Polymorphism in the immunoglobulin VH gene V1-69 affects susceptibility to rheumatoid arthritis in subjects lacking the HLA-DRB1 shared epitope

J. Vencovsky1, E. Zd’ársky, S. P. Moyes2, A. Hajeer3, S. Ruzicková1, Z. Cimburek, W. E. Ollier3, R. N. Maini2 and R. A. Mageed4,

Institute of Rheumatology, Na slupi 4, Prague and
1 Laboratory of Gene Expression, Charles University, Prague, Czech Republic,
2 Kennedy Institute of Rheumatology, London,
3 ARC Epidemiology Unit, Manchester and
4 Department of Immunology and the Centre for Rheumatology, University College London, London, UK


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objective. To investigate the contribution of polymorphism in the immunoglobulin heavy chain variable region V1-69 gene set to genetic susceptibility to rheumatoid arthritis (RA) in Czech and British patients.

Methods. We used V1-69 gene sequence-specific polymerase chain reaction (PCR) and restriction enzyme digestion to study polymorphism in the V1-69 gene set in germline DNA of 109 Czech and 159 British RA patients and 164 ethnically matched controls. Polymorphism was further studied by nucleotide sequencing of the V1-69 gene locus in germline DNA.

Results. We found that all patients and controls had at least one V1-69 gene copy. In the Czech RA cohort, the dimorphic nucleotide in codon 73 of V1-69 (GAA or AAA) was present in the homozygous form 73A/A in 31 of 109 (28.4%) RA patients vs 12 of 79 (15.2%) controls [odds ratio (OR)=2.22, P<0.001]. When the RA patients and controls were classified according to HLA shared epitope (SE) status, 73A/A was found in 18 of 76 (23.7%) SE+ patients compared with 13 of 38 (34.2%) SE- patients, four of 12 (18.2) SE+ controls and eight of 57 (14%) SE- controls. This suggests that homozygosity for the dimorphic sequence 73A contributed to susceptibility to RA in SE- Czech individuals (OR=3.2, P<0.001). The most striking observation was that none of the 38 SE- Czech patients, compared with 11 of 76 (14.5%) SE+ RA patients, three of 22 (13.6%) SE+ and 11 of 57 (19.3%) SE- ethnically matched controls, were homozygous for the alternative dimorphic sequence 73G/G (OR=9.1, P<0.05). These data, however, were not replicated in a Caucasoid British RA population.

Conclusion. The dimorphic sequence at codon 73 (73A/A) of the V1-69 gene contributes to genetic susceptibility in SE- Czech RA patients.

KEY WORDS: Immunoglobulins, Immunogenetics, Rheumatoid arthritis.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Susceptibility to rheumatoid arthritis (RA) is determined by the combined effects of environmental factors and alleles at multiple genetic loci [1]. Major histocompatibility complex (MHC) genes make a major contribution to genetic factors associated with susceptibility to RA in many populations [1, 2]. This association is based on the presence of a conserved sequence of amino acids in the third hypervariable region of the ß chain, named the ‘shared epitope’ (SE), which is found in 10 different HLA-DRB1 alleles (HLA-DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0409, *0410, *1402 and *1001) [3, 4]. However, this association can only explain ~37% of inheritance factors [5], and other genetic loci must exist.

A number of early studies suggested that polymorphism at immunoglobulin (Ig) constant region gene loci may contribute to susceptibility to RA [6, 7]. However, a more recent study contradicted the earlier observation of an association between Ig kappa chain and susceptibility to RA [8]. In contrast, restriction fragment length polymorphism (RFLP) studies of Ig variable region (IgV) genes, IgV germline gene cloning and sequencing provided evidence that a functionally important Ig heavy chain variable region (VH) gene (Humhv3005 gene) was deleted from both haplotypes in 24% of patients with RA but only 2% of normal subjects [9]. Although the precise functional significance of the association between IgV gene polymorphism and a complex inflammatory disease such as RA remains speculative, polymorphism of IgV genes was originally predicted to influence the expressed B-cell repertoire [10]. Interestingly, a number of recent studies provide some evidence that IgV gene polymorphism may indeed influence the B-cell repertoire and, consequently, the humoral immune response to self and infectious antigens. For example, Sasso et al. [11] showed that the percentage of IgD+ tonsillar B cells expressing an idiotypic marker of the V1-69 gene set recognized by the G6 monoclonal antibody [12], which is strongly associated with rheumatoid factor (RF) activity, was proportional to the number of germline copies of the V1-69 gene. In another study, the prevalence of a kappa light chain V gene allele (V{kappa}A) in Navajo Indians was shown to play a role in susceptibility to Haemophilus influenzae type b infection [13]. In this study, susceptibility was attributed to diminished rearrangement of the V{kappa}A2 gene due to nucleotide changes in the recombination signal sequence of the susceptibility allele.

