Centre for Rheumatology Research, Bloomsbury Rheumatology Unit, Department of Medicine, University College London, UK
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Abstract |
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Methods. Three groups of patients were tested for the presence of IgG, IgM and IgA anticardiolipin (aCL), antiphosphatidylinositol (aPI), antiphosphatidylglycerol (aPG) and antiphosphatidylserine (aPS) antibodies: (i) patients with primary APS (PAPS); (ii) patients with SLE and secondary APS; and (iii) patients with SLE without APS. First-degree relatives and spouses of patients with SLE/APS were also tested for circulating aPL.
Results. IgG aPL were particularly prevalent in patients with PAPS. IgG aPI and aCL were more prevalent in patients with PAPS than the IgM equivalents (P < 0.0001). Notably, none of the patients with PAPS had IgA aPL. A significantly higher number of relatives of patients with SLE/APS possessed IgG aPL than the normal controls. Except for aPG (P < 0.03), the prevalence of these antibodies in the relatives was not significantly different from patients with SLE/APS. The relatives also had significantly higher prevalence of IgG aPI, aPS and aCL antibodies than IgM aPL antibodies. In contrast, the prevalence of IgG aPL in the spouses was no different than in the healthy controls.
Conclusions. Genetic factors, shared by patients and their relatives, seem to have some effect on the prevalence of aPL in the subjects studied, while environmental factors shared by spouses appear to have no influence.
KEY WORDS: Systemic lupus erythematosus, Antiphospholipid syndrome/antibodies, First-degree relatives, Spouses.
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Introduction |
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Antibodies to phospholipids other than cardiolipin have been detected in patients with APS and SLE, some associated with certain clinical features [68]. Antiphospholipid antibodies (aPL) are also present in the normal population, unassociated with any disease [9]. The presence of aPL in the normal population indicates that some trigger factor must be present to induce disease.
Variability in the prevalence of SLE in some ethnic groups living in different parts of the world, and the presence of disease within families, suggest that both environmental and genetic factors may trigger SLE induction in susceptible individuals or induce autoantibody production [1012]. The question may be raised as to whether production of autoantibodies alone is induced through prevailing conditions in the local environment or individual genetic constitution. The trend has been to focus on aCL in the characterization of PAPS and SLE/APS. Other anionic phospholipids to which antibodies may be directed are not routinely used in laboratory tests.
We have determined the presence of antibodies to the anionic phospholipids, i.e. aCL, antiphosphatidylinositol (aPI), antiphosphatidylglycerol (aPG), and antiphosphatidylserine (aPS) first, in patients with PAPS, SLE with secondary APS (SLE/APS) and SLE alone, and secondly in the spouses and first-degree relatives of patients with SLE/APS. Our purpose was to determine whether environmental (as shared by the spouses) or genetic (as shared by the relatives) factors influence the prevalence of aPL antibodies.
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Patients and methods |
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Relatives and spouses
For the second part of the study, 43 healthy individuals were studied, 18 of whom were spouses and 25 were first-degree relatives of 14 patients with SLE/APS. Fifty healthy individuals (samples supplied by the National Blood Service, Colindale, UK) were included as normal controls (Table 1).
Methods
aCL, aPI, aPG and aPS enzyme-linked immunosorbent assay (ELISA)
Half the wells of microtitre ELISA plates (Polysorp, Nunc, Life Technologies, Paisley, UK) were coated with each of the following anionic phospholipids at 50 µg/ml: cardiolipin (CL), phosphatidylinositol (PI), phosphatidylglycerol (PG) and phosphatidylserine (PS) (all from Sigma, St. Louis, MO, USA) in ethanol, the other half with ethanol alone, and air dried overnight at 4°C. After blocking with 10% fetal calf serum (Sigma) in phosphate-buffered saline (10% FCS/PBS) for 1 h at 37°C and three washes with PBS, serum diluted 1:100 in 10% FCS/PBS was added in duplicate to both PL-coated and uncoated halves of the plates. After incubation for 1.5 h at 37°C and three washes with PBS, alkaline phosphatase-conjugated goat antihuman IgG, IgM or IgA (Sigma) was added at 1:1000 dilution, for 1 h at 37°C. Colour was developed by adding dinitrophenyl phosphate [1 mg/ml (Sigma) solution containing
1 M MgCl2 in bicarbonate buffer (BIC), pH9.6], after three washes with PBS and two with BIC. The plates were incubated at 37°C and read after 1 h at 405 nm (with reference 490 nm). Background values were obtained from wells containing no antigen, and their absorbances subtracted from all sample readings. Positive [IgG aCL = 126 IgG phospholipid units (GPLu); IgM aCL = 88 IgM phospholipid units (MPLu)] and negative control samples were included on each plate. The results were expressed as a percentage of the positive control: optical density (OD) (sample)/OD (positive control) x 100. Values >3 standard deviations (S.D.) above the mean of 50 healthy controls were deemed to be positive.
