WO2001012848A2 - Genetic marker for rheumatoid arthritis - Google Patents

Abstract

The present invention provides a method for determining tendency of an individual to develop rheumatoid arthritis (RA) and/or severity thereof, by detecting or measuring the presence of an FcηR gene or gene fragment or gene product thereof. The invention also provides diagnostic tools, antibodies and therapeutic composition.

Description

  
 



   Genetic Marker for Rheumatoid Arthritis
The present invention relates to a method and/or marker for determining the susceptibility to and/or potential severity of rheumatoid arthritis for use particularly, but not exclusively, for diagnostic, prognostic, drug screening or patient selection purposes; and a kit for use therefor.



  Background to the Invention
Rheumatoid arthritis (RA) is one of the commonest autoimmune diseases', but its aetiology remains elusive. RA is likely to arise from a complex interaction between multiple genetic susceptibility, persistence and severity factors, as well as environmental triggers. The genetic component of RA is well recognised and was established by familial aggregation studies, twin studies and segregation analyses2.



  Linkage has been demonstrated to the HLA locusl, particularly in patients with more severe disease3. RA susceptibility is associated with the inheritance of certain HLA class II   DRB1    alleles that share a conserved amino acid sequence in the third hypervariable region, at positions   70-744.    These RA associated alleles are collectively known as the rheumatoid or"shared epitope". Family studies suggest that   HLA-DRB1    contributes about one third of the genetic susceptibility to   RA5.   



  However, and despite many attempts with genes such as: TAP-1; TAP-2;   LMP-2;   
LMP-7; Major histocompatability complex class III C2; NRAMP-1; genes encoding for heat shock proteins; tumour necrosis factor and corticotropin-releasing hormone; -no other candidate genes have been consistently proven to be associated with RA.



  A marker gene that could be positively associated with RA would offer significant improvement in the diagnosis, prognosis and screening of candidate drugs for treating the disease and in the selection of patients likely to respond to a selected therapy. Moreover, a second marker gene used in combination with   HLA-DRB1    would offer the clinician and patient a significantly more reliable means of diagnosing and prescribing therapy than the current state of the art.  



  A characteristic feature of RA is the presence of rheumatoid factors and rheumatoid factor-containing immune complexes, both within the circulation and synovial fluide.



  Rheumatoid factors are autoantibodies that recognise the constant (Fc) region of IgG antibodies, and can be of any antibody isotype. IgG rheumatoid factor titres correlate with articular disease severity and the extra-articular manifestations of RA7 and IgG rheumatoid factors are thus thought to be highly pathogenic. Like other IgG immunoglobulins, IgG rheumatoid factors are bound by specialised receptors, the Fc gamma receptors (FcyRs), on immune effector cells.

   There are 3 classes of FcyRs, which vary in their cellular distribution and affinity for different IgG isotypes (reviewed   in9-1    In mice, it has become increasingly apparent that IgG triggered inflammatory responses require ligation of FcyRs on immune effector cells, as well as complement   activationl2-14.    The balance between activatory and inhibitory FcyRs is also   important 15.    For example,   H-2b FcyRIIB    knockout mice are susceptible to collagen-induced arthritis, whereas wild-type mice are   resistant   
Because of the important role of FcyRs in clearance of opsonised pathogens and immune complexes, polymorphisms in Class II and III FcyRs has been shown to play an important role in the pathogenesis of certain infectious and autoimmune diseases,

   including   SLEt7 18. FcyRIIIa    is the only intermediate affinity FcyR and is believed to play a pivotal role in the clearance of immune   complexesl9 zo.    The FcyRIIIA gene is polymorphic and a T to G substitution at nucleotide 559 results in a switch from phenylalanine to valine at amino acid position 158, which lies in the immunoglobulin-binding   domain21.    The presence of phenylalanine   (FcyRIIIa-158F      isoform)    results in a receptor with a low affinity for   IgGI    and IgG3 antibodies and immune complexes, whereas a valine (FcyRIIIa-158V isoform) results in a higher affinityreceptor
It is our hypothesis that FcyRIIIA variants might be directly associated with susceptibility to RA.

   The high affinity   FcyRIIIA-15 8V    allele could contribute to the development of RA in susceptible individuals by increasing the level of macrophage activation in response to immune complexes, thereby contributing to persistence of  inflammation and ultimately RA severity. We tested this hypothesis by examining the   frequency    of FcyRIIIA alleles and the corresponding genotypes in RA patients and controls, using two distinct racial groups.



  Our results have shown that a functional polymorphism in the intermediate affinity
IgG receptor, FcyRIIIA   (CD 16)    is associated with RA (odds ratio 1.7) and, in particular, seropositive nodular RA (odds ratio 2.8), a more severe disease subset.



  The generality of this association has been confirmed by studying two distinct racial groups. We believe that the intermediate affinity IgG receptor, FcyRIIIA represents a hitherto unidentified marker for the severity of RA and as such, finds use as a diagnostic, prognostic, patient selection and/or drug screening tool.



  Further investigations have shown a co-operative interaction between FcyRIIIA and   HLA-DRB1    alleles. Co-inheritance of at least one FcyRIIIA-158V allele and one
RA-associated   HLA-DRB1    allele results in a highly significant association with RA (odds ratio 8.2). FcyRIIIA thus proves a useful"susceptibility"or severity marker in
RA, particularly in conjunction with   HLA-DRB1    genotyping.



  Given the above, it can be seen that FcyRIIIA alleles serves as novel markers for not only establishing the tendency towards, but establishing the likely severity of RA.



  Statement of the Invention
According to first aspect of the invention there is provided a method for determining tendency of an individual to develop rheumatoid arthritis (RA) and/or severity thereof, comprising examining a blood or tissue sample derived from the individual, so as to detect or measure at least one selected gene FcyR gene or fragment or a gene product thereof.



