4.1. Role of IL-10 promoter polymorphism in susceptibility and clinical manifestation of SLE
Several evidences suggest that IL-10 could be a strong candidate gene influencing SLE susceptibility. IL-10 gene has been mapped to chromosome 1q31-32, which is a susceptibility region for SLE (LOD=3.79) [Johanneson et al., 2002]. It is also homologous to a murine SLE susceptibility region [Tsao et al., 1997]. More recently, Gateva et al., 2010 performed a large-scale replication study involving 1,310 cases and 7,859 controls, and identified 21 additional candidate susceptibility loci for SLE. Among the newly identified SLE loci is IL-10.
However, in spite of the considerable number of genetic studies performed, no definitive result about its involvement in SLE susceptibility was achieved. Some works showed significant associations between IL-10 microsatellites or SNPs with SLE susceptibility or with the development of certain clinical or immunological features [Rood et al., 1999; D’Alfonso et al., 2002; Schotte et al., 2004; Chen et al., 2006; Sung et al., 2006; Chong et al., 2006; Rosado et al., 2008] while other studies indicated that these polymorphisms did not appear to have any relevance in the disease [Alarcón-Riquelme et al., 1999; Van der Linden et al., 2000; Dijstelbloem et al., 2002; Guarnizo-Zuccardi et al., 2007]. The role of IL-10 genotypes has been recently reviewed by Lopez et al., 2010. The more recent association studies dealing IL-10 promoter polymorphisms for SLE susceptibility are summarized on Table 2.
With respect to microsatellite variants, different alleles of IL10.G have been reported to be associated with SLE incidence in various populations. Thus, frequency of IL10.G9 allele (21 CA repeats) was significantly decreased in European [D’Alfonso et al., 2000, D’Alfonso et al., 2002, Eskdale et al., 1997] and Mexican-American [Mehrian et al., 1998] SLE patients, whereas the long alleles IL10.G10, G11 and G13 (with a CA repeat number greater than 21) were significantly increased in Mexican-American [Mehrian et al., 1998], Italian [D’Alfonso et al., 2000, D’Alfonso et al., 2002] and British [Eskdale et al., 1997] patients respectively. On the contrary, an increase in IL10.G4 (short allele) was reported in Chinese patients [Chong et al., 2004] whereas no significant differences in IL10.G alleles were detected in other cohorts [Schotte et al., 2004, Alarcon-Riquelme et al., 1999; Johansson et al., 2002]. Recently, a large meta-analysis summarized the results focused on the role of IL-10 promoter polymorphisms for SLE susceptibility from 16 published case-control studies involving a total of 2391 SLE patients and 3483 controls (Nath et al., 2005). The results of the meta-analysis performed by Nath et al., 2005 showed a significant association between SLE and the G11 allele of IL10.G (OR=1.279, 95% CI; 1.027±1.593, P=0.028) in whole populations, and IL-10 promoter -1082G allele was associated with SLE in Asians (OR=1.358, 95% CI; 1.015±1.816, P=0.039). It has been reported that LPS-stimulated cells from individuals carriers of the IL10.G allele with 26 CA repeats presented higher IL-10 production than those from carriers of short alleles [Eskdale et al., 1998], suggesting that long alleles might be responsible for a high IL-10 production. Thus, accordingly to these data, high IL-10 producer genotypes (with more than 21 CA repeats) could be associated with SLE susceptibility, while presence of short alleles could confer a protective effect [Chen et al., 2006; D’Alfonso et al., 2002].
-1082; -819; -592
|Association of IL10.G11 allele in whole populations;|
Association of -1082G allele with SLE in Asians
|2391/3483||Meta-analysis||Nath et al., 2005|
|-592 A/C||No association with susceptibility|
Association of -592C with SLE activity
|350/330||Korean||Sung et al., 2005|
|IL10G||Increased G9 and decreased G8 in SLE|
Association of G13 with ACLA-IgM
Association of G8 with CNS lupus
|237/304||Taiwanese||Chen et al., 2006|
|-1082, -819, -592||Association of ACC/ACC with susceptibility to SLE||195/159||Thai||Hirankarn et al., 2006|
|-1082; -819; -592||No association||120/102||Colombian||Guarnizo-Zuccardi et al., 2007|
-1082; -819; -592
|No association of microsatellites|
Increased GCC in SLE
|116/51||Spanish||Rosado et al., 2008|
|-1082; -819; -592||Increased GCC in SLE||103/300||Polish||Sobkowiak et al., 2009|
|-1082; -819; -592|
|No association||110/138||Chinese||Yu et al., 2010|
|-1082; -819; -592||Increased ATA haplotype in sLE||172/215||Taiwanese||Lin et al., 2010|
|-1082||No association||157/126||Bulgarian||Miteva et al., 2010|
Association of IL-10 promotor polymorphisms - IL10G, IL10R, -1082G/A (rs1800896), -819C/T (rs1800871), -592A/C (rs1800872), with SLE. Association studies published after 2005 years are given only.
