Non-inherited maternal HLA alleles are associated with rheumatoid arthritis

S. Harney1,2,, J. Newton1,2, A. Milicic1, M. A. Brown1,2 and B. P. Wordsworth1,2

1 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN and
2 Nuffield Orthopaedic Centre, Oxford OX3 7LD, UK


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Rheumatoid arthritis (RA) is strongly associated with a series of HLA-DRB1 alleles that encode a conserved sequence of amino acids (70Q/R K/R R A A74) in the DRß1 chain, known as the shared epitope (SE). However 30% of patients are negative for DRB1*04 and 15% are SE-negative. Exposure to these alleles as non-inherited maternal antigens (NIMA) might explain this discrepancy. We undertook a family study to investigate the role of NIMA in RA.

Methods. One hundred families, including the RA proband and both parents, were recruited. HLA-DRB1 genotyping was performed using an allele-specific polymerase chain reaction by standard methods. The frequencies of NIMA and non-inherited paternal antigens (NIPA) were compared using contingency tables and a two-tailed P test. We then reviewed four previously published studies of NIMA in RA and conducted an analysis of the combined data

Results. We identified 36 families in which the proband was DRB1*04-negative and 13 in which the proband lacked the SE. There was an excess of DRB1*04 and SE NIMA (P=0.05) compared with NIPA. Combined analysis with previous studies showed that 53/231 mothers (23%) versus 25/205 fathers (12%) had a non-inherited DRB1*04 (P=0.003) and 30/99 mothers versus 18/101 fathers had a non-inherited SE allele (P=0.03).

Conclusion. A role for HLA NIMA in RA is suggested by these results.

KEY WORDS: Rheumatoid arthritis, Shared epitope, Non-inherited maternal alleles, HLA.


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a polygenic disorder to which the HLA region contributes about 30% of the genetic component. It is associated with certain HLA-DRB1 alleles encoding a conserved sequence of amino acids in the binding site of these antigen-presenting molecules, often referred to as the shared epitope (SE) [1]. The strongest associations are with certain HLA-DRB1*04 alleles (*0401, *0404, *0405 and *0408) but other SE-positive DRB1 alleles (*0101, *0102 and *1001) are also associated with RA. However, because about 30% of patients are negative for DRB1*04 and 15% are SE negative, a unifying pathological mechanism has proven elusive. Recent observations suggest that biologically relevant exposure to HLA antigens may occur during fetal development and/or subsequently through the persistence (perhaps for life) of maternal cells in the offspring (‘microchimerism’). It has been suggested that persistent microchimerism may be involved in the pathogenesis of certain autoimmune diseases, such as scleroderma and thyroiditis [2, 3]. Autoimmune thyroid disease is particularly interesting because, like RA, it occurs more commonly in women and there is an increased incidence post-partum. Thyroid disease is also more common in RA patients and their relatives than in the general population. We therefore feel that non-inherited maternal HLA antigens (NIMA) may also play a role in RA. Previous reports have not uniformly confirmed this hypothesis, but few have had adequate statistical power to reject it [47]. We combined all previous reports, both positive and negative, to see if this effect was real. Although this was not ideal, we felt confident pooling these studies as they were remarkably similar from a number of viewpoints: all had probands with RA defined by the 1987 American College of Rheumatology criteria; all patients were European and thus belonged to a population in which the importance of the DRB1*04 is well established; and all the DRB1 typing was performed using the same method. Additionally, if you pool only the French and Dutch data (i.e. data for mainland Northern Europeans), the results are significant for the DRB1*04 data. Overall, in these studies the number of Southern Europeans included was very small, and this was important as DRB1*01 plays a bigger role than in British Caucasians

We therefore undertook a study of 100 nuclear families to investigate the possible involvement of NIMA in RA.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One hundred nuclear families (RA proband and both parents) were recruited from the Oxfordshire and Southwest UK regions. All the probands fulfilled the 1987 American College of Rheumatology criteria for RA [8]. The probands and both parents were genotyped for HLA-DRB1 alleles, including DRB1*04 subtypes, by the polymerase chain reaction using sequence-specific primers. Samples that were positive for DRB1*04 were then subtyped for an additional nine primers. The potential role of NIMA was investigated by comparing the frequencies of DRB1*04 NIMA and SE NIMA with those of the corresponding non-inherited paternal antigens (NIPA). The results from four previous studies of the effects of NIMA in RA [47] were combined with those in our study for a combined analysis.

Statistical analysis
The frequencies of NIMA and NIPA were compared using contingency tables and a two-tailed P test. We used Mietinnen's method for calculating confidence intervals. In the SE studies there were only 13 families but 26 pieces of data were used as both parents were included. Following our reviewer's comments, we did homogeneity tests (Table 1Go) and found that our data set was different from the Dutch and French data sets, so we re-analysed their data separately.


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TABLE 1. Homogeneity tests

 


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We identified 36 families in which the proband was HLA-DRB1*04-negative and only 13 in which the proband was SE-negative. There was a non-significant excess of DRB1*04 NIMA compared with NIPA (Table 2Go) and a significant excess of SE-positive NIMA vs NIPA (P=0.05). The results of the combined analysis are shown in Table 2Go. There was a significant excess of DRB1*04 NIMA (23%) vs NIPA (12%), with an odds ratio of 2.1 (95% confidence interval 1.3–3.6) The incomplete data from some previous studies allowed us to undertake only a limited analysis of SE NIMA. Overall, 112 SE-negative probands were identified; a significant excess of mothers (30%) compared with fathers (18%) carried an SE-positive NIMA (P=0.04). As the French and Dutch data were remarkably similar and homogeneous when tests were done, we also combined these and found a similar significant result, with a P value of 0.01 (Table 3Go).


