Apolipoprotein H ("apoH" for protein; "APOH" for gene), also referred to as .beta.2-glycoprotein I, is a single chain glycoprotein of 326 amino acids as determined directly from purified protein (Lozier, J., et al., Proc. Natl. Acad. Sci. USA 81:3640 (1984)) and was subsequently confirmed by deduced am ino acid sequence by cDNA cloning and sequencing (Kristensen, T., et al., FEBS Lett. 289:183 (1991); Mehdi, H., et al., Gene 108:293 (1991); and Steinkasserer, A., et al., Biochem. J. 277:387 (1991)). The CDNA sequence predicts 345 amino acids which include 19 hydrophobic signal peptide residues not present in the mature protein. The apoH protein shows extensive internal homology with five consecutive homologous segments of about 60 amino acids each. These segments are referred to variously as: GP-I domains (because they were first found in .beta.2-glycoprotein 1 as described by Davie, E. W., et al., Cold Spring Harbor Symposium on Quantitative Biology Vol. Li pp. 509-514 (1986), Sushi domains as described by Ichinose, A., et al., J. Biol. Chem. 265:1341 (1990), SCRs (short consensus repeats), or CCP (complement control protein) repeats (Kristensen, T., et al., Federation Proc. 46:2463 (1987)). Such domains or repeats are commonly found in a number of complement component proteins, as well as in non-complement proteins. Based upon the predicted structure of the apoH protein from its cDNA sequence, there are 22 cysteine residues in human apoH. These positions are conserved in the apoH protein of bovine (Kato, H., et al., Biochemistry 30:11687 (1991)), rat (Aoyama, Y., et al., Nucl. Acids Res. 17:6401 (1989)), mouse (Nonaka, M., et al., Genomics 13:1082 (1992)) and dog (Sellar, G. C., et al., Biochem. Biophys. Res. Commun. 191:1288 (1993)). The apoH proteins of these species also consist of 5 GP-I domains as in humans. The human APOH gene has been localized on chromosome 17q23-24 and is expressed primarily in the liver (Steinkasserer, A., et al., Biochem. J. 277:387 (1991).
ApoH has been implicated in a variety of physiologic pathways including lipoprotein metabolism as described by Kamboh, M. I., et al., Adv. Lipid Res. 1:9 (1991), coagulation as described by Roubey, R. A. S., et al., Blood 84:2854 (1994) and in the production of antiphospholipid autoantibodies ("aPA") as described by Schousboe, I., et al., Blood 66:1086 (1985). ApoH also binds to platelets, mitochondria, heparin, DNA, and anionic phospholipids and has been shown to be involved in the blood coagulation pathway, platelet aggregation, and prothrombinase activity of platelets. ApoH is considered to be a required cofactor for anionic phospholipid antigen binding by the aPA found in sera of many patients with systemic lupus erythematosus ("SLE") and primary antiphospholipid syndrome ("APS") (see, for example, McNeil, H. P., et al., Proc Natl. Acad. Sci USA 87:4120 (1990); Galli, M., et al., Lancet 335: 1544 (1990); and Jones, J. V., et al., J. Rheumatol. 19:1397 (1992)), but it does not seem to be required for the reactivity of aPA associated with infections as described by Hunt, J. E., et al., Lupus 1:75 (1992). These studies suggest that the apoH-phospholipid complex forms the antigen to which aPA are directed as described by Cabral, A. R., et al., J. Autoimmunity 5:787 (1992) and Matsuura, E., et al., J. Exp. Med. 179:457 (1994). Recently, however, the presence of autoantibodies to phospholipid-free apoH has been shown in patients with primary APS (see, for example, Arvieux, J., et al., Clin. Exp. Immunol. 95:310 (1994); Cabiedes, J., et al., J. Rheumatol. 22:1899 (1995); Cabral, A. R., et al., J. Rheumatol. 22:1894 (1995)).