Studies from a number of laboratories, including ours, over a number of years have shown that the V1-69 gene set frequently encodes RF activity in mixed cryoglobulinaemia, natural RF in normals, in developing B cells in the foetus and in polyclonal RF in RA sera [1418]. At the nucleotide level, the gene is highly polymorphic, with at least 13 alleles in two subsets, 51p1-related and hv1263-related, which differ from each other by a maximum of six nucleotides in the coding region [19]. The nucleotide difference at codon 2 is a silent interchange (no amino acid difference in the protein) while the others result in amino acid replacements in CDR1 (position 33 alanine{leftrightarrow}threonine), CDR2 (50 glycine{leftrightarrow}arginine; 55 phenylalanine{leftrightarrow}leucine and 57 threonine{leftrightarrow}isoleucine) and FR3 (73 glutamic acid{leftrightarrow}lysine) (for clarity we refer herein to the FR3 dimorphic sequences as 73G and 73A). The 51p1-related and hv1263-related subsets are inherited as haplotypes, individuals having a total of 0–4 copies and 0–2 copies of genes from the two subsets respectively. In about 50% of individuals, there is a diploid dose of 3–4 51p1-related genes because of the prevalent two-gene haplotype created by gene duplication, which contains the 51p1-related variants 1 and 7 (Fig. 1Go) [20].



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FIG. 1.  A cartoon depicting the organization and gene number within the human VH locus on chromosome 14 and location of the V1-69 gene relative to other VH genes. The figure is based on the detailed map of VH gene organization in reference 20. Known functional genes are shown as black oval-shaped circles, pseudogenes as open rectangles and genes with open reading frames that have not previously been reported rearranged in vivo as triangles. Box A represents one of the seven known insertion polymorphisms (containing gene duplication). Fifty per cent of the population is known to have the insertion shown in box A with higher copy numbers of V1-69 genes. Although the precise location of the insertion is not known, it is suggested to be located in the region close to the V1-69 gene locus. Possible combinations of V1-69 alleles are indicated below the locus. 51p1-related alleles (51p1-r) are indicated by open ovals and hv1263-related (hv-1263-r) genes by black triangles to indicate lack of reported in vivo rearrangement. The predicted combinations of the most prevalent variants are indicated by the letters a, b, c and d in the bottom left part of the figure [19]. Approximate distance from the JH gene locus is indicated above the locus in kilobases (kb) of DNA.

 
On the basis of the strong association with RF activity, a specificity traditionally associated with RA, and the high degree of polymorphism in the V1-69 gene locus, we explored the contribution of this polymorphism to susceptibility to RA. We examined polymorphism in the V1-69 gene set in relation to RA susceptibility in two European Caucasoid populations, a cohort of Czech and British patients and in ethnically matched controls.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients and controls
Inheritance of V1-69 gene variants was studied in a cohort of 109 unrelated Caucasoid Czech RA patients (83 females and 26 males) attending the Institute of Rheumatology in Prague. To determine the consistency of 73G/A polymorphism in association with SE status in different populations, 159 Caucasoid RA British patients (ARC National Repository, Manchester, UK) was studied retrospectively. This cohort included more SE- individuals than would be expected from the normal prevalence in British RA patients (74 SE+ and 85 SE- patients; 119 females and 40 males). The mean age of the patients was 55 yr (range 19–84 yr) and all fulfilled the American College of Rheumatology criteria for RA [21]. The ethnically matched control groups comprised 79 Czech individuals (71 potential bone marrow donors and eight healthy Institute staff members; 57 females and 22 males) and 79 British individuals (48 female and 31 males; ARC National Repository). Of the Czech controls, 57 were SE- and 22 SE+. The British controls included 42 SE- individuals. To confirm the skewed prevalence of the 73G/A dimorphic sequences in SE- Czech RA patients, DNA from an additional 13 RA Czech patients, selected on the basis of being SE-, were studied retrospectively.

Polymerase chain reaction amplification
Genomic DNA was extracted from peripheral blood leucocytes by a modified salting-out protocol [22]. The V1-69 gene set was amplified from germline DNA by the polymerase chain reaction (PCR) using a primer complementary to the intron between the leader sequence and framework region 1 (FR1) in the coding region (positions -71 to -53; primer A, 5'-GAGGAAGGGATCCTGGT T-3') and an antisense primer to the 3' flanking region of the gene (position 297–318 of the DNA sequence; primer B, 5'-GGATGTGGGTTTTCACACTGTG-3') [19, 23]. The PCR products were purified by ethanol precipitation and used for restriction digestion to reveal dimorphism at codons 50, 57 and 73. Dimorphism at codons 33 and 55 was studied using the primary PCR products in a secondary nested PCR to introduce artificial restriction sites. The nested primers were primer C (complementary to nucleotide sequence 79–95, which encodes amino acids 27–32 in the FR1-CDR1; 5'-GGCACCTTCACAGCTAT-3'), which results in a PCR product with deletion of a G base from the third nucleotide position in codon 30, and primer D (antisense to the nucleotide sequence between nucleotide positions 165–183, which encodes amino acids 55–61 in CDR2; 5'-GCGTAGTTTGCTGTACTAA-3'), which replaces the first nucleotide at codon 56 (A{leftrightarrow}G) to allow digestion by the enzymes MslI and DdeI respectively (all locations according to reference 24). In each PCR, we included a negative control consisting of a PCR mix with no DNA template to confirm specificity.