  Preferably, the selected FcyR gene is FcyRIIIA   (CD16)    or a gene product thereof and more preferably the gene is   FcyRIIIA-158V    allele or variant or gene product thereof.  



  Preferably, the individual is human.



  A preferred method of the invention involves identification of selected FcyR gene transcripts and/or translation products and variants thereof and more preferably FcyR mRNAs. Alternatively or in addition, it involves determination of FcyR proteins.



  In the instance where the proteins are to be determined by the use of antibodies, monoclonal or polyclonal, said antibodies may be used in order to bind to FcyR proteins. Advantageously, additional antibodies may be used to amplify the nature of the reaction and/or antibodies tagged with suitable labels may also be used.



  In the instance where fluid is examined, a blood sample may be taken and the   amount/level    of FcyR allele or product thereof determined and ideally compared with a standard or normal levels. Once this is done, an elevated level of the FcyR allele and/or gene product (s) thereof in an individual would be indicative of the existence of, or severity of RA, and so appropriate action could be taken.



  It will be appreciated that the FcyR family of genes that have been identified in the invention as of particular use in detecting RA include the IIIA, IIB, IIC and   EB    families and that the method of the present invention includes any one or more genes from any one or more of the aforementioned closely related gene families.



  According to a further aspect of the invention there is provided the use of an FcyR genes, gene fragment and/or gene product thereof and/or probes thereto and/or proteins thereof and/or antibodies raised to such proteins for the determination of a predisposition or severity   of RA    in an individual.



  According to a yet further aspect of the invention there is provided use of an FcyR gene or genes, variants, fragments and/or derivatives thereof, products and/or proteins thereof, antibodies raised to such proteins or products for the manufacture of a medicament for diagnosing and/or treating RA.  



  Preferably, the selected FcyR gene is FcyRIIIA (CD 16) or a variant thereof and more preferably the   FcyRIIIA-158V    allele or variant thereof.



  Preferably, the individual is human.



  According to a yet further aspect of the invention there is provided a screening procedure for selecting a subject who is susceptible and/or has a predisposition to RA comprising examining a blood or tissue sample from the subject in order to determine whether an FcyR gene or gene fragment thereof is transcribed and/or translated.



  A preferred method includes the step of, depending on the results of the screening procedure, including or excluding the subject from trials to investigate the efficacy of a therapeutic.



  On the other hand the method may be used to identify subjects who are susceptible to or have a predisposition towards a severe form of RA so that such individuals can receive a suitable therapy. In such instances a therapeutic regime can be administered prior to the onset of severe RA symptoms.



  Preferably, the selected FcyR gene is FcyRIIIA   (CD 16)    or a variant thereof and more preferably the   FcyRIIIA-158V    allele or variant thereof.



  Preferably, the subject is human and optionally further includes any one or more of the features herein before described.



  It will be apparent that in the instance where the efficacy of a therapeutic, which is designed to treat particularly severe RA cases, is to be determined then individuals who are expressing the allele   FcyRIIIA-158V    or variant or fragment thereof and/or transcripts and/or translation products thereof, termed   FcyRIIIA-158V    genotype positive, will be selected. Conversely, individuals who do not express said allele  may also be selected. However, in some instances a mixed population of individuals may be selected according to the requirements of the screening program.



  According to a yet further aspect of the invention there is provided a method of identifying a patient that is responsive to a specific therapy programme comprising identifying subjects who express FcyR gene polymorphism and/or have transcript and/or translation products thereof.



  Preferably, the selected FcyR gene is FcyRIIIA   (CD 16)    or a variant thereof and more preferably the   FcyRIIIA-158V    allele or variant thereof.



  It will be appreciated that the method may be used to predict the pharmacogenomic response to a specific therapeutic agent. For example, treatment with   anti-tumour    necrosis factor a costs approximately ±8,000 per annum per patient. However, only about 50% of cases respond to such treatment. The method of the invention may be used to predict those patients who are likely to respond to, and benefit from, such treatment. Thus, a further benefit of the invention is in providing financial savings to the health service from targeted therapy and concomitantly preventing a patient from receiving unnecessary and non-beneficial treatment when they could be receiving an alternative therapeutic agent which could ameliorate their   condition/symptoms.   



  According to a yet further aspect of the invention there is provided a kit for determining the susceptibility and/or a predisposition of a subject to severe RA   and/or      responsiveness    of a subject suffering from RA to a specific therapy programme comprising a ligand which binds to or is specific for an FcyR gene, gene fragment thereof or transcript and/or translation products thereof.



  Preferably, the selected FcyR gene is FcyRIIIA   (CD 16)    or variants thereof and more preferably the   FcyRIIIA-158V    allele or variants thereof and more preferably the kit further includes any one or more of the features hereinbefore described.  



  Preferably, the kit comprises at least one product containing a polymorphic site of the   FcyRIIIA-158V    allele or variant thereof.



  Preferably, the kit comprises a FcyRIIIA-specific forward and/or reverse primers.



  According to a yet further aspect of the invention there is provided a method according to the invention, use according to the invention, a screening procedure according to the invention and a kit according to the invention further including in combination with an FcyR gene an   HLA-DRB1    allele, variant or fragment, or transcript and/or translation product thereof.



  According to a yet further aspect of the invention there is provided a therapeutic composition for use in the treatment   of RA    comprising a compound which is capable of interacting with at least one FcyR gene or gene fragment or transcript and/or translation product thereof..



  Preferably, the therapeutic composition is capable of preventing or blocking or disabling or inhibiting or inactivating the FcyR gene or gene fragment or transcript and/or translation product thereof.



  Preferably, the FcyR gene is FcyRIIIA   (CD16)    or a variant thereof and more preferably is the FcyRIIIA-158V allele or a variant thereof.



  According to a yet further aspect of the invention there is provided an antibody or antibody fragment raised against an FcyR gene product.