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TABLE 2. Comparison of NIMA and NIPA in DRB1*04- and SE-negative patients with RA

 

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TABLE 3. Data from Dutch and French data sets

 


    Discussion
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 Abstract
 Introduction
 Methods
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 Discussion
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HLA molecules are crucial in the definition of self and are key determinants of transplant success. Their role in susceptibility to RA is well established but the disease processes involved have not yet been clarified. Our results, combined with previous studies, strongly suggest that HLA NIMA may also be involved in RA.

It has already been shown that NIMA can influence immunologically mediated processes. Thus, Burlingham et al. [9] showed that graft survival for siblings mismatched with the donor for the NIMA haplotype was similar to that of graft survival in HLA-identical donors. It has been suggested that exposure to the NIMA haplotype in utero by transplacental transfusion may lead to tolerance. Maternal cells may persist in the fetus (and vice versa) in a process known as microchimerism, but whether this is necessary for tolerance to develop is unclear. Srivatsa et al. [10] recently demonstrated striking microchimerism in autoimmune thyroid disease, suggesting that it may play a significant role in autoimmunity. Microchimerism in thyroid disease is particularly interesting because of the high post-partum incidence of subclinical thyroiditis. Thyroid disease and RA have several parallels: both are more common in women and tend to flare or present post-partum. Nelson et al. [2] have highlighted the role of microchimerism in scleroderma. In contrast, Nelson [3] recently questioned whether microchimerism was just a remnant of healthy pregnancy. On the other hand, another article has reported a patient with a terminal thymic tumour that regressed after she received a stem-cell infusion of her daughter's cells, which were mismatched for NIMA. However, the mechanisms by which NIMA might influence susceptibility to RA are as unclear as the role HLA molecules play in the process. The mechanisms by which B cell unresponsiveness to NIMA is induced are not yet established [11]. Microchimerism is found frequently in the blood of healthy parous women, and healthy offspring often possess maternal cells. Although we have been able to demonstrate a significant excess of DRB1*04 and SE NIMA in RA families, this effect is relatively weak. We have shown that, with increased study numbers, this effect exists. However, to date there has been no large study in RA to prove this hypothesis definitively. As highlighted recently by Nelson, the role of NIMA in human health and disease is worth further investigation [2] and appears to play a significant role in the pathogenesis of RA.


    Acknowledgments
 
This work was supported by the Arthritis Research Campaign and the Grenville Bequest.


    Notes
 
Correspondence to: S. Harney, c/o Seddon Ward, Nuffield Orthopaedic Centre, Windmill Road, Headington, Oxford OX3 7LD, UK. E-mail: sineadharney{at}hotmail.com Back


    References
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 Abstract
 Introduction
 Methods
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 Discussion
 References
 

  1. Gregersen PK, Silver J, Winchester RJ. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205–13.[ISI][Medline]
  2. Nelson JL, Furst DE, Maloney S et al. Microchimerism and HLA-compatible relationships of pregnancy in scleroderma. Lancet 1998;351:559–62.[CrossRef][ISI][Medline]
  3. Nelson J. Microchimerism: expanding new horizons in human health or incidental remnant of pregnancy? Lancet 2001;358:2011–2.[CrossRef][ISI][Medline]
  4. ten Wolde S, Breedveld FC, de Vries RR et al. Influence of non-inherited maternal HLA antigens on occurrence of rheumatoid arthritis. Lancet 1993;341:200–2.[CrossRef][ISI][Medline]
  5. van der Horst-Bruinsma IE, Hazes JM, Schreuder GM et al. Influence of non-inherited maternal HLA-DR antigens on susceptibility to rheumatoid arthritis. Ann Rheum Dis 1998;57:672–5.[Abstract/Free Full Text]
  6. Barrera P, Balsa A, Alves H et al. Noninherited maternal antigens do not play a role in rheumatoid arthritis susceptibility in Europe. European Consortium on Rheumatoid Arthritis Families. Arthritis Rheum 2000;43:758–64.[CrossRef][ISI][Medline]
  7. Barrera P, Balsa A, Alves H et al. Noninherited maternal antigens do not increase the susceptibility for familial rheumatoid arthritis. European Consortium on Rheumatoid Arthritis Families (ECRAF). J Rheumatol 2001;28:968–74.[ISI][Medline]
  8. Arnett F, Edworthy SM, Bloch DA. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  9. Burlingham WJ, Grailer AP, Heisey DM et al. The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors. N Engl J Med 1998;339:1657–64.[Abstract/Free Full Text]
  10. Srivatsa B, Srivatsa S, Johnson KL, Samura O, Lee SL, Bianchi DW. Microchimerism of presumed fetal origin in thyroid specimens from women: a case–control study. Lancet 2001;358:2034–8.[CrossRef][ISI][Medline]
  11. Claas FH, Gijbels Y, van der Velden-de Munck J, van Rood JJ. Induction of B cell unresponsiveness to noninherited maternal HLA antigens during fetal life. Science 1988;241:1815–7.[ISI][Medline]
Submitted 19 March 2002; Accepted 28 June 2002





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