Department of Medicine II, Hokkaido University School of Medicine, Sapporo, Japan and
1 Lupus Research Unit, The Rayne Institute, St Thomas' Hospital, London, UK
Correspondence to:
A. Tsutsumi, Department of Medicine II, Hokkaido University School of Medicine, N15 W7, Kitaku, Sapporo 060, Japan.
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Abstract |
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Methods. This study comprised 88 Caucasoid patients with APS [57 with primary APS and 31 with APS secondary to systemic lupus erythematosus (SLE)]. Polymorphism assignment was determined by polymerase chain reaction followed by allele-specific restriction enzyme digestion (PCR-RFLP). The presence of anti-ß2GPI antibodies was detected by ELISA utilizing irradiated ELISA plates.
Results and conclusions. Anti-ß2GPI antibodies were present in 28 of 57 primary APS patients (49%) and in 19 of 31 secondary APS patients (61%). The allele containing valine247 was significantly more frequent in primary APS patients with anti-ß2GPI antibodies than in controls (OR=2.51, 95% CI 1.036.13, P=0.0396) or in primary APS patients without anti-ß2GPI antibodies (OR=2.92, 95% CI 1.167.39, P=0.0204). This tendency was not found in the secondary APS group. In conclusion, the ß2GPI polymorphism, valine/leucine247 , is correlated with anti-ß2GPI antibody production in patients with primary APS, and valine247 may be important in the formation of ß2GPI antigenicity.
KEY WORDS: Thrombosis, Genotype, Systemic lupus erythematosus, Anticardiolipin antibody, Conformational change.
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Introduction |
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The human ß2GPI gene is localized on chromosome 17q23-qter [9]. Protein structural polymorphisms, APOH*1, APOH*2, APOH*3W , APOH*3B and APOH*4, are detectable by isoelectric focusing and immunoblotting [10, 11]. Recently, the molecular basis of ß2GPI polymorphisms was also defined and three major polymorphisms (RsaI, Tsp509I and BstBI sites) have been identified [12, 13]. The significance of antigen polymorphisms in the production of autoantibodies or in the development of autoimmune diseases is not well understood. It may be that amino acid differences lead to differences in antigenic epitopes of a given protein. In particular, ß2GPI is thought to undergo conformational alteration upon interaction with phospholipids [14]. Amino acid differences of ß2GPI may affect the nature of conformational alterations induced by interaction with phospholipids. Therefore, polymorphism at or near the phospholipid binding site or the antigenic site may affect aCL production and the development of APS. ß2GPI RsaI, Tsp509I and BstBI genetic polymorphism sites locate on exon 7, 3 and 8 of the ß2GPI gene, respectively. Among these polymorphisms, ß2GPI RsaI polymorphism leads to a valine (RsaI site present)leucine (RsaI site not present) amino acid exchange at position 247 in domain 5 of ß2GPI, between phospholipid binding sites [5] and the potential site of the epitopes for anti-ß2GPI antibodies [6, 15]. Therefore, the position 247 polymorphism, or polymorphisms in linkage equilibrium, may affect the conformational change of ß2GPI and the exposure of the epitopes for aCL.
In this study, we analysed the genetic polymorphisms of ß2GPI in a British cohort of well-defined APS patients.
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Patients and methods |
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Among the APS patient group, 47 (53%) had a history of arterial thrombosis, 46 (52%) venous thrombosis, 31/78 (40%) recurrent pregnancy loss and 22 (25%) thrombocytopenia (some patients had more than one manifestation).
As a control group, 39 Caucasoid healthy individuals with no history of autoimmune, thrombotic or notable infectious diseases [median age 31 yr (2055), female:male 26:13] were studied.
ß2GPI gene polymorphisms
Genomic DNA was extracted from peripheral white blood cells using a standard phenolchloroform extraction procedure. Polymorphism assignment was determined by polymerase chain reaction followed by allele-specific restriction enzyme digestion (PCR-RFLP) using RsaI (Promega, Southampton, UK), Tsp509I (New England Biolabs Inc., Beverly, MA, USA) and BstBI (New England Biolabs Inc.), as described previously [12, 13].
Anti-ß2GPI ELISA
Serum samples were collected simultaneously and kept at -70°C. These patients were further subcategorized on the basis of the presence of anti-ß2GPI. The presence of anti-ß2GPI antibodies was detected by ELISA utilizing irradiated ELISA plates as described previously [17].
Statistics
Comparisons were expressed as odds ratios (OR) and P values determined by 2 test.
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Results |
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Discussion |
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Patients with secondary APS have other relevant autoantibodies and diseases, and the immunological dysfunctions underlying these conditions are more complex and multifactorial. As patients with secondary APS constitute a heterogeneous population, it is difficult to identify the role of a single factor. Therefore, patients with primary APS only may provide a more reliable study group for determining the effects of ß2GPI polymorphism in anti-ß2GPI antibody production, unless the polymorphisms are absolutely outstanding factors to overcome the clinical heterogeneity of secondary APS.
In our study, the presence of ß2GPI (valine247 ) correlated with anti-ß2GPI antibody production in patients with primary APS, although it is not a strong association as it was not confirmed if the multiple comparison was applied (data not shown). Individuals bearing this polymorphism might be prone to generate anti-ß2GPI antibodies. As RsaI polymorphism is located in the fifth domain, near the phospholipid binding site, and the potential site of anti-ß2GPI recognition, this polymorphism may be important in the formation of ß2GPI antigenicity. On the other hand, yet to be undefined polymorphisms in linkage disequilibrium with the RsaI polymorphism may be responsible for the induction of anti-ß2GPI antibodies.
We conclude that a ß2GPI RsaI polymorphism, valine/leucine247 , potentially correlates with anti-ß2GPI antibody production in patients with primary APS. However, ß2GPI valine247 is commonly present in apparently healthy populations, and the relatively small number of primary APS patients in this study (n=57) makes it difficult to draw a definite conclusion from this study. This observation needs to be confirmed by other studies with a larger number of primary APS patients. Furthermore, the correlation was not found in patients with secondary APS. The roles of other genetic and environmental variables should also be taken into account when considering anti-ß2GPI antibody production and APS development.
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References |
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