  Preferably, the antibody is raised to a gene product from an FcyRIIIA   (CD16)    gene product or a variant thereof and/or an   FcyRIIIA-158V    allele gene product or a variant thereof.  



  According to a yet further aspect of the invention there is provided use of an antibody raised against, an FcyR gene product in diagnosing RA severity and/or predisposition thereto and/or treatment thereof.



  Preferably the antibody has been raised against a gene product is from an FcyRIIIA   (CD16)    gene or a variant thereof and/or against an FcyRIIIA-158V allele gene product or a variant thereof.



  According to a yet further aspect of the invention there is provided a hybridoma cell line which produces an antibody as hereinbefore described more preferably the antibody produced is a monoclonal antibody.



  According to a yet further aspect of the invention there is provided a product for detecting the presence of an FcyR gene or gene fragment or gene product comprising a nucleic acid molecule of SEQ ID   NO:      1    and/or SEQ ID   NO:    2, derivatives or homologues thereof and optionally further including a label.



  Preferably, the product comprises an FcyRIIIA   (CD16)    gene or gene fragment or gene product thereof and/or is an FcyRIIIA-158V allele gene or gene fragment or gene productthereof.



  According to a yet further aspect of the invention there is provided an assay for the detection of sequence variation in nucleic acid target substrates comprising: i) providing a sample of nucleic acid to be assayed; ii) providing conditions for the denaturation of the nucleic acid; iii) incubating the nucleic acid sample with SEQ ID NO:   1    and SEQ ID   NO:    2; iv) providing reagents and conditions sufficient to PCR amplify the target
 nucleic acid substrate; and v) separating and analysing the products of the reaction in (iv) to detect said
 sequence variation.  



  According to a yet further aspect of the invention there is provided an assay for detecting or measuring the expression of a gene associated with RA comprising: i) providing a sample of nucleic acid to be assayed; ii) providing conditions for the denaturation of the nucleic acid; iii) incubating the nucleic acid sample with SEQ ID NO:   1    and SEQ ID   NO:    2; iv) providing reagents and conditions sufficient to PCR amplify the target nucleic
 acid substrate; and   v)    separating and analysing the products of the reaction in (iv) to detect said
 sequence variation.



  Preferably the nucleic acid used in the assays encodes an FcyR gene.



  Detailed Description of the Invention
An embodiment of the invention will now be described by way of example only with reference to the following Tables wherein:
Table 1 represents the characteristics of UK Caucasian and North Indian and
Pakistani RA patient populations;
Table 2 represents a comparison of FcyRIIIA-158V/F allele frequencies in UK
Caucasian and North Indian and Pakistani RA patients and racially matched healthy controls;
Table 3 represents a comparison of FcyRIIIA-158V/F genotype distributions in UK
Caucasian and North Indian and Pakistani RA patients and racially matched healthy controls;
Table 4 represents odds ratios for RA, highlighting interaction of the FcyRIIIA158V/F allele and the"shared epitope", comparing Caucasian and North Indian and
Pakistani RA patients and controls;

   and  
Table 5 represents comparison of   FcyRIIIA-158V    genotype distribution with the presence or absence of rheumatoid nodules in RA patients.



      I   
Materials and Methods
Study Design
This was an allelic association study where a functional polymorphism in FcyRIIIA was examined for a possible role in the development of RA. The study was performed in two well-defined racial groups, which were resident in the same geographical area of England, and comprised UK white Caucasians or individuals originating from North India or Pakistan. UK Caucasian was defined if at least three of the subject's grandparents were born in the"British Isles". North India and
Pakistan was defined as the states of Kashmir, Punjab, Haryana, Himachial, Pradesh and Delhi. A patient was attributed as North Indian or Pakistani if at least three of his or her grandparents were born in the defined area.



  Rheumatoid arthritis patients and controls
RA patients were recruited from the rheumatology outpatient clinics of three district general hospitals in central Birmingham, UK, as part of a cross-sectional study24. All patients who entered the study fulfilled the 1987 American College of Rheumatology criteria for   RA25    and were 16 years of age or older at the onset of their disease. All patients were assessed clinically and serologically using a standard   proforma.    The demographic details included age, sex, disease duration, use of disease modifying   anti-rheumatic    drugs (DMARDs), and radiographic evidence of erosions. Nodules were classified as present if found on examination or previously documented in the patient's medical records.  



  The same definitions were used for the racial origin of control subjects as for the RA patients.   UK    Caucasian controls were obtained from two sources: 98 healthy blood donors from central Birmingham and 26 healthy laboratory staff. North Indian and
Pakistani controls were obtained from the same local community as RA patients through"well woman"clinics at general practitioners'surgeries, Sikh temples and among hospital employees. Ethical approval was obtained from the local research ethics committee.



     Immunology   
Rheumatoid factor and anti-nuclear antibody (ANA) were assayed in the RA subjects at the hospital attended. A patient was defined as being rheumatoid factor positive (seropositive) if they had ever been documented to have a rheumatoid factor of  >  40
IU using a nephelometric assay.



  HLA-DRBI typing
DNA was extracted from EDTA anticoagulated blood using a salt precipitation   technique26. HLA-DRB1*01-16    types were determined by a PCR-based reverse line blot assay (Dynal   RELITM    SSO HLA DRB test). High resolution typing by sequencespecific PCR primers was performed to determine the   DRB 1 *04    subtypes   DRB1*0401-*0422.    The"shared epitope"was defined as   HLA-DRB1*01,    *0401,   *0404, *0405, *0408, *0409, *0410, *0413, *0416, *0419, *0421    or   *10,    in this study   FcyRIIIA    genotyping
We adopted a sequencing approach to genotyping the   FcyRIIIA-158V/F    polymorphism.

   A 199 base pair PCR product that contained the polymorphic site was amplified using a combination of previously published primer sequences23.