Conflicting results were also obtained after examining the possible association between SLE susceptibility and SNPs at -1082, -819, and -592 positions of IL-10 gene in the different populations in which they were investigated. The frequency of high IL-10 producers (carriers of -1082G allele or GCC haplotype) has been found to be increased in several works with Asian [Nath et al., 2005; Hirankarn et al., 2006] or European [Rosado o et al., 2008; Sobkowiak et al., 2009] patients, although most of the studies performed in Caucasian populations did not show significant associations [Lazarus et al. 1997, Guarnizo-Zuccardi et al, 2007;, Koss et al., 2000; Suárez et al., 2005; Dijstelbloem et al., 2002; Van der Linden et al., 2000; Crawley et al., 1999].
Rosado et al., 2008 found that the GCC haplotype frequency was significantly higher in Spanish patients with SLE. To assess the functional role of genotypes, they also quantified serum IL-10 levels from patients and controls and found higher serum IL-10 levels in patients. On the basis of these data, they suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in Spanish population. Similar are the results and final conclusion of the study performed by Sobkowiak et al., 2009; despite the higher prevalence of the GCC/GCC, GCC/ATA and ATA/ATA genotypes in SLE patients than in controls, they observed that only GCC/GCC genotype was significant more frequent in SLE. Hence, the conclusion suggests the GCC/GCC promoter genotype may contribute to SLE incidence in Polish patients. A very recent study investigated the association between of IL-10 promoter polymorphisms (-1082, -819 and -592) with SLE in a total of 172 Taiwanese patients and 215 controls reported an association of IL-10 ATA haplotype with SLE in Taiwanese population [Lin et al., 2010]. In another Asian population, an association of ACC/ACC haplotype of IL-10 in susceptibility to SLE has been observed by Hirankarn et al., 2006.
In this regard, we investigated the role of -1082A/G promoter polymorphism of IL-10 gene as risk factor for development and clinical manifestations of SLE in Bulgarian population. Our preliminary results did not reveal a significant association of -1082 SNP in IL-10 with SLE [Miteva et al., 2010]. New data for the distribution and the frequencies of the -1082A/G alleles and genotypes among the SLE patients and healthy controls are presented on Table 3. The results of our case-control study based on 157 patients with SLE and 166 unaffected control individuals showed that the genotype distribution is consistent with those published for other Caucasian type control cohorts [Lopez et al., 2010). We also found the prevalence of homozygous GG genotype in SLE cases (27%) compared to the controls (13%) with OR = 1.185 (95% CI = 0.58÷2.45), although the difference did not reach statistical significance (p=0.548). In addition, we observed an increased frequency of GG genotype compared to the reference AA genotype in patients with antiphospholipid synrom (APS) (27%) compared with patients without APS (14%) with OR = 2.750, 95% CI = 0.910 ÷ 8.347, p = 0.074). This suggests that carriage of a higher IL-10 producing genotype is a risk factor for antiphospholipid autoantibody production and APS appearance, thus having a modifying effect on the clinical presentation of the disease.
|AAn (%)||AGn (%)||AG+AAn (%)||AG+GGn (%)||GGn (%)||An (%)||Gn (%)|
Genotypic and allelic frequency of gene polymorphism at position -1082 A/G in IL-10 gene in SLE patients and controls
The presence of autoantibodies, mainly directed against nuclear antigens (ANAs), is one of the most characteristic features of SLE. The effect of IL-10 genotypes did not seem to be especially relevant, although it has been reported an increased prevalence of antibodies against several extractable nuclear antigens (anti-ENA) in patients with the allele IL-10.G9 [Eskdale, et al., 1997], and the presence of anti-Sm antibodies was found significantly overrepresented among patient carriers of G14 and G15 alleles and R2-G15 and R2-G14 haplotypes [Schotte et al., 2004]. An association of the carriage of low IL-10 producer alleles such as IL10.G13 allele with the presence of anticardiolipin IgM antibodies and IL10.G8 allele with neurological affectation has been reported in Taiwanese patients with SLE [Chen et al., 2006], results very similar to the data from our study.