Statistical analysis
Statistical analyses were performed using Biomedical Statistics application software (Italian version). Categorical analysis was performed by the chi-square test.
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Results |
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Relatives and spouses
Table 2 shows the prevalence (percentage of subjects with aPL levels above the mean + 3 S.D. of 50 normal controls) of IgG/M/A aPI, aCL, aPS and aPG antibodies in the first-degree relatives and spouses of patients with SLE/APS.
Figure 4 illustrates the prevalence of IgG aPL in patients with SLE/APS, their first-degree relatives and normal controls. There was no significant difference between the relatives and patients with SLE/APS regarding the prevalence of aPI, aCL and aPS. However, a difference occurred in aPG (P < 0.03). There was a significant difference between the relatives and normal controls (P < 0.0001 for aPI and aCL; P = 0.001 for aPS and aPG).
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Discussion |
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We have shown a high prevalence of IgG aPL in patients with PAPS. This is significantly higher than in patients with SLE/APS and SLE (Table 2, Fig. 1
). Although some differences were seen, there was no significant difference between the prevalence of IgG and IgM aPL in the patient groups (Table 2
). Interestingly, none of the patients with PAPS had IgA aPL.
We have also shown the presence of IgG aPL in the first-degree relatives of patients with SLE/APS. This was significantly different from the prevalence of IgG aPL in the healthy normal controls and generally not significantly different from patients with SLE/APS (Fig. 4). Although these individuals were considered normal, they have displayed some type of susceptibility to autoantibody production. It is of note that the genetic contribution to autoantibody production may vary between patients with PAPS, SLE/APS and SLE, and only the relatives and spouses of patients with SLE/APS were studied. As reviewed elsewhere, relatives of patients with autoimmune syndromes often have evidence of autoantibody production without evidence of clinical disease [15]. Mackworth-Young et al. found eight out of 101 (7.9%) relatives had aCL [16]. More recently, Goldberg et al. reported IgG aCL in 23% of relatives of aCL-positive patients [17].
The spouses of patients with SLE/APS showed a prevalence of IgG aPL not significantly different from normal controls. The prevalence of IgG aPL in the patients with SLE/APS was significantly different from their spouses (Fig. 5). These data indicate that although sharing environmental conditions with the patients with SLE/APS, the spouses were not predisposed to autoantibody production.
The prevalence of IgG aPL in the first-degree relatives of patients with SLE/APS was higher than in the spouses of patients with SLE/APS, suggesting that genetic predisposition to antibody production is likely to be more important than a shared environmental influence.
It was notable that IgA aPI were present in 42% of patients with SLE/APS, 19% of patients with SLE but 0% of patients with PAPS. In patients with SLE/APS and SLE these figures were the highest incidence of any of the IgG/M/A aPL tested. This observation may be interesting if there is some, as yet undiscovered, link between the presence of IgA aPI and SLE. The question may be raised as to whether SLE alone, or with APS as a secondary syndrome, has some influence on IgA aPI which is non-existent when APS is a primary syndrome. Further work would involve the determination of the clinical significance of IgA aPI in patients with SLE/APS and SLE, compared with patients with PAPS. The results underline the heterogeneity of the aPL detectable in human sera.
There is extensive literature exploring the genetic basis of SLE [1519]. Amongst the salient features are the fact that there is a 25% concordance rate for SLE amongst monozygotic twins but only 23% amongst dizygotic twins [19]. There have been attempts to link the inheritance of SLE to both major histocompatibility complex (MHC) and non-MHC genes. Genes likely to be involved in the development of SLE include those which regulate immune complex disposal, B cell signalling, regulation of apoptosis, antigen processing, T cell receptor and immunoglobulin structure. These topics have been reviewed in detail recently [20].
Many groups have reported on the prevalence of aPL and their association with clinical manifestations [6, 8, 21, 22]. The major comparison which can be drawn between these and our study is the high incidence of IgG aPL in the groups studied. Bertolaccini et al. reported a prevalence of IgG/M/A aPS and aPI in their patients with SLE/APS (LA/aCL positive) that was very similar to our own findings [21].
Our data show clearly that first-degree relatives of patients with SLE/APS have significantly higher levels of aPL than normal controls, indicating that genetic factors shared by patients and their relatives seem to have some effects on the prevalence of aPL in the subjects studied. However, the presence of autoantibodies does not result in overt manifestation of disease, suggesting that environmental triggers are required for disease development. The spouses of patients with SLE/APS do not have significantly higher levels of aPL than normal controls. The environmental factors alone, shared by patients and their spouses, appear to have no influence on the prevalence of aPL.
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Notes |
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References |
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