  Preferential amplification of FcyRIIIA, rather than the highly homologous   FcyRIIIB,    was confirmed by examination of the sequence at nucleotides 505 and 531. The  
FcyRIIIA-specific forward primer   (d-TCA    CAT ATT TAC AGA ATG GCA ATG
G) SEQ ID NO:   1    corresponded to nucleotides 449-473 and the reverse primer (d
CAG GAA ACA GCT ATG ACC CTT GAG TGA TGG TGA TGT TCA) SEQ ID   NO:    2 corresponded to nucleotides 610-590.

   A   30p1    PCR reaction was performed using 200ng DNA, 200nM of each primer,   200pM    dNTPs and   1.5mM      MgCl2.    The
PCR reaction was performed on a Techne PCR machine with   0.5U    Taq DNA polymerase (Promega) added at   95 C    to prevent primer-dimer formation. The PCR conditions were optimised to   95 C    for 5 minutes followed by 36 cycles of   95 C    for 30 seconds, annealing at   52 C    for 40 seconds and extension at   72 C    for 40 seconds, followed by a final extension step of   72 C    for 10 minutes. PCR products were visualised after electrophoresis on a 2% agarose gel, using ethidium bromide and UV transillumination.

   Fluorescent automated cycle sequencing of the PCR products was performed using a   dRhodamine    terminator reaction kit (Applied Biosystems).



  Electrophoresis was performed on polyacrylamide gels using the ABI   PRISM 377   
DNA Sequencer, and the sequence analysed utilising ABI   PRISME 377    sequencing software.



  SSCP   Methodfor genotping FcrRIIL   
A 199 base pair PCR product that contained the polymorphic site was initially amplified. The FcyRIIIA-specific forward primer was   d-TCA    CAT ATT TAC AGA
ATG GCA ATG G (SEQ ID   NO:    1) and the reverse primer was d-CAG GAA ACA
GCT ATG ACC CTT GAG TGA TGG TGA TGT TCA (SEQ ID NO: 2). A   30p1   
PCR reaction was performed using 200ng DNA, 200nM of each primer,   200uM    dNTPs and   1.5mM      MgCl2.    The PCR reaction was performed on a Techne PCR machine with   0.5U    Taq DNA polymerase (Promega) added at   95 C    to prevent primer-dimer formation.

   The PCR conditions were optimised to   95 C    for 5 minutes followed by 36 cycles of   95 C    for 30 seconds, annealing at   52 C    for 40 seconds and extension at   72 C    for 40 seconds, followed by a final extension step of   72 C    for 10 minutes. PCR products were visualised after electrophoresis on a 2% agarose gel, using ethidium bromide and LJV transillumination.  



  The PCR product then underwent ethanol precipitation using a standard technique, was   resuspended    in   6pL1    denaturing solution (94% formamide, 0.05% xylene cyanol and bromophenol blue), denatured at   95 C    for 5 minutes and directly placed on ice to prevent reannealing. The SSCP products underwent electrophoresis using a preprepared polyacrylamide gel (T=12%, C=3%) at   14 C,    600V, 25mA and 15W for 90 minutes. The PCR products were then visualised by silver staining.

   A single upper band   at¯225 base    pairs denotes FcyRIIIA-158FF individuals, a single lower band at   -195    base pairs denotes   FcyRIIIA-158VV    individuals and multiple bands   at-195-    225 base pairs indicates FcyRIIIA-158VF individuals.



  Statistical analysis
Statistical analyses were performed using the SPSS 8.0 for Windows, statistical package. The median and inter-quartile ranges or percentage of patients with a particular feature were calculated for each of the two racial groups of RA subjects.



     Non-parametric    analyses (Mann-Whitney U test for continuous variables and Pearson chi-square test for dichotomous variables) were used to compare the two different
RA populations. Two-sided P values below 0.05 were considered statistically significant throughout.



  Genotype and allele frequencies were compared using 3 x 2 and 2 x 2 contingency tables, respectively. The Pearson chi-square test was used for statistical comparisons.



  When numbers were too small to provide an accurate chi-square result, i. e. if there was an expected count of  <  5 in any cell, Fisher's exact test was used, which was estimated for 3 x 2 contingency tables using Monte Carlo methods.



  Odds ratios and their 95% confidence intervals (CI) were calculated to quantify the magnitude of the association between the FcyRIIIA-158V allele and RA, as an approximation of the relative risk. FcyRIIIA-158FF individuals were assumed to have no risk   of RA    for the analysis, that is an odds ratio of 1.0.  



  Results   
Characteristics of the UK Caucasian and North Indian and Pakistani RA patient    groups 249 RA patients from two racial groups (141 UK Caucasian, 108 North Indian and
Pakistani) and 237 ethnically matched healthy controls (124 UK Caucasian, 113
North Indian and Pakistani) were included in the study. The demographic details and characteristics of the RA patients from each racial group are shown in Table 1. There were no statistically significant differences in age, sex, disease duration or the proportion that were rheumatoid factor or ANA positive, between the two groups.



  There were increased prevalences of nodules and"shared epitope"positive individuals (at least one RA-associated   DRB1    allele) in the UK Caucasian compared to the North Indian and Pakistani RA group, which were statistically significant. All
UK Caucasian and 96% of the North Indian and Pakistani patients had erosive disease, and erosions were not considered further in the analysis.



     Distribution of FcrRIIlA genotypes and association with RA   
All RA patients and controls were genotyped for the   FcyRIIIA-158V/F    polymorphism. There was no departure from Hardy-Weinberg equilibrium in either control group. There was a statistically significant increase in the frequency of the   FcyRIIIA-158V    allele in both UK Caucasian and North Indian and Pakistani RA patients compared to their respective controls (UK Caucasian P = 0.028, North
Indian and Pakistani P = 0.050, both races combined P = 0.003). There was no significant difference in allele frequencies between the two racial groups for either the RA or control populations. We therefore included results of combining the racial groups in the analysis. The genotype distributions are shown in Table 3.

