite repeat: these are mainly CA repeats and length polymorphisms become increasingly common as the length increases above 20 CA repeats. The distribution of CA repeats is random and frequent, occurring approximately once every 50 kb. However, only one in five C A repeats is highly polymorphic meaning that informative polymorphisms only occur once every 250kb. These microsatellite repeats are most easily identified by amplification using the polymerase chain reaction with flanking oligonucleotide primers. Minisatellite repeat or VNTR (variable number of terminal repeats): these are biased to telomeres (the ends of chromosomes) and are short G+C rich sequences from 9-64 bp in length that are organized in tandem repeats from 100 bp20 kbp in length. These commonly show length polymorphisms caused by variation in the number of tandemly repeated elements. The different alleles can be detected by Southern blotting. CentiMorgan: unit of recombination—1 cM is equivalent to one chromosome cross-over for every 100 meioses. Units of recombination can be roughly translated into genetic distances with 1 cM being equivalent to approximately 1 million base pairs of DN A, although some regions of the genome are more liable to recombination than others. Superantigen: these are products of certain bacteria and viruses which activate large numbers of T cells by virtue of their promiscuous binding properties. They bind to MHC Class II molecules outside their antigen-binding groove and are not restricted to particular allelic subtypes. Similarly, they bind to the f5 chain of T cell receptors in such a way that T cells expressing the products of particular families of V(3 genes are activated. Mice carry endogenous retroviruses, some of which encode superantigens, and when these are expressed early in ontogeny the result is deletion of whole families of T cell receptors. There is no evidence to date that the human T cell repertoire is similarly influenced by the expression of endogenous superantigens.
705
CONFERENCE REPORT
IMMUNOGENETICS OF RHEUMATOID ARTHRITIS AND THE ARTHRITIS AND RHEUMATISM COUNCIL'S NATIONAL REPOSITORY BY M. J. WALPORT, W. E. R. OLLIER* AND A. J. SILMAN* Rheumatology Unit, Department of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN; and *ARC Epidemiology Research Unit, Stopford Building, University of Manchester, Oxford Road, Manchester Ml 3 9PT
conferred by gene products in the major histocompatibility locus and that 200 families should be sufficient to detect almost any significant disease susceptibility locus. HOW STRONG IS THE GENETIC BASIS OF RA? The most powerful method of addressing this question is a comparison of the prevalence of RA in monozygotic and dizygotic twins. Alan Silman, Manchester, presented preliminary results of the ARC's national RA twin study. Monozygotic twin pairs, containing at least one member with RA, were identified during March and April 1989 both from outpatient clinics and by means of an extensive media campaign. The main surprise of the meeting was the low concordance figure amongst the monozygotic twins, subsequently confirmed at 15%, comparable to the Finnish twin registry study but much lower than the frequently quoted figures from the earlier studies by Lawrence (Table I). There was considerable discussion about the meaning of these results which, taken at face value, imply only a small genetic contribution to disease, which could be explained by major histocompatibility complex (MHC) genes alone. Why are these concordance rates so much lower than those of previous studies? Two possibilities, both of which are probably partly correct, are: (i) a tendency to over-ascertain twins concordant for disease during previous studies; and (ii) the inclusion in the present study of twins in whom the diseased proband only suffered from mild disease, in which disease susceptibility genes may play a much smaller part, as discussed below.
