SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Primary antiphospholipid syndrome (APS) is formally classified by the presence of antiphospholipid antibodies, recurrent thrombosis, and/or pregnancy morbidity in the absence of any underlying full-blown systemic autoimmune disease. However, systemic manifestations in patients with primary APS have been recently reported, as has the presence of serologic markers in common with systemic lupus erythematosus (SLE). In spite of similarities between the 2 diseases, only a minority of cases of primary APS evolve into full-blown SLE, even after a long followup period. The aim of this study was to investigate whether the analysis of SLE susceptibility genes may provide at least a partial explanation for such a discrepancy.

Methods

One hundred thirty-three patients with primary APS classified according to the Sydney criteria and 468 healthy control subjects from the same geographic area were recruited. We genotyped 3 single-nucleotide polymorphisms (SNPs) in IRF5 (rs2004640, rs2070197, and rs10954213), 4 SNPs in STAT4 (rs1467199, rs3821236, rs3024866, and rs7574865), 2 SNPs in BANK1 (rs10516487 and rs3733197), and 1 SNP in BLK (rs2736340).

Results

STAT4 and BLK displayed a strong genetic association with primary APS (for rs7574865, odds ratio [OR] 2.19, P = 5.17 × 10−7; for rs2736340, OR 2.06, P = 1.78 × 10−6), while a weak association with IRF5 and no association with BANK1 were observed.

Conclusion

The presence of a strong genetic association with only a few SLE susceptibility genes and the absence of a more complex gene association may contribute to the lack of cases of full-blown SLE developing in patients with primary APS, in spite of the clinical and serologic similarities between SLE and primary APS.

Antiphospholipid syndrome (APS) is characterized by the persistent positivity of antibodies against phospholipid-binding proteins in the presence of recurrent arterial/venous thrombotic events and/or pregnancy morbidity. Although APS was originally described in patients with systemic lupus erythematosus (SLE), more than 50% of cases of APS are now recognized in patients without any underlying full-blown systemic autoimmune disease (e.g., primary APS) (1).

Understanding of the clinical spectrum of primary APS has evolved in the past years, with a gradual recognition that it is not merely an autoimmune thrombophilic disorder but, rather, a more systemic disease having several clinical manifestations in common with SLE (1). The similarity of the 2 diseases is further supported by additional biologic findings, i.e., 1) the role of complement activation and, at least for some manifestations, the involvement of inflammatory processes in APS pathogenesis (1), 2) the recent demonstration of the occurrence of antichromatin (i.e., antinucleosome) autoantibodies at medium/high titers in a large proportion of patients with primary APS (2), and 3) the fact that antiphospholipid antibodies (aPL) or false-positive results of a biologic test for syphilis due to aPL may represent the first marker of autoimmunity in some patients with SLE, even years before development of full-blown disease (3). Nevertheless, the 2 diseases are formally distinct clinical entities, and more importantly, a relatively small proportion of cases of primary APS evolve into full-blown SLE, even after a long followup period (1).

Recently, several genes involved in SLE susceptibility have been identified and confirmed. Of those, particularly IRF5 (4–7) and STAT4 (5, 8–10) have been confirmed in several studies and are clearly associated with lupus. For BLK and BANK1, replication studies are under way (11, 12). In lupus, IRF5 displays an allelic odds ratio (OR) of 1.67, while STAT4 has an OR of 1.54, BLK an OR of 1.39, and BANK1 an OR of 1.38. In a study of the genetic association of IRF5 with SLE in Mexicans (13), the prevalence of homozygosity for the risk haplotype among lupus patients was high, with a high frequency of the risk haplotype in American Indian and Mestizo populations (20% and 31%, respectively) as compared with Europeans (9%) (13). The homozygous risk genotype could confer an OR of nearly 10. Therefore, because the frequencies of risk variants can vary across populations, the frequencies may also vary across diseases that are clinically closely related to SLE, such as primary APS. Based on this, we speculated that the analysis of SLE susceptibility genes may provide insight into both the similarity and the incomplete overlapping between the 2 diseases.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients with primary APS.

The study group comprised 133 patients with primary APS who were recruited from the University of Milan and University of Brescia clinics; all patients provided informed consent, and the study was approved by the institutional review board. Only patients with primary APS classified according to the Sydney criteria were included (14). Antiphospholipid antibodies were detected as described previously (2). A total of 468 healthy control subjects from the same geographic area were also included (2).

Methods.