   Odds ratios and 95% CI were calculated to estimate the risk   of RA    in individuals who possessed at least one   FcyRIIIA-158V    allele and these are also shown in Table 3 (UK Caucasian
P = 0.051, odds ratio 1.6,95% CI 1.0-2.6; North Indian and Pakistani P = 0.023, odds ratio 1.9,95% CI 1.1-3.2; both races P = 0.003, odds ratio 1.7,95% CI 1.2  2.5). This suggests a causative effect of the   FcyRIIIA-158V    allele or a nearby linked gene.

 

  In addition, we calculated the odds ratios for association between the FcyRIIIA-158V allele and seropositive nodular RA (Table 3), which was taken to represent a severe
RA subset (UK Caucasian P = 0.044, odds ratio 2.2,95% CI 1.0-5.1; North Indian and Pakistani P = 0.285, odds ratio 3.3,95% CI both races P = 0.020, odds ratio 2.8,95% CI 1.2-6.4). There was a stronger association with more severe disease in the UK Caucasian and combined racial group, but the patient numbers were not high enough to reach statistical significance in the North Indian and
Pakistani group.



  Interaction   of the FcrRIIL4-158V/F polymorphism and th 



     Association of FcyRIIIA with rheumatoidfactor and rheumatoid nodules in RA   
In RA patients, homozygosity for the FcyRIIIA-158V allele was associated with the development of nodules (Table 5). Forward stepwise logistic regression analyses (dependent variable: nodules; independent variables: disease duration, FcyRIIIA158VV, rheumatoid factor,"shared epitope"positivity, smoking and race) revealed that   FcyRIIIA-158VV    was the only significant independent predictor of nodules (odds ratio 4.9,95% CI 1.1-21.4)   other than race.   



  There was no association between the FcyRIIIA-158V allele and the presence or a high titre of rheumatoid factor, defined as greater than the   75th percentile,    in the RA patients.



  We have demonstrated that a functional polymorphism in FcyRIIIA is associated with
RA and, in particular, seropositive nodular RA, a subset characterised by more severe articular and extra-articular disease. We have confirmed the generality of this association by studying two distinct racial groups.



  Activated macrophages are the predominant infiltrating cell type found in rheumatoid synovium, pannus and nodules Macrophages play important roles in   phagocytosis    and clearance of immune complexes, mediated through binding to   FcyRs30.      FcyRIIIa    has recently been demonstrated to have an intermediate affinity for immune complexes, between   FcyRI    and FcyRIIa, and is thought to be the major receptor involved in their   clearancel9, zo. Cross-linking FcyRs    results in macrophage activation and release of pro-inflammatory cytokines, reactive oxygen species and proteolytic enzymes6. Monokines, such as IL-1 and   TNFa,    dominate the cytokine profile of RA and mediate most of the synovial inflammation and articular.

   We believe that the high affinity FcyRIIIa-158V isoform increases the level of macrophage activation within the joint through enhanced binding of IgG rheumatoid factors and immune complexes. In turn, this may result in the release of chemokines, such as   MCP-131,    which further increase macrophage recruitment into the joint.  



  Release of M-CSF following FcyR ligation could promote monocyte survival inside the joint   by      protecting    monocytes from   apoptosis32.    Continued macrophage activation by immune complexes or IgG rheumatoid factors may then promote persistence of inflammation or increase articular disease severity.



  The extra-articular manifestations of RA appear to be restricted to sites where connective tissue macrophages express   FcyRIIIa,    such as rheumatoid nodules, alveoli, liver, pericardium, lymphoid tissue, bone marrow and salivary glands
Circulating IgG rheumatoid factors form dimers, which pass freely into the extravascular   space.    They do not fix complement well and thus under normal circumstances are thought to mediate little tissue damage8. However, if the high affinity FcyRIIIa-158V isoform is expressed by these connective tissue macrophages, they may be more likely to bind circulating IgG rheumatoid factor dimers, resulting in macrophage activation and subsequent tissue damage that would not otherwise have occurred.

   This hypothesis is substantiated by the increased risk of developing nodules in RA individuals homozygous for the FcyRIIIA-158V allele.



  We have also demonstrated that susceptibility to RA in the North Indian and
Pakistani population is determined by co-inheritance of at least one FcyRIIIA-158V allele and the"shared epitope". If an individual inherits only one of these two   "susceptibility"genes    in isolation, there appears to be no excess risk   of RA    (Table 4).



  The synergistic interaction between these two genetic loci may partly explain the inconsistent association between the"shared epitope"and less severe forms of RA.



  It also provides another example of epistasis in a complex human genetic trait35 and may explain why linkage to the FcyR locus has not been demonstrated in a recent sibpair studyl. HLA-DRB1 and FcyRIIIA lie on different chromosomes and their interaction cannot be explained in terms of linkage disequilibrium. It is therefore most likely to occur at the functional level.



  There has been some controversy relating to the precise association between the "shared epitope"and RA. The"shared epitope"is undoubtedly associated with the  most severe forms of   RA particularly    seropositive disease38, erosive   disease39,    the need for early joint   surgery,    extra-articular organ involvement including   nodules and Felty's syndrome.    Such an association between the"shared epitope" and RA has been observed in most racial groups, with the exception of blacks and   Hispanics4243. However,    association between the"shared epitope"and RA in community based   studies44¯46    or in unselected patients presenting with early arthritis47 has not always been demonstrated, or is weak in comparison to hospital based patient populations.

