We examined the frequency of the G-463A MPO gene polymorphism in patients with SLE and matched controls and SLE patients with LN versus those without LN. To our knowledge, this is the first study to investigate the association between MPO polymorphisms and SLE/LN. Our results indicate that the G-463A MPO SNP is not significantly linked with development of lupus, but is highly significantly associated with development of proliferative nephritis in African American patients with lupus. The results were opposite to our preconceived hypothesis in that it was the A allele that was associated with lupus nephritis, in contrast to antineutrophil cytoplasmic antibody (ANCA) vasculitis that is associated with the G allele. The enhanced association of LN with individuals of the AA genotype versus GA versus GG genotype supports the significance of modulation of MPO expression in the pathogenesis of lupus nephritis in African Americans.
The etiology of SLE remains unknown, but is believed to be multifactorial, resulting from complex interactions between genetic, hormonal, immunologic, and environmental factors13,14
. Practically, every compartment of the immune system has been reported to be abnormal in lupus13,14
. However, the etiology of these abnormalities remains largely unresolved. Studies of animal models and recent human studies indicate a 2-phase pathogenesis in lupus. The first phase is the loss of tolerance leading to autoimmunity characterized by autoantibody production26
. Both murine models and humans can progress to this stage of “lupus” without ever developing clinically significant signs or symptoms (i.e., first-degree relatives of lupus patients, sle1 mice and C3H/lpr mice)26,27
. The second phase is the progression to end-organ involvement, with renal disease being the most serious and prevalent26
. Genetic predispositions to the first phase (autoimmunity) are required for disease, but may not influence development of the second phase (autoimmune disease). The development of the first phase of disease on a “proinflammatory” genetic background, we propose, is crucial for development of end-organ involvement. As an example, we have previously reported an association of a SNP in the inducible nitric oxide synthase gene (iNOS), resulting in enhanced NO production, leads to a 3–4-fold increased risk of developing lupus in African American women28
Most polymorphisms are phenotypically benign in the normal host. However, in the appropriate environmental and genetic context, a polymorphism may influence susceptibility and/or outcome in a disease, often by acting upon one or more pathways unaffected by the primary defect26
. Therefore, for a given population, polymorphisms may act as susceptibility genes and modify the clinical expression of the disease. Several SNP have been analyzed for linkage with the development of SLE and/or LN. Karassa, et al
examined the role of the Fcγ-receptor IIa polymorphism in susceptibility to SLE and LN29
. A metaanalysis of 17 studies, comparing 1405 patients with LN and 1709 without LN, concluded that the Fcγ-RIIa polymorphism represents a significant risk factor for SLE, but not for LN29
. In another metaanalysis, Karassa, et al
also found that the Fcγ-RIII A-V/F158 polymorphism has a statistically significant effect on the development of LN, but only a trend for the development of SLE30
. These results are consistent with the 2-phase model of lupus disease development. Quintero-Del-Rio, et al
found 2 SLE susceptibility loci, SLEN2 and SLEN3, in Black patients with SLE that are significantly associated with LN by linkage analysis. These loci were not associated with LN in White patients, emphasizing that ethnic differences likely exist in the genetics of SLE24
. The identity of the genes responsible for this linkage effect remains unknown.
Interpretation of data on the potential role of this MPO gene polymorphism in the development of LN in African American patients with SLE is limited by the lack of published information on the functional significance of the polymorphism on MPO-mediated reactions relevant to autoimmune diseases. The in vitro
data supporting the functional relevance of this polymorphism are strong31
. The G to A base difference is located in the promoter region within an Alu repeat. PPAR-γ binds the Alu, inducing MPO when added with MCSF, while suppressing MPO when cultured with GMCSF in macrophages. The estrogen receptor alpha binds −463 A preferentially, and estrogen blocks PPAR-γ actions, especially on the MPO A allele with the stronger ER binding site. The MPO G allele, however, is not the higher expressing allele in all cell types and biologic settings. The GA genotype is associated with 1.6 to 2.5-fold higher MPO mRNA levels than GG in primary human peripheral blood mononuclear cells (PBMC). It is in macrophages that the GG genotype is associated with 4.6 to 7-fold higher MPO levels than GA9
. Thus, the MPO A allele can be higher or lower expressing than the MPO G allele, depending on the cell type, inflammatory state, presence of PPAR-γ ligands, and estrogen levels9
. In LN, the intrinsic immune cell, the mesangial cell, as well as infiltrating macrophages and neutrophils all can express MPO. Systemic or PBMC MPO expression may also not reflect the expression of MPO in the kidney.