Genotyping was performed using TaqMan predesigned single-nucleotide polymorphism (SNP) genotyping assays (Applied Biosystems, Foster City, CA). All patients and control subjects were European Italians. We genotyped 3 SNPs in IRF5: rs2004640, rs2070197, and rs10954213. These SNPs are known to be part of the risk haplotype of IRF5 in SLE (4, 6). We also genotyped 4 SNPs in STAT4 (rs1467199, rs3821236, rs3024866, and rs7574865), 2 SNPs in BANK1 (rs10516487 and rs3733197), and 1 SNP in BLK (rs2736340). The SNP in BLK is a proxy for rs13277113 (D′ = 0.95, r2 = 0.87), previously described as a risk factor for SLE (11).

Statistical analysis.

Statistical analyses were performed using UNPHASED version 3.0.13 software (Frank Dudbridge, MRC Biostatistics Unit, Cambridge, UK), and ORs were calculated using PLINK version 1.05 software (free Software Foundation, Boston, MA). Power was calculated with Quanto version 1.2.3 software (Jim Gauderman and John Morrison, University of Southern California, Los Angeles, CA) at a level of significance of α = 0.05, under an additive model for the OR previously identified for SLE (4, 11, 12, 14) and the allelic frequencies in control subjects given an approximate disease prevalence of 0.0005.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

As shown in Table 1, 3 of 4 STAT4 SNPs (rs3821236, rs3024866, and rs7574865) and the BLK SNP displayed a significant association with primary APS, with ORs ranging from 1.21 to 2.19. We also observed a weak association between primary APS and 2 IRF5 SNPs (rs2070197 and rs10954213). In contrast, no association at all was observed with any of the BANK1 SNPs (Table 1). Both associations for IRF5 became nonsignificant after Bonferroni correction.

Table 1. Genotype and allele frequencies of STAT4, BLK, BANK1, and IRF5 in patients with primary APS*
Gene/SNPGenotype frequency, no. (%)χ2PAllele frequency, no. (%)χ2POR (95% CI)Power, %
  • *

    See Patients and Methods for descriptions of the manner in which allelic odds ratios (ORs) and power were calculated. APS = antiphospholipid syndrome; SNP = single-nucleotide polymorphism; 95% CI = 95% confidence interval.

  • Not significant after Bonferroni adjustment.

STAT4           
 rs1467199GGCGCC  GC    
  Cases (n = 108)7 (6)47 (44)54 (50)2.670.262361 (28)185 (72)1.340.24691.21 (0.87–1.70)5.4
  Controls (n = 422)29 (7)148 (35)245 (58)  206 (24)638 (76)    
 rs3821236AAAGGG  AG    
  Cases (n = 127)10 (8)56 (44)61 (48)14.930.00057376 (30)178 (70)14.660.00012851.86 (1.35–2.56)99.4
  Controls (n = 410)17 (4)119 (29)274 (67)  153 (19)667 (81)    
 rs3024866CCCTTT  CT    
  Cases (n = 129)15 (12)55 (43)59 (46)4.890.086685 (33)173 (67)5.120.023551.41 (1.04–1.92)92.3
  Controls (n = 410)31 (8)149 (36)230 (56)  211 (26)609 (74)    
 rs7574865TTGTGG  TG    
  Cases (n = 121)13 (11)62 (51)46 (38)26.311.93 × 10−788 (36)154 (64)25.205.17 × 10−72.19 (1.60–2.99)100.0
  Controls (n = 414)22 (5)127 (31)265 (64)  171 (21)657 (79)    
BLK           
 rs2736340TTCTCC  TC    
  Cases (n = 130)22 (17)56 (43)52 (40)24.514.74 × 10−6100 (39)160 (62)22.811.78 × 10−62.06 (1.52–2.77)60.7
  Controls (n = 395)20 (5)144 (36)231 (58)  184 (23)606 (77)    
BANK1           
 rs3733197GGGAAA  GA    
  Cases (n = 127)57 (45)63 (50)7 (6)1.810.403771177 (70)77 (30)0.420.51360.90 (0.66–1.22)61.5
  Controls (n = 434)192 (44)202 (47)40 (9)  586 (68)282 (32)    
 rs10516487GGGAAA  GA    
  Cases (n = 124)62 (50)54 (44)8 (6)1.100.57441178 (72)70 (28)0.620.43090.88 (0.64–1.20)66.8
  Controls (n = 433)207 (48)185 (43)41 (9)  599 (69)267 (31)    
IRF5           
 rs2004640TTGTGG  TG    
  Cases (n = 122)35 (29)61 (50)26 (21)1.180.55225131 (54)113 (46)0.760.38270.88 (0.66–1.17)48.9
  Controls (n = 401)109 (27)187 (47)105 (26)  405 (50)397 (50)    
 rs2070197CCCTTT  CT    
  Cases (n = 124)4 (3)21 (17)99 (80)7.740.02084629 (12)219 (88)4.950.0261.69 (1.06–2.69)96.6
  Controls (n = 420)2 (0.5)57 (14)361 (86)  61 (7)779 (93)    
 rs10954213AAAGGG  AG    
  Cases (n = 120)50 (42)57 (48)13 (11)4.400.11059157 (65)83 (35)3.870.049031.27 (1.01–1.46)44.9
  Controls (n = 397)140 (35)183 (46)74 (19)  463 (58)331 (42)    