   This has led to suggestions that the"shared epitope"is a marker of severity or persistence of inflammation, rather than susceptibility to RA per se. There also appears to be a complex dosage effect between the number of "shared epitope"alleles inherited, the presence of specific alleles such as 0401 or 0404, certain allelic combinations, for example 0401/0404, and the risk of developing RA or specific RA features
A significant proportion of synovial macrophages express HLA class II antigens and are thought to be capable of antigen presentation. The"shared epitope"lies at one side of the HLA class II antigen-binding site4, which results in the   HLA-DRB1    alleles varying in their affinity for different antigens.

   The   HLA-DRB1    alleles associated with RA are thus believed to be more likely to present pathogenic antigen (s) to T-cells and initiate autoimmunity. The autoantigen in RA remains elusive, but may be derived from a self-peptide (s) or alternatively a common pathogen (s) that either cross-reacts with a self-peptide or mimics the MHC-binding motif49. The high affinity   FcyRIIIa-158V    isoform may result in more efficient presentation of pathogenic peptides by at least three mechanisms. Firstly, increased uptake of IgG-containing immune complexes or opsonised pathogens may increase the number of antigens processed within the macrophage, resulting in enhanced presentation of a wide range of epitopes.

   Secondly, macrophage activation may result in upregulation of MHC class   50u51      and co-stimulatory    molecule expression, such as CD86 and CD8052, that may increase antigen presenting function. Thirdly, it has been proposed that macrophage activation may lead to an hyper-responsive MHC  class II signalling pathway, which results in a reduced threshold for antigen presentation53.



  The association that we have demonstrated may be with either FcyRIIIA itself or a closely linked gene. FcyRIIIA lies in a 600 kb gene cluster at chromosome lq22, which contains all of the class II and class III FcyRs54. Previous studies have shown an association between FcyRIIIA and   SLE55    or lupus   nephritis23 s6. FcyRIIA    is the closest gene to FcyRIIIA in this gene cluster.

   Some studies have shown an association between FcyRIIA and either SLE   itself57,    or a subgroup with lupus nephritis, in certain ethnic groups, but not others   In the    two studies in which both FcyRIIIA and FcyRIIA were examined in the same populations, however, an association with either SLE or nephritis was only demonstrated for FcyRIIIA and not   FcyRIIA55-56.    This suggests that FcyRIIIA and FcyRIIA are not in linkage disequilibrium, but does not exclude the possibility of an FcyR RA susceptibility haplotype.   FcyRIIa    could also play a role in RA pathogenesis, but it does not bind monomeric IgG or IgG dimers efficiently and is thought to be less important in the clearance of immune complexes.

   The major differences described between the two   isoforms    of FcyRIIa also predominantly affect the binding of IgG2, which is an uncommon autoantibody   isotype   
Further confirmation of the role of   FcyRIIIA    in RA susceptibility and severity is needed from longitudinal studies, and from community-based populations in additional geographical areas and racial groups. The interaction between FcyRIIIA and specific   HLA-DRB1    alleles also requires further clarification as it may ultimately improve our understanding of RA pathogenesis. The FcyRIIIA-158V/F polymorphism in combination with the"shared epitope"may prove to be a useful marker of severe RA and could highlight a group of patients that require more aggressive therapy in the early stages of disease.  



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  Table 1. Characteristics of UK Caucasian and North Indian and Pakistani RA patient populations.
EMI28.1     





   <SEP> UK <SEP> NorthIndianandPakistaniRARA <SEP> 
<tb>  <SEP> Median <SEP> (inter-quartile <SEP> range) <SEP> or <SEP> % <SEP> Median <SEP> (inter-quartile <SEP> range) <SEP> or <SEP> %
<tb>  <SEP> n <SEP> = <SEP> 141 <SEP> n <SEP> = <SEP> 108
<tb> Age <SEP> (years) <SEP> 53 <SEP> (44-60) <SEP> 52 <SEP> (42-57)
<tb> Sex <SEP> 82% <SEP> female <SEP> 85% <SEP> female
<tb> Disease <SEP> duration <SEP> (years) <SEP> 6 <SEP> (3-12) <SEP> 7 <SEP> (2-13)

  
<tb> Rheumatoid <SEP> factor <SEP> positive <SEP> 71% <SEP> 74%
<tb> Nodulespresent <SEP> 31% <SEP> 4% <SEP> f
<tb> ANA <SEP> positive <SEP> 21% <SEP> 20%
<tb> #Shared79%47%#positive <SEP> 
<tb> Joint <SEP> erosions <SEP> 100% <SEP> 96%
<tb> 
Two-sided P values were calculated to determine if there was any significant   di$erence    between the two racial groups using the   Mann-Whitney    U test for continuous data and the Pearson   chi-square    test for dichotomous data.



     0.05#P <      
Table 2. Comparison of   FcyRIUA-158V/F ailele frequencies in    UK Caucasian and North Indian and
Pakistani RA patients and racially matched healthy controls.
EMI29.1     


<tb>



   <SEP> P <SEP> value'
<tb>  <SEP> Controls <SEP> RA <SEP> (total <SEP> alleles)
<tb>  <SEP> UKCaucasian
<tb> 158 <SEP> V <SEP> 028 <SEP> 037 <SEP> 0.028
<tb> 158 <SEP> F <SEP> 0.72 <SEP> 0.63
<tb> North <SEP> Indien <SEP> andpakzstmi
<tb> 0.270.360.050158V <SEP> 
<tb> 158 <SEP> F <SEP> 0.73 <SEP> 0.64
<tb>  <SEP> BothRaces
<tb> 0.280.370.003158V <SEP> 
<tb> 158 <SEP> F <SEP> 0.72 <SEP> 0.63
<tb>  1 Two-sided P values were calculated   from 2 X 2    contingency tables using the Pearson chi-square test to   detenmne    the difference in allele frequencies between RA and control subjects from each racial group.



