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Rheumatology 1999; 38: 721-723
© 1999 British Society for Rheumatology

Correlation between ß2-glycoprotein I valine/leucine247 polymorphism and anti-ß2-glycoprotein I antibodies in patients with primary antiphospholipid syndrome

T. Atsumi, A. Tsutsumi, O. Amengual1, M. A. Khamashta1, G. R. V. Hughes1, Y. Miyoshi, K. Ichikawa and T. Koike

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.


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. ß2-Glycoprotein I (ß2GPI) exon 7 polymorphism leads to a valine–leucine amino acid exchange at position 247 in domain 5 of ß2GPI, between the phospholipid binding site and the cryptic site of the epitopes for anti-ß2GPI antibodies. Therefore, position 247 polymorphism may affect the conformational change of ß2GPI and the exposure of the epitopes for anticardiolipin antibodies (aCL) (= anti-ß2GPI antibodies). In this study we analysed the genetic polymorphism of ß2GPI in a British cohort of well-defined antiphospholipid syndrome (APS) patients.

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 1.03–6.13, P=0.0396) or in primary APS patients without anti-ß2GPI antibodies (OR=2.92, 95% CI 1.16–7.39, 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.


   Introduction
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 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Antiphospholipid antibodies (aPL), detected by immunological or coagulation methods, are pertinent as markers of the antiphospholipid syndrome (APS), an acquired thrombophilic disorder characterized by venous and arterial thrombosis [1]. In 1990, it was reported that most of the anticardiolipin antibodies (aCL) from patients with APS bound to cardiolipin in the presence of an `aCL cofactor', namely ß2-glycoprotein I (ß2GPI), also called apolipoprotein H [2–4]. While the phospholipid binding site is present within the fifth domain of ß2GPI [5], the epitope for representative aCL binding seems to be located in the fourth domain [6]. Matsuura et al. [7] and Roubey et al. [8] showed that aCL recognize ß2GPI in the absence of cardiolipin when ß2GPI was coated onto polystyrene plates where oxygen was introduced by radiation, thereby implying that aCL can bind not only cardiolipin–ß2GPI complex, but also ß2GPI alone. Thus, it has been hypothesized that the interaction of ß2GPI with phospholipids or an oxidized plastic surface allows exposure of cryptic epitopes, which are consequently recognized by aCL (= anti-ß2GPI antibodies) [7].

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.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
The study population comprised 88 Caucasoid patients with APS [57 with primary APS and 31 with APS secondary to systemic lupus erythematosus (SLE)] who fulfilled the proposed criteria for the APS [1, 16] [median age 41 yr (16–67); female:male 78:10].

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 (20–55), female:male 26:13] were studied.

ß2GPI gene polymorphisms
Genomic DNA was extracted from peripheral white blood cells using a standard phenol–chloroform 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 {chi}2 test.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Anti-ß2GPI antibodies were present in 28 of 57 primary APS patients (49%) and in 19 of 31 secondary APS patients (61%). Each allele frequency of three examined ß2GPI polymorphisms is shown in Table 1Go. There were no differences in the frequencies of Tsp509I/BstBI polymorphisms between controls and any group of patients. In contrast, the allele containing the RsaI site was significantly more frequent in primary APS patients with anti-ß2GPI antibodies than in the controls (OR=2.51, 95% CI 1.03–6.13, P=0.0396) or in primary APS patients without anti-ß2GPI antibodies (OR=2.92, 95% CI 1.16–7.39, P=0.0204). This tendency was not evident in the secondary APS group.


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  		TABLE 1. 
 

   Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
In this study, the allele frequency of valine247 was significantly increased in those with anti-ß2GPI antibodies, when we analysed patients with primary APS.

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.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1.  Hughes GRV. The antiphospholipid syndrome: ten years on. Lancet 1993;342:341–4.[Web of Science][Medline]
  2.  Galli M, Comfurius P, Maassen C et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:952–3.
  3.  Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990;336:177–8.[Web of Science][Medline]
  4.  McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that induces a lipid-binding inhibitor of coagulation: ß2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990;87:4120–4.[Abstract/Free Full Text]
  5.  Hunt JE, Krilis S. The fifth domain of ß2-glycoprotein I contains a phospholipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol 1994;152:653–9.[Abstract]
  6.  Igarashi M, Matsuura E, Igarashi Y et al. Human ß2-glycoprotein I as an anticardiolipin cofactor determined using deleted mutants expressed by a Baculovirus system. Blood 1996;87:3262–70.[Abstract/Free Full Text]
  7.  Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize ß2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457–62.[Abstract/Free Full Text]
  8.  Roubey RAS, Eisenberg RA, Harper MF, Winfield JB. `Anticardiolipin' autoantibodies recognize ß2-glycoprotein I in the absence of phospholipid. J Immunol 1995; 154:954–60.[Abstract]
  9.  Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ. Complete nucleotide and deduced amino acid sequence of human ß2-glycoprotein I. Biochem J 1991;277:387–91.
  10. Kamboh MI, Ferrell RE, Sepehrnia B. Genetic studies of human apolipoprotein. IV. Structural heterogeneity of apolipoprotein H (ß2-glycoprotein I). Am J Hum Genet 1988;42:452–7.[Web of Science][Medline]
  11. Kamboh MI, Wagenknecht DR, McIntyre JA. Heterogeneity of the apolipoprotein H*3 allele and its role in affecting the binding of apolipoprotein H (ß2-glycoprotein I) to anionic phospholipids. Hum Genet 1995;95:385–8.[Web of Science][Medline]
  12. Steinkasserer A, Dörner C, Würzner R, Sim RB. Human ß2-glycoprotein I: molecular analysis of DNA and amino acid polymorphism. Hum Genet 1993;91:401–2.[Web of Science][Medline]
  13. Sanghera DK, Kristensen T, Hamman RF, Kamboh MI. Molecular basis of the apolipoprotein H (ß2-glycoprotein I) protein polymorphism. Hum Genet 1997;100:57–62.[Web of Science][Medline]
  14. Matsuura E, Igarashi M, Igarashi Y et al. Molecular studies on phospholipid-binding sites and cryptic epitopes appearing on ß2-glycoprotein I structure recognized by anticardiolipin antibodies. Lupus 1995;4(suppl. 1):S13–7.
  15. Ichikawa K, Khamashta M, Koike T, Matsuura E, Hughes GRV. Reactivity of monoclonal anticardiolipin antibodies from patients with the antiphospholipid syndrome to ß2-glycoprotein I. Arthritis Rheum 1994;37:1453–61.[Web of Science][Medline]
  16. Harris EN. Antiphospholipid antibodies. Br J Haematol 1990;74:1–9.[Web of Science][Medline]
  17. Amengual O, Atsumi T, Khamashta M, Koike T, Hughes GRV. Specificity of ELISA for antibody to ß2-glycoprotein I in patients with antiphospholipid syndrome. Br J Rheumatol 1996;35:1239–43.[Abstract/Free Full Text]
Submitted 15 December 1998; revised version accepted 5 March 1999.
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