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All of the investigated SNPs have previously been shown to strongly associate with SLE in European and non-European populations, and some are known to be functional. The STAT4 SNPs rs3821236 and rs7574865 confer susceptibility to SLE independently, and the risk varies in North European and South European populations (14). SNP rs1467199, which is located close to STAT1, was the only SNP shown to be associated with SLE in a Mexican pediatric population (14).

When we began the study, we were aware that the number of patients with primary APS was small and most likely did not provide enough power to identify a genetic association with any of the polymorphisms. For this reason, we were not surprised when we observed a weak association of IRF5 with primary APS and a lack of association with BANK1 (Table 1). In contrast, we were surprised to observe a strong genetic association of STAT4 and BLK with primary APS (Table 1). Indeed, the OR for the genetic association of the STAT4 risk SNP rs7574865 (2.19, P = 5.17 × 10−7) was even higher than those observed for SLE; similarly, genotypic and haplotype associations we identified for STAT4 were found to be comparable with those observed in SLE (8, 9, 14). BLK also exhibited a strong association with primary APS (OR 2.06, P = 1.78 × 10−6).

The weak genetic association with IRF5 is worth mention. IRF5 is one of the strongest and most consistent genetic associations described for SLE, after HLA. Here, we observed a weak association with 2 SNPs, one of which is the tag SNP for the risk haplotype of IRF5 in lupus, rs2070197. The other is a functional SNP, the 3′-untranslated region polyadenylation site SNP rs10954213. This SNP is clearly the main SNP responsible for the high IRF5 expression correlating with allele A in the SLE risk haplotype. The genetic association of this SNP alone with SLE is not strong. We believe that the genetic association between primary APS and IRF5 cannot be considered true until we replicate it in a new set of European patients with primary APS and control subjects, particularly because the IRF5 SNP most strongly associated with lupus across most studies, rs2004640, did not show any association in primary APS, and the associations with the other IRF5 SNPs became nonsignificant after Bonferroni correction.

Furthermore, before initiating this study, we analyzed the statistical power of the sample according to the ORs identified for SLE. Interestingly, although the sample had a power of 96.6% to detect IRF5 SNP rs2070197, this genetic association was not observed in primary APS. Alternatively, the power to detect BLK was only 60.7% considering the ORs identified for lupus, but this genetic association with primary APS was strong. This supports primarily our conclusion that IRF5 is not associated with primary APS, while the result for BANK1 remains inconclusive until new samples are genotyped.

To our knowledge, this investigation is the first to analyze risk alleles and genotypes of SLE-associated genes in a well-studied set of patients with primary APS. Clearly, STAT4 and BLK could have an important role in the development of this disease. Conversely, the lack of association with BANK1 and IRF5 could explain in part why these patients show a limited clinical presentation and suggests differences in the pathways leading to disease.

Interestingly, our group and other investigators have observed an additive effect between STAT4 SNPs and IRF5 SNPs that increased the risk of lupus (9, 14). Thus, it appears that the lack of association of IRF5 with primary APS could imply that at least 1 genetic risk factor for lupus is not generally observed in patients with primary APS. It was recently shown that STAT4 SNP rs7574865 is associated with increased sensitivity to interferon-α (IFNα) signaling and lower levels of IFNα in serum, while the opposite was observed for the IRF5 risk genotypes (15). At present, it is unknown whether abnormal plasma levels of IFNα can be identified in patients with primary APS, or whether cells from patients with primary APS show an IFN signature.

In our set of patients with primary APS, we did not observe an association with BANK1, and therefore, as in the case of STAT4 and IRF5, a second genetic factor for the development of SLE was not observed in patients with primary APS in general. Of course, a few patients in our cohort may carry the risk alleles for IRF5 and BANK1; in this regard, followup of patients carrying the risk genotypes of all 4 genes might be of interest. Further studies involving larger cohorts should be performed before arriving at a definitive conclusion regarding the implications of the analyzed genes in primary APS susceptibility.