Although the structural domains of apoH which bind to anionic phospholipids are unknown, studies have proposed that the expressed fifth domain of apoH carries the potential binding site for anionic phospholipids and anticardiolipin antibodies ("aCL") and it may be critical for lipid-protein interaction (see, Hunt, J. E., et al., Proc. Natl. Acad. Sci. USA 90:2141 (1993) and Hunt, J. E., et al., J. Immunol. 52:653 (1994). As stated above, the precise location of the apoH site which binds to anionic phospholipids has not been delineated prior to the present invention.
ApoH exhibits genetically determined structural polymorphism as revealed by isoelectric focusing ("IEF") and immunoblotting as described by Kamboh, M. I., et al., Am. J. Hum. Genet. 42:452 (1988). Three common alleles, APOH*1, APOH*2 and APOH*3 control the expression of six phenotypes, designated 1-1, 2-1, 2-2, 3-1, 3-2 and 3-3. A fourth allele, APOH*4, has been observed only in populations of African ancestry. Three IgG1.sub.K monoclonal antibodies ("mAb"), 3G9, 1B4, and 3D11, have been produced to human apoH as described by Wagenknecht, D. R., et al., Thromb. Haemost. 69:361 (1993). In contrast to mAb 3G9 and 1B4, which recognize free and phospholipid-bound apoH and which react with all apoH allelic isoforms, the mAb 3D11 recognizes only one form of the APOH*3 allele, called APOH*3.sup.W (allele called APOH*3.sup.B not recognized), that does not bind to anionic phospholipids (see, Kamboh, M. I., et al., Hum. Genet. 95:385 (1995)). Therefore, plasma samples reacting with mAb 3D11 could be either homozygous H3.sup.W /H3.sup.W or heterozygous H3.sup.W /H3.sup.B.
Systemic lupus erythematosus ("SLE") is a chronic inflammatory disease affecting the connective tissues with a pathogenesis believed to involve abnormalities of the immune system. SLE is associated with the generation of numerous autoantibodies directed against various cell components. This autoimmune disorder affects women more than it does men. Overall reported prevalence of SLE is approximately 50-75 per 100,000. The incidence of SLE peaks during the ages of 15 and 45 and this excess is attributable to females, who outnumber males by 5:1. The female to male frequency ratio is particularly striking between ages 15 and 45, in that it reaches 12-15:1.
Antiphospholipid autoantibodies are a heterogeneous group of autoantibodies including most commonly a lupus anticoagulant ("LAC") and anticardiolipin antibodies ("aCL") which are directed against negatively charged phospholipids. The prevalence of aCL in the general population have been reported to be as low as 1.7%. In contrast, the frequency of antiphospholipid autoantibodies (including aCL and LAC) in SLE patients varies between 20 and 60%. Although a high frequency of patients with SLE may have these autoantibodies, only approximately one-third will have a clinical manifestation associated with the presence of these autoantibodies, including a thrombotic event, fetal loss, or thrombocytopenia. The presence of antiphospholipid autoantibodies in SLE patients has been associated with recurrent deep vein thrombosis and other thrombotic complications, including pulmonary, renal, and retinal thrombosis, as well as Budd-Chiari syndrome. In addition, associations between antiphospholipid autoantibodies and arterial thrombosis including cerebral, retinal and peripheral artery have been reported. Recurrent fetal losses, usually occurring in the second and third trimester, felt to be due in part to thrombosis of the placental vessels and subsequent infarction resulting in placental insufficiency and ultimately fetal loss have also frequently been reported in association with antiphospholipid autoantibodies.
Due to the critical nature of the SLE disease, there is an urgent need for better diagnostic methods for early detection of the disease, and screening methods to detect genetic carriers of the disease. Additionally, there is a critical need for an effective treatment, and even more preferably, a cure for the underlying genetic defect. There further remains a need for means to understand the underlying mechanism for the production of antiphospholipid autoantibodies in SLE patients. All these needs are addressed by the present determination of the structural mutations in the APOH gene which affect binding of negatively charged phospholipids and the determination that one of those mutations provides protection from the occurrence of SLE.
The disclosures of all publications referenced above and throughout this application below are hereby incorporated in their entirety herein by reference.
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