PRINCIPLES OF GENETIC ANALYSIS OF DISEASE-AFFECTED SIBLING PAIRS The reason for studying sibling pairs who are both already suffering from disease is that environmental influences become irrelevant, since any such influences have already exerted their effect. The principle of the genetic analysis is to search for regions of the genome showing higher than expected sharing of polymorphic alleles between diseased siblings (Fig. 1). In order to accomplish this task, there are two requirements: (i) a genetic map of the entire human genome; and (ii) characterization of highly polymorphic loci regularly spaced throughout the genome, such that there is a high probability that any individual will be heterozygous at each locus. The required resolution of the genetic map requires identification of a highly polymorphic locus spaced approximately 15 cM (or 15 megabases of DNA) or less apart throughout the genome. Two types of polymorphic locus are proving particularly useful, minisatellite repeats and microsatellites (see glossary). The rapid pace of analysis of the human genome means that these mapping requirements will soon be met. This approach was illustrated by Dr Francois Cornelis, working in Professor John Bell's laboratory in Oxford, who presented estimates which suggested that 50 families with affected sib pairs would be sufficient to detect any locus which conferred disease susceptibility with a similar strength to that
MHC ASSOCIATIONS WITH RA Much of the meeting was spent in discussion of what is already known about disease susceptibility genes for RA. Several speakers described the current state of knowledge of MHC associations with RA. WHAT IS THE MHC ASSOCIATION WITH RA? Numerous studies have demonstrated that DR1 and particular subtypes of DR4 (DRB 1*0401 (Dw4), DRB 1*0404/8 (Dwl4), DRB 1*0405 (Dwl5)) were most strongly associated with RA. Subsequent sequencing of these alleles [3, 4] revealed that they shared a highly conserved DRB1 sequence of amino acids in the third hypervariable region (amino acid positions 70-74), namely QKRAA or QRRAA. It was
Submitted 28 June 1992; accepted 3 July 1992. Correspondence to M. J. Walport.
© 1992 British Society for Rheumatology
0263-7103/92/010710 + 05 $08.00/0 701
Downloaded from http://rheumatology.oxfordjournals.org/ at Carleton University on March 16, 2015
THE Arthritis and Rheumatism Council's (ARC) National Repository of lymphocytes from multicase arthritis families formed the focus for a discussion meeting about the immunogenetics of rheumatoid arthritis (RA) recently held at Holly Royde Conference Centre, Manchester University, organized by Professor Alan Silman and Dr Bill Oilier. The core of the National Repository is a collection of Epstein-Barr virus-transformed lymphocyte cell lines from sibling pairs, in which both members suffer from well-documented RA, and from other non-affected family members. This collection is well underway and it is planned to extend it to include cell lines from sibling pairs with juvenile chronic arthritis, systemic lupus erythematosus (SLE) and other rheumatic diseases. The Repository will form a unique resource for analysing the genetic basis of rheumatic disease.
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
702
ab Q —|I—
1
1 ac
cd
O ad
be
a, b, c and d are alleles at a locus not linked to disease susceptibility. The probability of the diseased siblings (denoted by blackened symbols) sharing allele a is 50%.
cd
O be
bd
be
a, b, c and d are alleles at a locus which is linked to disease susceptibility. The disease susceptibility gene is linked to allele b. The probability of the diseased siblings sharing allele b is greater than 50%. Note that the third sibling without disease also shares allele b—this could be because that individual has not been exposed to the appropriate environmental trigger or because that individual has not inherited other necessary disease susceptibility genes in addition to allele b. Fig. 1.—Principle of studying disease-affected sibling pairs to locate regions of the genome containing disease susceptibility genes. The best loci to use to study allele sharing are those expressing a high degree of polymorphism and heterozygosity. Problems may arise if there is limited polymorphism. For example, if both parents expressed allele b, then the probability of allele b being present in any two siblings by chance is 75%, and it is impossible to discern whether the paternal or maternal b allele is linked to the disease susceptibility gene.
suggested that this may form the basis of a shared DRB1 epitope involved in conferring RA susceptibility [5]. Further population studies have indicated that amino acid position 86 may also be critical for this epitope, and DR4/DR1 variants having a glycine (G) at position 86 are thought to be more associated with RA [6]. The mechanism of the association of Class II molecules with disease is not understood, but presumably follow from the physiological activity of Class II molecules in presentation of peptide antigens to T cell receptors and in the selection of the T cell repertoire within the thymus during ontogeny. It follows from these activities that association of particular Class II molecules with disease could follow from: (i) the selective ability of certain Class II molecules to present as antigens particular peptides leading to autoimmunity; and (ii) thymic selection of populations of potentially pathogenic T cells.
WHAT IS THE PREVALENCE OF RA IN DIFFERENT PARTS OF THE WORLD? A related question to that of the MHC association is to establish the prevalence of disease in different populations. Alan Silman (Manchester) reported that a community-based RA prevalence study in the Yoruba of Nigeria confirmed previous anecdotal reports of low RA prevalence, with no RA cases identified in the 2000 subjects studied. One possible explanation for this low RA prevalence may be the low frequency of DRB1 alleles carrying the shared RA epitope. If DRBl*0102 is excluded from the shared epitope and DRBl*10 included, only 14.5% of those studied possessed the shared epitope. Wendy Thomson (Manchester) also presented RA prevalence and immunogenetic data for Hong Kong Chinese. The prevalence of RA in Hong Kong (0.35%) is lower than that seen in the UK. Of the cases diagnosed as RA 55.6% were typed as DRB1 *04 and were predominantly the DRBl*0405 variant.
