Because of the important role of β2
GPI in the production of APA and the observation that β2
GPI-mediated immune response in patients with autoimmune diseases may lead to atherosclerosis 18
, it is important to understand the role of APOH
genetic variation in relation to autoimmune diseases and associated premature CVD. A priori, one might expect that genetic variation in elements which control APOH
expression (promoter region) can be associated with the disease risk. Promoter sequences are potential sources of polymorphisms affecting gene expression and phenotypic variation 33
. Promoter variants may potentially alter the affinities of existing protein-DNA interactions or recruit new proteins to bind to the DNA, altering the specificity and kinetics of the transcription process. To our knowledge, this is the first study to evaluate the role of APOH
promoter SNPs in relation to SLE and related phenotypes, including lupus nephritis and subclinical CVD.
Our study revealed a significant association for the −643T>C SNP with SLE risk in the Pittsburgh sample (P = 0.014). However, this association was not confirmed in a second relatively small sample from Chicago (P = 0.919) and furthermore, the significance level decreased in the combined Pittsburgh + Chicago sample, (P = 0.025) despite increase in the sample size and power. This suggests that either the effect of the −643T>C SNP on SLE risk is small which is difficult to reproduce in all samples or this association is due to chance. The latter assumption was confirmed in the haplotype analysis where one haplotype that carried the defining −643C allele (H3 haplotype in ) was not associated with SLE risk. On the other hand, our six-site haplotype analysis yielded a significant difference in overall haplotype distribution between SLE cases and controls (P = 0.009). The haplotype (H7 haplotype) with the most striking difference between cases and controls (P = 1.21 × 10−5) had a frequency of less than 2% in the total sample suggesting that only a small number of individuals were predicted to carry this haplotype. Notably, while no example of this haplotype was observed in 888 control chromosomes based on EH program, 4.5% of the patients carried this haplotype. H7 haplotype was not defined by a particular allele, although it carried the minor alleles for three APOH promoter SNPs (−643T>C, −1190G>C and −759A>G). The minor alleles at these three SNPs do not appear to be causative, as the presence of the −643C allele in H3 haplotype alone or the presence of the −1190C and −759G alleles together in H2 and H5 haplotypes did not demonstrate significant association. It appears that the simultaneous presence of these three alleles affect the risk of SLE in an additive fashion as demonstrated by the H7 haplotype. The apparently additive effects of these alleles were also obvious in the gene expression assay where the H7 haplotype was associated with greatest effect on luciferase activity. Alternatively, the unique H7 haplotype is a marker for a functional variant present in APOH or a nearby gene. Sequencing of the individuals who carry the haplotype H7 may help to identify putative functional variant(s).
The −643T>C SNP showed convincing association with the presence of carotid plaque among SLE patients as it was found to be associated in two independent samples from Pittsburgh and Chicago. In the combined Pittsburgh + Chicago SLE sample the association was more significant than observed in individual samples from each site (adjusted OR = 0.35, P
= 0.002). The accumulation of oxLDL is believed to initiate the process of atherosclerosis and β2
GPI is known to inhibit the uptake of oxLDL by macrophages in vitro
while it promotes the influx of oxLDL in the presence of APA33
. Several lines of evidence suggest that autoimmune vascular inflammation and oxidative stress may promote the formation of oxLDL/ β2
GPI complexes in the arterial wall as seen in SLE and APS patients 18
. Since many SLE patients are positive for APA, it is likely that low β2
GPI expression associated with the −643C allele may retard the influx of oxLDL to macrophages in the presence of APA and thus provide protection against plaque formation. Alternatively, there may be another mechanism underlying the effect of −643C allele on carotid plaque formation or it may simply be in strong LD with another variant, especially given that another APOH
promoter variant (−32A allele), which is also associated with low APOH
, was associated with increased risk for carotid plaque after adjusting for covariates (OR = 2.63, P
= 0.031). Additional studies in large data sets may help to delineate the role of APOH
promoter SNPs in relation to carotid plaque formation.
Renal disease is a major cause of morbidity in SLE patients. Some studies reported an increased production of APA in patients with Lupus nephritis , including anti-β2
GPI and anti-oxLDL 34–36
. We found that −1219G>A SNP may potentially affect the lupus nephritis risk (age-adjusted OR = 0.36, P
= 0.016) and this effect seems to be independent from APA occurrence given that this SNP did not show significant association with any of the APA examined in our sample. Rather, we found significant association of the −38G>A SNP with the occurrence of APA (P
= 4.57 × 10−4
), aCL (P
= 0.018), LAC (P
= 0.001) and anti-β2
= 0.002) in SLE cases but not in controls using multiple regression under dominant model. It is possible that the −38G>A SNP may be interacting with other factors that are involved in SLE etiopathogenesis to show its effect on APA in an autoimmune background. It is reasonable to expect that some factors would specifically show their effects on APA in association with disease status, thus contributing to the increased prevalence of APA in patients with autoimmune diseases as compared to the general population. Alternatively, the APOH
variants may have modest effects on the occurrence of APA which may be difficult to reproduce in all samples. Finally, given the very low MAF of −38G>A SNP, we cannot exclude the possibility of its spurious association.
The power for most of the examined SNPs in this study was adequate to detect SLE disease - related risk. For four SNPs (−1190G>C, −759 A>G, − 700C>A, −643T>C), we had 80% power to detect ORs between 1.50 and 1.55. For the −1219G>A and −32C>A SNPs, we had 80% power to detect an OR of 1.61 and 1.75, respectively. The power for the less common SNPs (−1284C>G and −38G>A) with MAF < 0.05 was low.
In summary, we present evidence that individual APOH promoter SNPs as well as haplotypes may be involved in the etiology of SLE and especially the risk for autoimmune-mediated CVD. Our results merit further investigation of APOH in independent large SLE case-control samples with subclinical phenotype information.