Enzymatic digestion and detection of polymorphism
PCR products obtained with primers A and B were studied for dimorphism at codons 50, 57 and 73 by digestion with enzymes MnlI, RsaI and HinfI respectively. MnlI cuts DNA at the GGAG(N)7 (N=any nucleotide) sequence and therefore cuts V1-69 variants that have nucleotide G at the first base position of codon 50, which encodes glycine. The enzyme does not cut the sequence when the G is replaced by A (encodes arginine). Thus, a complete cut by MnlI indicates homozygosity for a sequence encoding glycine at this position in the protein product. Completely uncut DNA, in contrast, indicates homozygosity for the alternative sequence, which encodes arginine. Partially cut DNA implies heterozygosity. RsaI cuts DNA at the GTAC sequence and distinguishes between sequences that have C from those that have T at the second nucleotide base of codon 57 (Fig. 2Go). As with MnlI, a complete cut by RsaI implies homozygosity for a sequence encoding threonine, completely uncut DNA implies homozygosity for the alternative sequence encoding isoleucine and a partial cut implies heterozygosity. HinfI cuts at the GANTC sequence and thus distinguishes between V1-69 sequences that have G from alternative sequences that have A at the first base position of codon 73. The enzyme thus cuts 73G but not 73A sequences. Again, homozygosity is indicated either by a complete cut or completely uncut DNA and heterozygosity by partial cutting (Fig. 2Go). For the restriction digestions, 6 µl of the PCR products were digested with 4 U of MnlI (New England Biolabs, Beverly, MA, USA), 2 U of RsaI or HinfI (Amersham, MGP Zlin, Czech Republic) for 1 h at 37°C. The restriction patterns were studied on 2.5% agarose gels containing ethidium bromide.



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FIG. 2.  Restriction digestion of PCR-amplified V1-69 genes. The sizes of the different fragments are indicated at each side of the figure, based on the fragments obtained by digesting pBR322 with MspI. Lane 1 is an example of V1-69 DNA amplified using primers A and B. Lanes 2–4 show restriction digests of PCR-amplified V1-69 cut with RsaI. In lane 2 the DNA sample is uncut, indicating homozygosity for the variant codon ATA (encodes isoleucine in the protein). In lane 3, a partial cut implies heterozygosity (AC/TA). In lane 4, complete DNA digestion with RsaI implies homozygosity for the variant codon ACA (encodes threonine). Lanes 5–7 show digestion of DNA samples with HinfI to assess dimorphism at codon 73. In lane 5 the DNA is uncut, implying homozygosity for 73A variants. The partial digestion in lane 6 implies heterozygosity. In lane 7, the complete cut of the DNA implies homozygosity for 73G variant(s).

 
DNA from the secondary nested PCR with primers C and D was used to reveal dimorphism at codons 33 and 55 by digestion with enzymes MslI and DdeI respectively. MslI cuts at the CAYNNNNRTG sequence (Y represents a pyrimidine base, R a purine base and N any base) and thus cuts sequences that have G but not the alternative sequences that have A at the first nucleotide position of codon 33. The codon GCT encodes alanine while ACT encodes threonine. DdeI cuts at a CTNAG sequence in sequences with T but not C at the first nucleotide position of codon 55. The codon TTT encodes leucine while CTG encodes phenylalanine. In each case, 3 µl of the PCR products was digested with 1.5 U of MslI or 2 U of DdeI (New England Biolabs) for 1 h at 37°C. The digests were separated on 10 or 15% polyacrylamide gel. Homozygosity and heterozygosity were assessed as for codons 50, 57 and 73 (Fig. 3Go).



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FIG. 3.  The main germline nucleotide codons that distinguish the different variants of V1-69 gene set. Sequences of the prototype 51p1 and hv1263 genes are shown in bold. The first 13 alleles are designated by the numbers assigned by Sasso et al. [19] on the basis of RFLP analysis and probing with CDR1 and CDR2 sequence-specific oligonucleotide probes. The three new sequences discovered in the present study are provisionally given the numbers 3w, 4w and 12w. Although it is likely that these sequences represent new variants, the possibility that they represent PCR artefacts is not excluded and further evidence that these represent real new variants will need to be provided. Codon numbering and the location of the FR and CDR (shown only in part) are according to Kabat et al. [38]. Nucleotides identical to those of the germline genes are indicated by a dash, while changes are indicated according to whether they are silent changes (small letters) or replacement changes (capital letters). Also indicated above the 51p1 and below the hv1263 prototype sequences (only when different from 51p1) are the predicted amino acids at each position. To simplify the nomenclature of the encoded VH chains and clarify the relationship between the coding regions of the different alleles, the predicted amino acid sequence of each allele is assigned a number. Variants 1 and 5, 2 and 11, 3 and 9 and 10 and 13 have identical coding region sequences but are found on different TaqI-cut RFLP fragments due to non-coding region sequence variations and are therefore considered to represent different variants [19]. The corresponding VH chains of the possible new variants—3w, 4w and 12w—are given the numbers 6w, 7w and 9w because they have the closest predicted amino acid similarity to the VH chains 6, 7 and 9 respectively. By random assortment, variants 1 and 7 were found to be in linkage disequilibrium and we suggest that they occur on the same haplotype representing a recent duplication of the gene.