Our studies of NO in lupus indicate that African American lupus patients produce significantly higher levels of NO than do Caucasians, and SNP in the iNOS gene are only linked with lupus in African Americans28,32
. MPO has been shown to suppress the induction of iNOS gene expression due to consumption of low levels of NO required for iNOS induction33
. These 2 findings may partly provide a mechanistic explanation for the G-463A MPO polymorphism association with LN in African Americans in our analysis in that potentially low renal MPO expression may leave NO toxicity unopposed.
The G-463A MPO gene polymorphism is reported to be associated with susceptibility to several diseases including acute promyelocytic leukemia8,34
, multiple sclerosis35,36
, lung cancer25
, digestive tract cancer37
, Alzheimer disease38-40
, coronary artery disease41,42
, and MPO-ANCA-associated vasculitis in women43
. Of interest, in most of these studies, it was the high producing G allele linked with disease25,34,37,42-44
. In contrast, Mäkelä, et al
found that GA/AA genotypes were associated with increased severity of atherosclerosis and larger aortic lesions than GG genotypes45
, and Reynolds, et al39
found that in a Finnish population, the MPO A allele enhanced Alzheimer disease risk by 3.8-fold and also the MPO AA genotype was associated with selective mortality in men. Finally, Pope, et al40
found that the AA allele was a risk factor for cognitive decline in a cohort of 2350 adults. These latter reports are consistent with our findings of an association of the A allele and LN.
The association of the apparent low producing MPO allele with development of LN in African American patients with lupus suggests that increased MPO expression is antiinflammatory and/or protective in LN. There are known mechanisms by which overexpression of MPO can be protective. Experiments in MPO-deficient mice indicate that T cell-mediated disease models are aggravated in MPO knockout mice, whereas more acute inflammatory models show protection in the MPO knockouts36,46-49
. The MPO-hydrogen peroxide-chloride system can act as an antagonist to the NO/peroxynitrite pathway3,4
. Further, MPO-derived chloramines have antiinflammatory effects. Taurine chloramines can inhibit cytokine production by leukocytes and impair and/or block expression of monocyte chemoattractant protein-1, free radical generation, and NO synthesis in macrophages7
. In addition, the MPO-hydrogen peroxide-chloride system can down-regulate NADPH oxidase activity3
. Conversely, NO, generated either by endothelial NOS at low concentrations or by iNOS at high concentrations, can inhibit MPO activity, albeit by different concentration-dependent mechanisms3
There are limitations to our study, including differences in the 3 cohorts studied. The CLU study is an inception cohort and thus individuals in this cohort may develop LN in the future22
. We also included only patients with biopsy-proven Class III-V nephritis to insure only patients with LN were included. We excluded patients with abnormal urinalyses that had not had biopsies. The Lupus Multiplex Registry is a nationwide recruitment study to determine the genetics of lupus24
. Again, only patients with biopsy-proven LN were included as LN. This cohort is older and has a higher prevalence of renal disease than the CLU cohort. Despite these differences, the association between LN and the MPO SNP was similar. The final cohort, the Sea Island cohort, comprised African Americans living on the Sea Islands of South Carolina. These individuals, referred to as “Gullah,” are highly genetically homogeneous with minimal genetic admixture, with common ancestral origin from Sierra Leone and the Ivory Coast23
. The genetic homogeneity of this unique African American population offers advantages in defining genetic links with lupus and LN. The prevalence of the A allele is higher in the Gullah, although not significantly, than in the other 2 cohorts. The A allele, however, is significantly associated with LN in this cohort as in the other 2 cohorts. As with any association study, it is possible we are not studying the actual disease-associated polymorphism. Polymorphisms tend to be in linkage disequilibrium and thus the G-463A MPO polymorphism may not be pathogenic but in linkage dis-equilibrium with another SNP that is pathogenic. Formal analysis of linkage of SNP within the MPO promoter is not currently available.
In conclusion, studying the G-463A MPO gene polymorphism in SLE patients and controls, our results indicate that this SNP does not appear to be a risk factor for lupus, but the MPO A allele is significantly linked with developing proliferative LN in African Americans in a gene dose-dependent manner. Further studies will address whether the SNP is linked with progression of renal disease via followup of the CLU and Sea Island cohorts and the influence of this SNP on local expression of MPO in the lupus kidney. Finally defining the pathogenic role of MPO expression and its impact on lupus nephritis will allow better treatment and prevention strategies of the disease.