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ab
Several papers at the meeting addressed the question of whether HLA-DR molecules bearing the shared epitope really are the relevant disease susceptibility genes for RA located in the MHC. An important method for answering this question is to study different ethnic populations. Results were presented from diverse populations including two studies of Hong Kong Chinese patients (Alex So, London; Wendy Thomson, Manchester), the Yoruba of Nigeria (Alan Silman, Manchester), Zimbabweans (Shirley Cutbush, London), South African blacks (Paul Wordsworth, Oxford) and two studies of Greek caucasoids (Jerry Lanchbury, London; Bill Oilier, Manchester). The results of each of these studies showed consistent associations of disease with the shared epitope, however, this association appears to be much weaker in some populations than others. This is particularly the case in southern Europe. Initial studies of Greek RA patients had failed to show any association with DR4, and a weak association with DR1 [7] and similar results had been reported from Israel [8, 9]. Over 50% of patients with RA were negative for the 'susceptibility haplotype'. In Greeks, a third DR product, DRwlO, with the third AHVR sequence, RRRAA (containing one amino acid substitution distinguishing the sequence from that in DR4 and DR1) appears to be associated with disease susceptibility. Jerry Lanchbury, London, presented a study of 100 consecutive attendees with RA at a clinic in Ioannina—the three sequences comprising the shared epitope, QKRAA, QRRAA and RRRAA, had a relative risk for disease in this population of 3.8 with a prevalence of 43.5% in the patients compared with 17% amongst the controls. Similar results were reported from an Athenian population by Bill Oilier. The significance of the weaker association of disease in these populations with the shared epitope is uncertain—it is possible that disease is milder in these populations or alternatively that the particular cohorts of patients studied had milder disease (see below).
WALPORT ETAL.: IMMUNOGENETICS OF RA AND THE ARC'S NATIONAL REPOSITORY
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TABLE I PREVIOUS STUDIES OF THE PREVALENCE OF RA IN MONOZYGOTIC VERSUS DIZYGOTIS TWINS
Monozygotic twins
Dizygotic twins
No. of pairs
% Concordance
No. of pairs
% Concordance
Notes
Reference
97 20 73
32 30 12.3
188 73 173
9 5 3.5
Literature summary ARC survey Finnish twin registry
[1] [1] [2]
These data provide strong support for the idea that the presence of the shared epitope in the third AHVR of the HLA-DRBl*0101 and 0401 alleles is associated with the presence of severe RA. However, this does not answer the question of whether the association is with disease progression or susceptibility. On the one hand it is possible that there is a single aetiology for many cases of early polyarthritis and the presence of the shared epitope in the third AHVR predisposes to disease progression to severe RA. The alternative hypothesis is that there are many aetiologies to polyarthritis, one of which is marked by the possession of the shared epitope, and whose clinical expression is severe RA. DO BOTH MHC HAPLOTYPES MATTER? A further complexity to the association of Class II products with RA came from observations that both alleles at DR may matter. Felty's syndrome is associated with a very high prevalence of DR4, consistently over 90% in the majority of studies. Jerry Lanchbury (London) found that 40 of 43 patients with Felty's were DR4 positive, and 13 of these were DR4homozygotes. Amongst these DR4 homozygotes there was a marked excess of Dw4/Dwl4 heterozygotes. Dwl4 haplotypes only appeared to play a role in combination with Dw4 suggesting that independent synergistic mechanisms may be involved in development of Felty's. Very similar results were presented by Paul Wordsworth, Oxford, who found that DR4 homozygosity was increased both in severe RA and also in Felty's syndrome, and again there was an excess of Dw4/Dwl4or Dw4/Dwl5 heterozygosity. There was also an increase in DR4/DR1 heterozygosity amongst patients with severe RA. What is the explanation for heterozygous associations with DR molecules each bearing the shared epitope, but differing in other polymorphic regions of the molecule? Two explanations that were discussed were: (i) the possibility that the range of pathogenic T cells selected within the thymus is greater; and (ii) that the range of 'pathogenic' peptides presented is increased. THE T CELL RECEPTOR IN RA Now that the main MHC associations with RA appear to have been defined, the focus of attention is switching to analysis of the T cell receptors to which Class II molecules may be presenting peptide antigen, or possibly superantigen (see glossary). How may the T cell receptors influence disease susceptibility? At germ-line level there may be differences in the inher-