 

HLA typing
HLA-DRB1 typing was performed by PCR (British Society for Histocompatibility, Bristol, UK) as described [25]. The localizations, sequences, lengths, melting temperatures, and specificities of primers were as published [26]. The resolution of this typing system was set to equal serological DR typing for the allelic series DR1 to DR18. Twenty-three reactions were set for each typing and results were visualized on 2% agarose gels. A similar approach was used for subtyping the HLA-DRB1*04+ individuals, using a kit (Dynal, Oslo, Norway).

Cloning and sequencing of PCR products
PCR products obtained with primers A and B were separated by electrophoresis on 1% low-melting point agarose gels and bands excised from the gel and DNA was purified using an Ultrafree-MC filter unit (Millipore, Watford, Herts, UK). The DNA was purified by ethanol precipitation, ligated into the Invitrogen cloning vector (pCR II; Invitrogen, Leek, The Netherlands) and used to transform Escherichia coli strain INVaFí. Six to 14 of the bacterial colonies from nine patients were sequenced. Sequence information was processed and compared with existing sequences in the Genbank/EMBL databases using Lasergene software (DNAstar, Madison, WI, USA).

Statistical analysis
Odds ratios (OR) were calculated to evaluate susceptibility to RA on the basis of the observed frequency of dimorphic sequences of the V1-69 gene set in patients and controls. The {chi}2 test with Yates’ correction was used to analyse differences in proportions. The data were analysed for consistency with the Hardy–Weinberg equilibrium for population frequencies using Pearson's {chi}2 test on the basis of the observed and expected frequencies of variants of the V1-69 gene set with one degree of freedom.


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 Abstract
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 Methods
 Results
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 References
 
HLA-DR typing and HLA-DR4 subtyping
Fifty one of the 109 Czech RA patients (46.8%) were HLA-DR4+, and 43 (39.4%) of these carried a single DR4 allele (Table 1Go). Forty-six patients had ‘susceptible’ DR4 alleles (HLA-DRB1*0401, 0404/8, 0405), while five had non-susceptible DR4 alleles (i.e. SE-). HLA-DR1 alleles were present in 30 patients, of whom five were also DR4+. Other alleles with the SE were represented rarely: two patients were positive for HLA-DRB1*1402 and one for HLA-DRB1*1001. All three patients were heterozygous and the other alleles were either DR4+ (two patients) or DR1+ with the SE. In the ethnically matched Czech controls, nine individuals (11.4%) were DR4+ and all were SE+. There were also 15 (19%) DR1+ individuals, of whom two were also DR4+. Fifty-three of the 74 SE+ British RA patients (68%) were HLA-DR4+, and 34 (46%) of these carried a single allele. A further 29 patients (39.3%) were HLA-DR1+, and 12 of these were also DR4+. Of the 37 SE+ British controls, 27 (73%) were DR4+ and 11 (28%) were DR1+; four of the latter were also DR4+.


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TABLE 1.  HLA genotype of Czech and British RA patients and ethnically matched controls included in the study

 

Polymorphism of the V1-69 gene
PCR amplification of the V1-69 gene was successful in all patients and controls. Subsequent amplification, cloning and nucleotide sequencing of DNA from nine Czech RA patients confirmed that only V1-69 gene sequences were amplified. In agreement with previous studies, the PCR and restriction digestion data meant that at least one V1-69 gene copy was present in the germline of all the individuals studied and no subjects were found to be homozygous for V1-69 null alleles [19].

Analysis of sequence dimorphisms at codons 33 (GCT{leftrightarrow}ACT), 50 (GGG{leftrightarrow}AGG), 55 (TTT{leftrightarrow}CTT) and 57 (ACA{leftrightarrow}ATA) revealed some differences between the Czech patients and matched controls, but none were statistically significant (Table 2Go). The only statistically significant difference was in the prevalence of individuals whose V1-69 genotype consisted only of variants that were 73A-positive. Thirty-one Czech RA patients (28.4%) but only 12 (15.2%) ethnically matched controls were 73A/A. In contrast, only 11 Czech patients (10.1%), compared with 14 ethnically matched controls (17.7%), were homozygous for the 73G dimorphic sequence (73G/G). Statistical analyses suggested that 73A homozygosity made a contribution to RA susceptibility in the Czech population (OR=2.22; P<0.001) (Table 2Go).