We have not yet identified all genetic risk factors in SLE or all gene–gene or gene–environment interactions contributing to the risk of SLE. Testing the known lupus genes in patients with primary APS will reveal interesting differences and similarities and will present the possibility for clinical studies. Prospective followup of these patients will reveal those in whom SLE may eventually develop and demonstrate whether the genetic correlations observed in this study and the associated conjectures are indeed correct.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Alarcón-Riquelme had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Yin, Borghi, Tincani, Meroni, Alarcón-Riquelme.

Acquisition of data. Yin, Tincani, Meroni, Alarcón-Riquelme.

Analysis and interpretation of data. Yin, Delgado-Vega, Meroni, Alarcón-Riquelme.

Recruitment of patients. Tincani.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Dr. Sandra D'Alfonso for providing some of the Italian control samples used in this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  • 1
    Shoenfeld Y, Toubi E. APS and SLE: are they separate entities or just clinical presentations on the same scale? Curr Opin Rheumatol 2009. In press.
  • 2
    Andreoli L, Pregnolato F, Burlingame RW, Allegri F, Rizzini S, Fanelli V, et al. Antinucleosome antibodies in primary antiphospholipid syndrome: a hint at systemic autoimmunity? J Autoimmun 2008; 30: 517.
  • 3
    Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 2003; 349: 152633.
  • 4
    Graham RR, Kozyrev SV, Baechler EC, Reddy MV, Plenge RM, Bauer JW, et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat Genet 2006; 38: 5505.
  • 5
    Harley JB, Alarcon-Riquelme ME, Criswell LA, Jacob CO, Kimberly RP, Moser KL, et al. Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nat Genet 2008; 40: 20410.
  • 6
    Kozyrev SV, Lewen S, Linga Reddy PM, Pons-Estel BA and the Argentine Collaborative Group, Witte T and the German Collaborative Group, Junker P, et al. Structural insertion/deletion variation in IRF5 is associated with a risk haplotype and defines the precise IRF5 isoforms expressed in systemic lupus erythematosus. Arthritis Rheum 2007; 56: 123441.
  • 7
    Demirci FY, Manzi S, Ramsey-Goldman R, Minster RL, Kenney M, Shaw PS, et al. Association of a common interferon regulatory factor 5 (IRF5) variant with increased risk of systemic lupus erythematosus (SLE). Ann Hum Genet 2007; 71 ( Pt 3): 30811.
  • 8
    Remmers EF, Plenge RM, Lee AT, Graham RR, Hom G, Behrens TW, et al. STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus. N Engl J Med 2007; 357: 97786.
  • 9
    Sigurdsson S, Nordmark G, Garnier S, Grundberg E, Kwan T, Nilsson O, et al. A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5. Hum Mol Genet 2008; 17: 286876.
  • 10
    Taylor KE, Remmers EF, Lee AT, Ortmann WA, Plenge RM, Tian C, et al. Specificity of the STAT4 genetic association for severe disease manifestations of systemic lupus erythematosus. PLoS Genet 2008; 4: e1000084.
  • 11
    Hom G, Graham RR, Modrek B, Taylor KE, Ortmann W, Garnier S, et al. Association of systemic lupus erythematosus with C8orf13-BLK and ITGAM-ITGAX. N Engl J Med 2008; 358: 9009.
  • 12
    Kozyrev SV, Abelson AK, Wojcik J, Zaghlool A, Linga Reddy MV, Sanchez E, et al. Functional variants in the B-cell gene BANK1 are associated with systemic lupus erythematosus. Nat Genet 2008; 40: 2116.
  • 13
    Reddy MV, Velazquez-Cruz R, Baca V, Lima G, Granados J, Orozco L, et al. Genetic association of IRF5 with SLE in Mexi-cans: higher frequency of the risk haplotype and its homozygozity than Europeans. Hum Genet 2007; 121: 7217.
  • 14
    Abelson AK, Delgado-Vega AM, Kozyrev SV, Sanchez E, Velazquez-Cruz R, Eriksson N, et al. STAT4 associates with SLE through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk. Ann Rheum Dis 2008. E-pub ahead of print.
  • 15
    Kariuki SN, Kirou KA, MacDermott EJ, Barillas-Arias L, Crow MK, Niewold TB. Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-α in lupus patients in vivo. J Immunol 2009; 182: 348.