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IS THE DRB1 ASSOCIATION WITH PRESENCE OR SEVERITY OF RA? This question has been addressed in three ways by study of: (i) the prevalence of MHC gene products in patients with early disease; (ii) measurement of MHC haplotype sharing in affected sibling pairs with disease of differing severity; and (iii) correlation of MHC gene products with disease progression. Initial studies suggested that HLA-DR4 was mainly associated with severe disease, for example the prevalence of HLADR4 in RA patients studied in different settings showed a progressive increase as follows: general practice, hospital clinic, extra-articular features, Felty's syndrome. Three presentations (Paul Emery, Birmingham; Wendy Thompson, Manchester; Chris Deighton, Newcastle) focused on the characterization of populations of patients with early inflammatory polyarthritis and reached broadly similar conclusions. There was no particular excess of HLA-DR4 or 1 amongst patients presenting within a few months of onset of an inflammatory polyarthritis. Features which correlated with disease progression and with the presence of HLADR4 and 1 were the early onset of erosions and the presence of rheumatoid factor. Chris Deighton, Newcastle, presented the results of a study examining the frequency of HLA haplotype sharing in sib-pairs with RA, stratified according to the severity of the disease. The expected frequency of sharing of haplotypes amongst sibling pairs is two haplotypes 25%, one haplotype 50% and no haplotypes 25%. Amongst sibling pairs with mild RA there was no significant change from the expected frequency of haplotype sharing. In contrast, amongst a cohort of 15 sibling pairs with severe disease 50% shared both MHC haplotypes. Nick de Vries (Nijmegen) and Bernhard Lang (Regensburg) presented data on the association of HLA-DR4 with disease progression and severity. The prevalence of HLA-DR4 rose in parallel with the number of ACR criteria for the presence of RA, an indirect measure of disease severity. De Vries presented data showing that presence of the shared sequence in the third allelic hypervariable region was associated during 3 years of follow-up with greater increase in numbers of erosions and increase in X-ray score than in a matched cohort of rheumatoid patients not possessing this DRB sequence. Similarly, Lang found that HLA-DR4 was associated with greater progression in erosions, more extra-articular features and the development of rheumatoid nodules.
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
DOES GENETIC VARIATION WITHIN THE TcR LOCI DETERMINE DISEASE SUSCEPTIBILITY? This question has been addressed at the population level by study of polymorphisms located within the TcR loci identified by Southern blotting and no clear answer has emerged. This is not surprising considering the large size of the TcR loci (approximately 600 kb for TcR and 1000 kb for TcR-a) and our lack of knowledge of the extent of linkage disequilibrium between genes encoded in these regions. However, this is one of the first questions that can be answered by use of material from the affected sib pairs in the ARC repository, and suitable polymorphic marker loci within the TcR loci are available to undertake this work. One surprising finding that was presented by Nick de Vries (Nijmegen) at the meeting, based on a popula-
CONCLUSIONS AND A PLEA TO THE READER The major contribution of molecular genetics to medicine to date has been the characterization of the molecular basis of monogenic disorders. The majority of common chronic inflammatory diseases are probably mediated by a combination of disease susceptibility genes, coupled with environmental factors. Methods are now available to tackle polygenic disorders and a vital part of this work is the collection of material from informative families. The ARC National Repository is an important step in this direction. The search for sibling pairs with RA juvenile arthritis or SLE is a major task and will require international collaboration. If you are looking after sibling pairs affected by one of these diseases, please contact: Irene Smith, Family Studies Coordinator, ARC Epidemi-
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GENE INTERACTIONS IN ANIMAL MODELS The complexity of the problems to be addressed was illustrated by Peter Gregersen (New York) who reviewed the complex interactions between the MHC and the repertoire of T cell receptors in the induction of the collagen-induced arthritis in mice. This disease can be induced by immunization of susceptible animals with native collagen in Freund's adjuvant and its pathogenesis is dependent on a cellular and T cell-dependent humoral response. Disease can be transferred from mouse to mouse using T cell clones positive for T cell receptors of the V(38 family. However susceptibility to disease is also linked to the MHC (I-Aq). It is of great interest that the SWR strain of mice has I-Aq and yet is not susceptible to collagen arthritis and the explanation for this lack of susceptibility is almost certainly that the SWR has deletion of a block of T cell receptor variable genes including V(38 [10, 11]. In mice, the expressed T cell receptor repertoire is influenced by the expression of genes encoding endogenous 'superantigens' (see glossary). One such is called Mlsa (minor lymphocyte stimulating system of the mouse) and is associated with deletion of VP8 TcR. Expression of Mlsa in susceptible strains of mice is associated with marked protection against induction of collageninduced arthritis [12].