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TABLE 2.  V1-69 variant prevalence in Czech RA patients and ethnically matched controls

 

Pattern of inheritance of V1-69 polymorphism in relation to DR4/DR1 status
Eighteen (23.7%) of the DR4+ and/or DR1+ patients were 73A/A compared with four (18.2%) ethnically matched controls. The proportion of individuals who were either 73G/G or 73A/A was similar in the patients and controls (Table 3Go). In contrast, 13 (39.4%) of the DR4- and/or DR1- RA patients were 73A/A but none was 73G/G. Among the DR4- and/or DR1- normal subjects, eight (14%) were 73A/A and 11 (19.3%) 73G/G. The data were consistent with Hardy–Weinberg equilibrium ({chi}2=6.48, P<0.01). The calculated OR for patients homozygous for the 73A (i.e. 73A/A and not 73A/G or 73G/G) was 3.98. In the SE- patients, which included the DR4+ without the SE, the OR decreased to 3.19 (P<0.001). The risk of RA in DR4- and/or DR1- patients who were not homozygous for 73G (i.e. either 73A/A or 73G/A) was high (OR=7.81, P<0.05). When the DR4+ patients without the SE were considered among the DR4- and/or DR1- group, the risk factor was even higher (OR=9.1; P<0.05). To confirm the skewed low prevalence of 73G/G in SE- Czech patients, genomic DNA from a further 13 SE- Czech RA patients was analysed. Only one patient within this group was found to be 73G/G while two were 73A/A and 12 73G/A. The skewed prevalence of the dimorphic sequences 73A/A and 73G/G was not replicated in the English cohort (Table 4Go).


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TABLE 3.  Prevalence of V1-69 73G/A variants in Czech individuals grouped according to HLA-DR haplotype and SE status

 

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TABLE 4.  Prevalence of V1-69 73G/A variants in Czech and British RA patients and ethnically matched controls in relation to HLA-DR and SE status

 

Sequence analysis of V1-69 variants in Czech RA patients
To confirm the specificity of the PCR and to study the pattern of inheritance of the variants, genomic DNA from nine Czech patients was amplified, cloned and sequenced. Six to 14 bacterial colonies from each of the nine cloned PCR products were sequenced in both directions. The sequences confirmed the PCR–RFLP analyses and no inconsistencies were observed. In seven individuals the nucleotide sequences were identical to the known variants, with variations only at positions that constitute the basis for the known variants [19] (Fig. 4Go). In DNA from two of the Czech patients (patients 34 and 198), however, three possible new sequences were found (Figs 3Go and 4Go). However, as these three new sequences could represent PCR artefacts, further studies are needed to confirm that the three new sequences represent true variants. These variants are, therefore, provisionally given the numbers 4w, 3w and 12w to correspond with those originally identified by RFLP analysis [19]. Because the numbering of the V1-69 variants is based on the original RFLP analysis, some variants have identical coding regions but differ in their non-coding region and have different TaqI-generated fragments. Consequently, it is not possible to ascertain exactly which V1-69 variant was represented by some of our cloned nucleotide sequences. Nevertheless, on the basis of the linkage disequilibrium between some of the variants, the sequencing data showed that the most prevalent hv1263-related variant was variant 3/9 and the most prevalent 51p1-related variant was variant 5 in the nine subjects whose V1-69 genes were cloned (Fig. 4Go).



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FIG. 4.  Summary of sequence analysis of the V1-69 alleles isolated from the Czech RA patients reported in this study. The sequences were determined by cloning the genes from genomic DNA, using primers A and B, from nine RA patients. Six to 14 colonies were sequenced in both directions. Although the entire sequence of the coding regions was determined, only the dimorphic codons are shown for brevity. Numbering of the heavy-chain variants is as in Fig. 3Go. Codon numbering and demarcation of the FR (framework region) and CDR (complementarity-determining region) are according to Kabat et al. [38]. Numbering of variants is according to Sasso et al. [19], as described in the legend to Fig. 3Go. The three new sequences, in part related to the hv1263 set, were observed in the DNA from patients 34 and 198 (sequences 4w, 3w and 12w).

 


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Genetic susceptibility to RA is determined by the combined effects of alleles at multiple genetic loci, including HLA and non-HLA loci [1, 2, 5]. The nature and pathophysiological contribution of most allelic variants are largely unknown, but for HLA molecules they are likely to involve differential antigen binding and presentation to T cells [27]. Examples in support of this prediction include the preferential binding of peptides from self antigens, such as the 70-kDa heat shock protein HSP73 and acetylcholine receptor {alpha}-derived peptides to RA-associated HLA-DR molecules [27, 28]. An alternative perspective on genetic susceptibility is that the inheritance of particular alleles relates to RA severity rather than susceptibility [29]. This model predicts that RA is a heterogeneous disease representing different entities that are related to the inheritance of different combinations of susceptibility alleles [30].