  In addition there was no significant difference in the allele frequencies between the two races for either the
RA or control groups.    Table 3. Comparison of Fc&gamma;RIIIA-158V/F    genotype distributions in UK Caucasian and North Indian and
Pakistani RA patients and racially matched healthy controls.
EMI30.1     


<tb>



   <SEP> Seropositive
<tb>  <SEP> Controls <SEP> RA <SEP> Odds <SEP> Ratio'Nodular <SEP> RA <SEP> Odds <SEP> Ratio'
<tb>  <SEP> No. <SEP> (%) <SEP> No. <SEP> (%) <SEP> (95% <SEP> Cl) <SEP> No. <SEP> (%) <SEP> (95% <SEP> Cl)
<tb>  <SEP> n=124n=141n=36UKCaucasian <SEP> 
<tb> 158 <SEP> VV <SEP> 11 <SEP> (9) <SEP> 21 <SEP> (15) <SEP> 1.6 <SEP> (1. <SEP> 0-2.6) <SEP> t <SEP> 9 <SEP> (25) <SEP> 2. <SEP> 2 <SEP> t
<tb> 158 <SEP> VF <SEP> 48 <SEP> (39) <SEP> 63 <SEP> (45) <SEP> 15 <SEP> (42)
<tb> 158 <SEP> FF <SEP> 65 <SEP> (52) <SEP> 57 <SEP> (40) <SEP> 12 <SEP> (33)
<tb>  <SEP> n=113n=108n=4NorthIndian <SEP> 
<tb>  <SEP> andPakistani
<tb> 158 <SEP> VV <SEP> 8 <SEP> (7) <SEP> 10 <SEP> (9) <SEP> 1.9 <SEP> 2 <SEP> (50) <SEP> 3.3 <SEP> (0.3-32.

   <SEP> 5)
<tb> 158 <SEP> VF <SEP> 46 <SEP> (41) <SEP> 58 <SEP> (54) <SEP> 1 <SEP> (25)
<tb> 158 <SEP> FF <SEP> 59 <SEP> (52) <SEP> 40 <SEP> (37) <SEP> 1 <SEP> (25)
<tb>  <SEP> BothRaces <SEP> n <SEP> = <SEP> 237 <SEP> n <SEP> = <SEP> 249
<tb> 158 <SEP> W <SEP> 19 <SEP> (8) <SEP> 31 <SEP> (12) <SEP> 1.7 <SEP> (1. <SEP> 2-2.5) <SEP> # <SEP> 11 <SEP> (28) <SEP> 2.8 <SEP> (1.2-6. <SEP> 4)#
<tb> 158 <SEP> VF <SEP> 94 <SEP> (40) <SEP> 121 <SEP> (49) <SEP> 16 <SEP> (40)
<tb> 158 <SEP> FF <SEP> 124 <SEP> (52) <SEP> 97 <SEP> (39) <SEP> 13 <SEP> (32)
<tb>  'Odds ratio and 95% confidence   intervals    (CI) to estmate risk of RA in   Fc7RinA-158W or-I58VF    individuals in comparison to   FcyRMA-158FF    individuals, assuming the latter have an odds ratio of 1.0.



     0.05#P <        Table 4. Odds ratios for the interaction between the Fc&gamma;RIIIA-158V allele and the #shared epitope# (SE) in predicting development of RA.   
EMI31.1     





  UK <SEP> Caucasian <SEP> N. <SEP> Indian <SEP> and <SEP> Paklstani <SEP> Both <SEP> Races
<tb> Comparison <SEP> Group1 <SEP> Reference <SEP> Group <SEP> Odds <SEP> Ratio <SEP> Odds <SEP> Ratio <SEP> Odds <SEP> Ratio
<tb> IIIA-158V <SEP> alleel/SE <SEP> IIIA-158V <SEP> allele/SE <SEP> (95% <SEP> CI) <SEP> (95% <SEP> CI) <SEP> (95% <SEP> CI)
<tb> Effect <SEP> SE <SEP> in <SEP> absence <SEP> IIIA-158V <SEP> -/+ <SEP> -/- <SEP> 2.1(0.8-5.0) <SEP> 1.6(0.6-4.0) <SEP> 1.8(1.0-3.4)
<tb> Effect <SEP> IIIA-158V <SEP> in <SEP> absence <SEP> SE <SEP> +/- <SEP> -/- <SEP> 1.0(0.3-2.7) <SEP> 1.0(0.5-2.2) <SEP> 1.0(0.5-1.9)
<tb> Joint <SEP> Effect <SEP> IIIA-158V <SEP> and <SEP> SE <SEP> +/+ <SEP> -/- <SEP> 3.5(1.4-8.6)# <SEP> 8.2(2.4-27.7)# <SEP> 4.1(2.1-7.9)#
<tb>    1The comparison adn reference groups indicate how the odds ratios were calculated, 

   RA and control patients were compared in all cases.



  #P  <  0.05       Table 5. Comparison of Fc&gamma;RIIIA-158V/F genotype    distributions with the presence or absence of rheumatoid nodules in RA patients.
EMI32.1     





   <SEP> RA <SEP> + <SEP> nodules <SEP> RA-nodules <SEP> Odds <SEP> Ratio'
<tb>  <SEP> (95%CI)No.(%)No.(%) <SEP> 
<tb>  <SEP> N=36N=79UKCaucasian <SEP> 
<tb>  <SEP> 158 <SEP> VV <SEP> 9 <SEP> (25) <SEP> 7 <SEP> (9) <SEP> V: <SEP> 1.6 <SEP> 
<tb>  <SEP> 158 <SEP> VF <SEP> 15 <SEP> (42) <SEP> 37 <SEP> (47) <SEP> W: <SEP> 3.6 <SEP> (1. <SEP> 1-11.8)#
<tb> 12(33)158FF <SEP> 35(44)
<tb>  <SEP> andPakista 

Claims

Claims 1. A method for determining tendency of an individual to develop rheumatoid arthritis (RA) and/or severity thereof, comprising examining a blood or tissue sample derived from the individual, so as to detect or measure at least one FcyR gene or fragment or a gene product thereof.