tion study, was that a DNA restriction fragment length polymorphism, demonstrated using a Vp8 probe, appeared to correlate with disease severity in a cohort studied at the time of presentation and 3 years later. WHAT IS THE TcR REPERTOIRE IN RA? If there was limited heterogeneity of expression of T cell receptors at sites of inflammation in a disease mediated by T cells, this would be an attractive target for therapy. The results of initial experiments in animals with models of autoimmune diseases looked extremely promising in this respect and candidate TcR were identified in experimental autoallergic encephalomyelitis [13, 14], collagen arthritis [11, 15] and murine diabetes [16]. The results in outbred humans appear different and less encouraging. An initial report from Paliard and colleagues [ 17] showed an excess of TcR bearing receptors expressing |3-chains encoded by the Vpi4 family, but this has not been confirmed by others. Alex So and Claudio Lunardi (Hammersmith Hospital) and Jerry Lanchbury (Guy's Hospital) presented their own findings and reviewed the current state of knowledge of the repertoire of TcR expressed in RA. The following conclusions seemed to be generally accepted: (i) that in certain patients the expressed TcR repertoire in synovium is different from that of peripheral blood; (ii) in some patients there is expression of dominant clonotypes within synovium but that these differ from one subject to another. There are many important caveats in the interpretation of all such data. The stage at which disease is studied may be extremely important—it may be that restricted TcR expression may only be a feature of early disease. Indeed, it was shown some years ago by Pelton and colleagues [18] that in advanced RA, synovial tissue looks and behaves like a lymph node. They demonstrated an anamnestic response to a number of common infectious and immunizing stimuli occurring within joints, demonstrating that the specificity of recruitment of lymphocytes to synovial tissue may have been lost (if it ever existed). Other important factors that will require careful control experiments are MHC typing and disease severity.
ited repertoire between different individuals. At the somatic level, factors which could be important are: (i) the T cell receptor repertoire selected within the thymus by interactions with Class I and II molecules associated with self-peptides; (ii) selective usage of T cells bearing particular sets of receptors in particular diseases; and (iii) stimulation by superantigens of T cells expressing receptors expressing (3-chains derived from particular Vp gene families. It has become possible to address each of these questions following a great expansion in knowledge of the molecular structure of the TcR genes. The development of novel techniques for sequencing large numbers of T cell receptors using modifications of the polymerase chain reaction has allowed much of this work to take place.