Immunoglobulins, like T-cell receptors for antigen, are involved in antigen recognition. In addition, membrane immunoglobulins are involved in antigen uptake for processing and presentation by B cells to T cells and in shaping the B-cell repertoire through their role in maintaining the pool of recirculating B cells [31, 32]. The deletion, or duplication, of functionally important IgV genes is therefore likely to influence the primary B-cell repertoire and may contribute to disease susceptibility. However, until recent years there has been a scarcity of evidence for the involvement of IgV genes in diseases in general and in RA specifically. This stems at least partly from the high degree of linkage equilibrium within the IgV gene loci, which creates a great degree of haplotype diversity in humans [33].

The purpose of the present study was to determine if deletion or an increased copy number of genes which encode RF, a specificity traditionally associated with RA, in association with particular HLA haplotypes in clinically and ethnically defined subpopulations can contribute to RA susceptibility. The data presented in the present study reveal that the V1-69 gene was present in all individuals studied and therefore homozygous absence of the V1-69 locus is not a susceptibility factor for RA. Nevertheless, the data reveal an intriguing association between the pattern of inherited variants of the V1-69 gene set and susceptibility to RA in SE- Czech individuals. Our approach to studying the polymorphism of the V1-69 gene set relied on PCR of the gene variants in the germline, restriction digestion and germline gene sequencing. The sequencing revealed three possible new variants (all hv1263-related) from two patients. However, more experiments are under way to confirm that the new sequences represent true variants and exclude the possibility that the new sequences are PCR artefacts. Nevertheless, the pattern of combination of the otherwise standard alternative nucleotide substitutions at the polymorphic sites distinguished the possible new dimorphic sequences from the 13 variants known previously [19].

In the mostly Caucasian populations studied previously, the most prevalent alleles of V1-69 genes were variant 5, which is 73G, and the linked pair of variants 1 and 7, which are 73G and 73A respectively. On the basis of these previous observations, it is possible to assume that nearly all cases of 73G/G are homozygous for variant 5 because all other 73G-positive alleles are rare (excluding variant 1). Therefore, the decreased prevalence of 73G/G in Czech RA patients strongly suggests decreased prevalence of variant 5 homozygotes. However, the prevalence of the variant pair 1+7 may not necessarily have increased in the Czech RA patients because there was also an increase in the prevalence of RA patients with 73A/A. These results, therefore, suggest that the shift from 73G/G to 73A/A in Czech patients is likely to be due to an increased frequency of hv1263-related genes in addition to a contribution from the solitary variant 7 gene, seen previously in only one of 96 haplotypes [19]. Together with the observation that the frequency of 33A-positive variants was increased in Czech RA patients, this suggests that the 73A/A shift is due in part to the increased frequency of V1-69 variants having both 33A and 73A, i.e. variants 8, 10 and 13.

A recent cellular study revealed that differences in the number of inherited copies of 51p1-related variants (dimorphic sequences mostly with 73G) could have functional implications. Thus, a direct relationship was revealed between the number of inherited 51p1-related gene copies and the proportion of IgD+ B cells expressing a 51p1-related cross-reactive idiotype recognized by the G6 monoclonal antibody in human tonsils [11]. One straightforward interpretation of our data would therefore be that increased susceptibility to RA in the SE- Czech patients could be related to a higher frequency of V1-69 genes encoding 73A heavy chains. Because codon 73 encodes an amino acid in the FR3, outside the conventional antigen-binding site, its significance may not be for the amino acid it encodes but rather as a marker for V1-69 genes whose CDR1 or CDR2 encodes specificities relevant to RA susceptibility. In addition, the result also indicates that these individuals would have lower numbers of, or no (in 73A/A individuals), B cells expressing the 73G dimorphic sequences in the primary B-cell repertoire. This could be important because hv1263-related 73A variants, despite having open reading frames, have been expressed disproportionately less than the 51p1-related genes in the adult and foetal repertoires in vivo [20, 34]. Consequently, this would suggest that the higher number of B cells expressing 51p1-related 73G variants in normal individuals could serve useful regulatory immunological functions. In this context it is noteworthy that cells expressing the 51p1-related cross-reactive idiotype recognized by the G6 monoclonal antibody localize to the mantle zone of secondary follicles in human tonsils, where, it has been suggested, they capture and present antigens in immune complexes to T cells [35]. G6+ IgM+ IgD+ B cells might therefore help maintain peripheral T-cell tolerance through the presentation of self peptides to activated T cells in the absence of costimulatory molecules, or through the production of T-lymphocyte-inhibitory cytokines, such as transforming growth factor ß [36].

An alternative interpretation of the association between the increased frequency of 73A variants in SE- Czech RA patients is that increased susceptibility in this group is due to linkage disequilibrium of 73A variants with a nearby disease gene(s) on chromosome 14. This possibility may be relevant to the finding that the gene duplication involving alleles 1 and 7 represents part of an 80-kilobase multigene chromosomal insertion in 50% of the population [20].