2. A method according to Claim 1 wherein the FcyR gene is FcyRIIIA (CD16) or gene product thereof.

3. A method according to Claim 2 wherein the FcyRIIIA is an FcyRIIIA-158V allele or variant or gene product thereof.

4. A method according to any preceding claim wherein the individual is human.

5. A method according to any preceding claim including wherein the gene product comprises a gene transcript or gene translation product.

6. A method according to Claim 5 wherein the gene transcript is FcyR mRNA or a fragment thereof and the translation product is a FcyR protein or fragment thereof.

7. A method according to either Claim 5 or 6 wherein the FcyR protein or fragment thereof is detected or measured using an antibody or a fragment thereof.

8. A method according to any preceding claim wherein an amount/level of the FcyR gene or gene product thereof in the individual is compared with a standard.

9. The use of an FcyR gene, gene fragment and/or gene product thereof and/or probes thereto and/or proteins thereof and/or antibodies raised to such a protein for the determination of a predisposition or severity of RA in an individual.

10. Use of an FcyR gene or genes, variants, fragments and/or derivatives thereof, products and/Qr proteins thereof, antibodies raised thereto for the manufacture of a medicament for diagnosing and/or treating RA.

11. Use according to Claim 10 further including any one or more of the features of Claims 2-8.

12. A screening procedure for selecting a subject who is susceptible and/or has a predisposition to RA comprising examining a blood or tissue sample from the subject in order to determine whether an FcyR gene or gene fragment thereof is transcribed and/or translated.

13. A screening procedure according to Claim 12 further including the step of, depending on the results of the screening procedure, including or excluding the subject from trials to investigate the efficacy of a therapeutic.

14. A screening procedure according to Claim 12 further including the step of identifying subjects who are susceptible to, or have a predisposition towards, a severe form of RA so that such individuals can receive a therapeutic prior to the onset of severe RA symptoms.

15. A screening procedure according to any one of Claims 12-14 further including any one or more of the features of Claims 2-8.

16. A method of identifying a patient who is responsive to a specific therapy programme comprising identifying subjects who express FcyR gene polymorphism and/or have transcript and/or translation products thereof.

17. A method according to Claim 16 further including any one or more of the features of Claims 2-8.

18. A kit for determining the susceptibility and/or a predisposition of a subject to RA and/or reponsiveness of a subject suffering from RA to a specific therapy programme comprising a ligand which binds to or is specific for identifying an FcyR gene, or fragment thereof, or transcripts and/or translation product thereof.

19. A kit according to Claim 18 further including any one or more of the features of Claims 2-8.

20. A kit according to either Claim 18 or 19 further comprising at least one product containing a polymorphic site of the FcyRIIIA-158V allele or variant thereof.

21. A kit according to any one of Claims 18-20 comprising a FcyRIIIA-specific forward (SEQ ID NO: 1) and/or reverse primer (SEQ ID NO: 2).

22. A method according to any one of Claims 1,9,10,12,16 or 18 further including, in combination with at least one FcyR gene, product or variant thereof an HLA-DRB1 allele, variant or fragment, or transcript and/or translation product thereof.

23. A therapeutic composition for use in the treatment of RA comprising a compound which is capable of interacting with at least one FcyR gene or gene fragment or gene product or transcript and/or translation product thereof.

24. A composition according to Claim 23 that is capable of preventing or blocking or disabling or inhibiting or inactivating an FcyR gene or gene fragment or transcript and/or translation product thereof.

25. A composition according to Claim 24 wherein the FcyR gene includes an FcyRIIIA (CD16) or a variant thereof and/or includes an FcyRIIIA-158V allele or a variant thereof.

26. An antibody or antibody fragment raised against an FcyR gene product.

27. An antibody according to Claim 26 wherein the gene product is an FcyRIIIA (CD16) gene product or a variant thereof and/or is an FcyRIIIA-158V allele gene product or a variant thereof.

28. Use of an antibody raised against an FcyR gene product in diagnosing RA severity and/or predisposition thereto and/or treatment thereof.

29. Use according to Claim 28 wherein the gene product is an FcyRIIIA (CD 16) gene product or a variant thereof and/or is an FcyRIIIA-158V allele gene product or a variant thereof.

30. A hybridoma cell line which produces an antibody according to either Claim 26 or 27.

31. A product for detecting the presence of an FcyR gene or gene fragment or gene product comprising a nucleic acid molecule of SEQ ID NO: 1 and/or SEQ ID NO: 2, derivatives or homologues thereof and optionally further including a label.

32. A product according to Claim 31 wherein the FcyR gene or gene fragment or gene product is an FcyRIIIA (CD 16) gene or gene fragment or gene product thereof and/or is an FcyRIIIA-158V allele gene or gene fragment or gene product thereof.

33. A assay for the detection of sequence variation in nucleic acid target substrates comprising: i) providing a sample of nucleic acid to be assayed; ii) providing conditions for the denaturation of the nucleic acid; iii) incubating the nucleic acid sample with SEQ ID NO: 1 and SEQ ID NO: 2; iv) providing reagents and conditions sufficient to PCR amplify the target nucleic acid substrate; and v) separating and analysing the products of the reaction in (iv) to detect said sequence variation.

34. An assay for detecting or measuring the expression of a gene associated with RA comprising: i) providing a sample of nucleic acid to be assayed; ii) providing conditions for the denaturation of the nucleic acid; iii) incubating the nucleic acid sample with SEQ ID NO: 1 and SEQ ID NO: 2; iv) providing reagents and conditions sufficient to PCR amplify the target nucleic acid substrate; and v) separating and analysing the products of the reaction in (iv) to detect said sequence variation.

35. An assay according to either Claim 33 or 34 wherein said nucleic acid encodes an FcyR gene.