WALPORT ETAL.:
IMMUNOGENETICS OF RA AND THE ARC'S NATIONAL REPOSITORY
ology Research Unit, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT. Tel: 061-275 5622, Fax 061-275 5043
REFERENCES
1. Aho K, Koskenvuo M, Tuominen J, Kaprio J. Occurrence of rheumatoid arthritis in a nationwide series of twins. J Rheumatol 1986;13:899-902. 2. Lawrence JS. Rheumatoid arthritis—nature or nurture. Ann Rheum Dis 1970;29:357-79. 3. Gregersen PK, Shen M, Song Q-L et al. Molecular diversity of HLA-DR4 haplotypes. Proc Natl Acad Sci USA 1986;83:2642-6. 4. Cairns JS, Curtsinger JM, Dahl CA, Freeman S, Alter BJ, Bach FH. Sequence polymorphism of HLA
DRbetal alleles relating to T-cell-recognized determinants. Nature 1985;317:166-8. 5. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. Arthritis Rheum 1987; 30:1205-13. 6. Gao XJ, Olsen NJ, Pincus T, Stastny P. HLA-DR alleles with naturally occurring amino acid substitutions and risk for development of rheumatoid arthritis. Arthritis Rheum 1990;33:939^6. 7. Papasteriades CA, Kappou ID, Skopouli FN, Barla MN, Fostiropoulos GA, Moutsopoulos HM. Lack of HLA-antigen association in Greek rheumatoid arthritis patients. Rheumatol Int 1985;5:201-3. 8. Schiff B, Mizachi Y, Orgad S, Yaron M, Gazit E. Association of HLA-Aw31 and HLA-DR1 with adult rheumatoid arthritis. Ann Rheum Dis 1982;41:403-4. 9. Brautbar C, Naparstek Y, Yaron M et al. Immunogenetics of rheumatoid arthritis in Israel. Tissue Antigens 1986;28:8-14. 10. Behlke MA, Chou HS, Huppi K, Loh DY. Murine T cell receptor mutants with deletions of b-chain variable region genes. Proc Natl Acad Sci USA 1986;83:767-71. 11. HaqqiTM, Banerjee S, Jones WLef a/. Identification of T-cell receptor V beta deletion mutant mouse strain AU/ssJ (H-2q) which is resistant to collageninduced arthritis. Immunogenetics 1989;29:180—5. 12. Anderson GD, Banerjee S, Luthra HS, David CS. Role of Mls-1 locus and clonal deletion of T cells in susceptibility to collagen-induced arthritis in mice. J Immunol 1991;147:1189-93. 13. Acha-Orbea H, Mitcell DJ, Timmermann L et al. Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell 1988;54:263-73. 14. Urban JL, Kumar V, Kono DH etal. Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cell 1988;54:577-92. 15. Haqqi TM, Anderson GD, Banerjee S, David CS. Restricted heterogeneity in T-cell antigen receptor V beta gene usage in the lymph nodes and arthritic joints of mice. Proc Natl Acad Sci USA 1992;89:1253-5. 16. Reich E-P, Sherwin RS, Kanagawa O, Janeway Jr CA. An explanation for the protective effect of the MHC Class II I-E molecule in murine diabetes. Nature 1989;231:326-8. 17. Paliard X, West SG, Lafferty JA et al. Evidence for the effects of a superantigen in rheumatoid arthritis. Science 1991 ;253:325-9. 18. Pelton BK, Harvey AR, Denman AM. The rheumatoid synovial membrane participates in systemic anti-viral immune responses. Clin Exp Immunol 1985;62:657-61.
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GLOSSARY Genetic polymorphism: inherited variation in the sequence of a particular locus, present at a frequency in a population of greater than 1%. Microsatellite repeat: these are mainly CA repeats and length polymorphisms become increasingly common as the length increases above 20 CA repeats. The distribution of CA repeats is random and frequent, occurring approximately once every 50 kb. However, only one in five C A repeats is highly polymorphic meaning that informative polymorphisms only occur once every 250kb. These microsatellite repeats are most easily identified by amplification using the polymerase chain reaction with flanking oligonucleotide primers. Minisatellite repeat or VNTR (variable number of terminal repeats): these are biased to telomeres (the ends of chromosomes) and are short G+C rich sequences from 9-64 bp in length that are organized in tandem repeats from 100 bp20 kbp in length. These commonly show length polymorphisms caused by variation in the number of tandemly repeated elements. The different alleles can be detected by Southern blotting. CentiMorgan: unit of recombination—1 cM is equivalent to one chromosome cross-over for every 100 meioses. Units of recombination can be roughly translated into genetic distances with 1 cM being equivalent to approximately 1 million base pairs of DN A, although some regions of the genome are more liable to recombination than others. Superantigen: these are products of certain bacteria and viruses which activate large numbers of T cells by virtue of their promiscuous binding properties. They bind to MHC Class II molecules outside their antigen-binding groove and are not restricted to particular allelic subtypes. Similarly, they bind to the f5 chain of T cell receptors in such a way that T cells expressing the products of particular families of V(3 genes are activated. Mice carry endogenous retroviruses, some of which encode superantigens, and when these are expressed early in ontogeny the result is deletion of whole families of T cell receptors. There is no evidence to date that the human T cell repertoire is similarly influenced by the expression of endogenous superantigens.
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