One caveat to the finding of an association between V1-69 gene polymorphism and RA is that examination of a British cohort did not replicate the association. Instead, a trend towards a reduced frequency of 73A variants in the SE+ patients compared with SE- patients and controls was seen. Although strict inclusion criteria were applied to ensure the genetic homogeneity of the Czech cohort, it is still possible that we may have overlooked some relevant factors (e.g. hidden population stratifications) that may have resulted in a spurious association. Alternatively, the observed association between V1-69 gene polymorphism and susceptibility to RA in the Czech population could indicate that the combination of genetic and environmental factors conferring susceptibility to RA varies in different populations.

Association between RA susceptibility and other (non-HLA) loci has been reported previously. For example, analysis of a microsatellite marker adjacent to the transcription element a (TEA) in the T-cell receptor complex indicated that, in a group negative for DRB1*04 and DRB1*01 alleles, the relative risk of acquiring RA increased (>13) or decreased (<0.07) depending on the inherited microsatellite allele adjacent to the TEA locus [30]. Furthermore, association of RA with allelic variants within the T-cell receptor {alpha} locus with no influence from RA-associated HLA-DR alleles was also observed [37]. Therefore, to confirm the relevance of V1-69 gene polymorphism to RA susceptibility, large numbers of patients and controls, preferably including multicase families from different European Caucasoid populations, need to be studied. Furthermore, studies are required to address the functional implications relating to how sequence dimorphism in a single IgV gene locus could contribute to susceptibility to a complex disease such as RA.


    Acknowledgments
 
We wish to thank Drs Hugh O. McDevitt and Frances Hall (Department of Microbiology and Immunology, University of Stanford Medical School, Stanford, USA) for invaluable comments and critical evaluation of the results. We thank Ms Jenny Head (Department of Epidemiology and Public Health, University College London) for advice on the statistical protocols used in the study. This research was supported in part by grant 039893/Z/93 from The Wellcome Trust, UK and grants VS96129 from the Czech Ministry of Education, Youth and Sports and 302/94/0939 from the Grant Agency of the Czech Republic. SPM, RAM and RNM are supported by the Arthritis Research Campaign.


    Notes
 
Correspondence to: R. A. Mageed, Department of Immunology, The Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T 4JF, UK. Back


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Wordsworth P, Bell JI. The immunogenetics of rheumatoid arthritis. Springer Semin Immunopathol1992;14:59–78.[ISI][Medline]
  2. Stastny P. Association of the B-cell alloantigen Drw4 with rheumatoid arthritis. N Engl J Med1978;298:869–71.[Abstract]
  3. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis—an approach to understanding the molecular genetics of rheumatoid arthritis susceptibility. Arthritis Rheum1987;30:1205–13.[ISI][Medline]
  4. Winchester R, Dwyer E, Rose S. The genetic basis of rheumatoid arthritis: the shared epitope hypothesis. Rheum Dis Clin N Am1992;18:761–83.[ISI][Medline]
  5. Deighton CM, Walker DJ, Griffiths ID, Roberts DF. The contribution of HLA to rheumatoid arthritis. Clin Genet1989;36:178–82.[ISI][Medline]
  6. Ollier W, Thomson W, Welch S, De Lange G, Silman A. Chromosome 14 markers in rheumatoid arthritis. Ann Rheum Dis1988;47:843–8.[Abstract]
  7. Moxley G. DNA polymorphism of immunoglobulin kappa confers risk of rheumatoid arthritis. Arthritis Rheum1992;32:634–7.
  8. Singh R, Han J, Moxley G. Polymerase chain reaction-based genotyping of the constant segment of immunoglobulin kappa shows weak or no association with rheumatoid arthritis. Arthritis Rheum1995;38:1526–7.[ISI][Medline]
  9. Yang PM, Olsen NJ, Siminovitch KA et al. Possible deletion of a developmentally regulated heavy chain variable region gene in autoimmune diseases. Proc Natl Acad Sci USA1990;86:7907–11.
  10. Ben-Neriah Y, Cohen JB, Rechavi G, Zakut R, Givol D. Polymorphism of germ-line immunoglobulin VH genes correlates with allotype and idiotype markers. Eur J Immunol1981;11:1017–20.[ISI][Medline]
  11. Sasso EH, Johnson T, Kipps TJ. Expression of the immunoglobulin VH gene 51p1 is proportional to its germline gene copy number. J Clin Invest1996;97:2074–80.[Abstract/Free Full Text]
  12. Mageed RA, Dearlove M, Goodall DM, Jefferis R. Immunogenic and antigenic epitopes of immunoglobulins. XVII. Monoclonal antibodies reactive with common and restricted idiotopes on the heavy chain of human rheumatoid factor. Rheumatol Int1986;6:179–83.[ISI][Medline]
  13. Feeney AJ, Atkinson MJ, Cowan MJ, Escuro G, Lugo G. A defective V{kappa} A2 allele in Navajos which may play a role in increased susceptibility to Haemophilus influenzae type b disease. J Clin Invest1996;97:2277–82.[Abstract/Free Full Text]
  14. Crowley JJ, Goldfien RD, Schrohenloher RE et al. Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins. J Immunol1988;140:3411–8.[Abstract/Free Full Text]
  15. Shokri F, Mageed RA, Richardson P, Jefferis R. High frequency expression and modulation of autoantibody associated cross-reactive idiotypes in CD5 expressing B-lymphocytes from patients with chronic lymphocytic leukaemia. Scand J Immunol1993;37:673–9.[ISI][Medline]
  16. Thompson KM, Randen I, Børretzen, Førre Ø, Natvig JB. Variable region gene usage of human monoclonal rheumatoid factors derived from healthy donors following immunisation. Eur J Immunol1994;24:1771–8.[ISI][Medline]
  17. Schroeder HW, Wang JY. Preferential utilization of conserved immunoglobulin heavy chain variable gene segments during human fetal life. Proc Natl Acad Sci USA1990;87:6146–50.[Abstract]
  18. Shokri S, Mageed RA, Tunn E, Bacon PA, Jefferis R. Qualitative and quantitative expression of VHI associated cross-reactive idiotopes within IgM rheumatoid factor from patients with early synovitis. Ann Rheum Dis1990;49:150–4.[Abstract]
  19. Sasso EH, Willems van Dijk K, Bull AP, Millner EC. A fetally expressed immunoglobulin VH1 gene belongs to a complex set of alleles. J Clin Invest1993;91:2358–67.[ISI][Medline]
  20. Cook GP, Tomlinson IM. The human immunoglobulin VH repertoire. Immunol Today1995;16:237–42.[ISI][Medline]
  21. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum1988;31:315–24.[ISI][Medline]
  22. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res1988;16:1215.[ISI][Medline]
  23. Børetzen M, Randen I, Zd’ársky E, Føre Ø, Natvig JB, Thompson KM. Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions. Proc Natl Acad Sci USA1994;91:12917–21.[Abstract/Free Full Text]
  24. Chen PP, Liu M-F, Glass CA, Sinha S, Kipps TJ, Carson DA. Characterization of two immunoglobulin VH genes that are homologous to human rheumatoid factors. Arthritis Rheum1989;32:72–6.[ISI][Medline]
  25. Olerup O, Zetterquit H. HLA-DR typing by PCR amplification with sequence-specific primers PCR-SSP in 2 hours: An alternative to serological DR typing in clinical practice including donor–recipient matching in cadaveric transplantation. Tissue Antigens1992;39:225–35.[ISI][Medline]
  26. Olerup O, Zetterquist H. DR Low-resolution PCR-SSP typing—a correction and an up-date. Tissue Antigens1993;41:55–6.[ISI][Medline]
  27. Hawke S, Matsuo H, Nicolle M et al. Cross-restriction of a T cell clone to HLA-DR alleles associated with rheumatoid arthritis: clues to arthritogenic peptide motifs. Arthritis Rheum1999;42:1040–50.[ISI][Medline]
  28. Auger I, Escola JM, Gorvel JP, Roudier J. HLA-DR4 and HLA-DR10 motifs that carry susceptibility to rheumatoid arthritis bind 70-kDa heat shock proteins. Nature Med1996;2:306–10.[ISI][Medline]
  29. Weyand CM, McCarthy TG, Goronzy JJ. Correlation between disease phenotype and genetic heterogeneity in rheumatoid arthritis. J Clin Invest1995;95:2120–6.[ISI][Medline]
  30. Gomolka M, Menningre H, Saal JE et al. Immunoprinting: various genes are associated with increased risk to develop rheumatoid arthritis in different groups of adult patients. J Mol Med1995;73:19–29.[ISI][Medline]
  31. Zhong G, Sousa CR, Germain RN. Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen–major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro. J Exp Med1997;186:673–82.[Abstract/Free Full Text]
  32. Lam KP, Kühn R, Rajewsky K. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell1997;90:1073–83.[ISI][Medline]
  33. Walter MA, Cox DW. Nonuniform linkage disequilibrium within a 1,500-kb region of the human immunoglobulin heavy-chain complex. Am J Hum Genet1991;49:917–31.[ISI][Medline]
  34. Schroeder HW Jr, Hillson JL, Perlmutter RM. Early restriction of human antibody repertoire. Science1987;238:791–3.[ISI][Medline]
  35. Carson DA, Chen PP, Kipps TJ. New roles for rheumatoid factor. J Clin Invest1991;87:379–83.[ISI][Medline]
  36. Wahl SD, Hunt A, Wong HL et al. Transforming growth factor-b is a potent immunosuppressive agent that inhibits IL-1 dependent lymphocyte proliferation. J Immunol1988;140:3026–31.[Abstract/Free Full Text]
  37. Cornelis F, Hardwick L, Flipo RM et al. Association of rheumatoid arthritis with an amino acid allelic variation of the T cell receptor. Arthritis Rheum1997;40:1387–90.[ISI][Medline]
  38. Kabat EA, Wu TT, Reid-Miller M, Perry HM, Gottesman KS. Sequence of proteins of immunological interest. Bethesda, MD: US Public Health Service, 1991.
Submitted 2 November 2000; Accepted 18 October 2001





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