Considering increased circulating levels of IL-10 have been consistently reported in the sera of patients with SLE, it is possible that different cytokine production may not only influence the autoantibody production, but also the clinical presentation of the disease. However, there were no definitive data on the association of IL-10 polymorphisms and specific clinical manifestations, probably due to the heterogeneity of the disease. For instance, renal involvement has been associated with both high (GCC) [Lazarus et al., 1997, Zhu et al., 2005] and low (ATA) [Mok et al., 1999] IL-10 producer genotypes. High prevalence of neuropsychiatric [Rood et al., 1999; Chen et al. 2006] and cardiovascular disorders [Fei et al., 2004] has been reported in patients with low genetic production whereas high IL-10 production has been linked to an increased incidence of serositis, hematological disorder [Chong et al.], SLICC/ACR Damage Index [Sung et al., 2006] and presence of discoid or mucocutaneous lesions [Suárez et al., 2005; Alarcón-Riquelme et al.,1999]. This last association was supported by the increased frequency of the high producer -1082G allele observed in patients with discoid lupus erythematosus [Suárez et al., 2005; Van der Linden et al., 2000] and by the fact that cutaneous manifestations improved in SLE patients under anti IL-10 monoclonal antibody treatment [Llorente et al., 2000]. In conclusion IL-10 promoter SNPs alone have not exhibits strong association with SLE susceptibility, but their role cant’ be excluded. Each polymorphism in regulatory regions of gene, may either directly influence gene expression or indirectly via tight linkage with other polymorphisms occurring elsewhere in the same or in other cytokine gene. A particular combination of SNPs on cytokine genes in individual genotype has different impacts on induced cytokine production. Miteva and Stanilova investigate the combined effect of -1082A*G in IL10 and +16974A*C in IL12B SNPs on induced cytokine production by stimulated peripheral blood mononuclear cells isolated from healthy donors (Miteva, Stanilova, 2008). Results demonstrated that the production of IL-10 from PBMC depended on both, -1082А*G in IL10 and +16974A*C in IL12B polymorphisms and the presence of high producer IL-12p40 genotype led to diminished production of IL-10 determined by -1082*G-allele of SNP in IL10. In the same vein, we suppose that individuals with genotype which combine SNPs responsible for higher production of IL-10 simultaneously with lower production of TGF-β1 should be more susceptible to SLE.
4.2. Role of TGB-β1 genetic polymorphisms in susceptibility and clinical manifestation of SLE
Regarding the association between decreased TGF-β1 serum levels and the development of autoimmunity, the mechanisms which control the concentration of TGF-β1 in plasma are under extensive investigations. The concentration of both latent complex and active TGF-β1 in plasma has been shown to be predominantly under genetic control [Grainger et al., 1999]. In light of these findings TGF-β1 gene is a functional candidate gene for genetic predisposition in systemic lupus erythematosus.
The presence of polymorphisms in the TGFB1 locus may indicate predisposition to diseases, such as systemic lupus erythematosus that have been linked here and elsewhere [Caserta et al., 2004; Lu et al., 2004] to the circulating levels of TGF-β1. To test this hypothesis, we performed a population based case-control study to investigate the association of -509C/T polymorphism of the TGFB1 gene with susceptibility to SLE [Manolova et al., 2012]. In this investigation, the change at position -509C/T in the TGFB1 gene (rs1800469) was studied using RFLP-PCR among 147 cases with SLE and 134 normal Bulgarian subjects. The genotype distribution and allele frequencies of -509C/T SNP in gene promoter of TGFB1 among SLE patients and healthy donors are presented in Table 4.
|CCn (%)||CTn (%)||CT+CCn (%)||CT+TTn (%)||TTn (%)||Cn (%)||Tn (%)|
Genotypic and allelic frequency of gene polymorphism at position -509 in TGFB1 gene in SLE patients and controls
The genotype distribution for TGFB1 -509C/T polymorphism was in agreement with Hardy-Weinberg equilibrium among cases (χ2=2.00; p=0.367) and controls (χ2=2.237; p=0.326). Homozygous CC genotype was found in 32.2% of patients and 36.8% of control subjects, heterozygous CT genotype was observed in 53% of SLE patients and 42.5% of controls, homozygous TT genotype was detected in 14.8% of cases and 21.6% of controls. There were no significant differences in the genotype (p=0.155) and allele (p=0.694) frequencies of -509C/T polymorphism of the TGFB1 gene between SLE patients and controls. However, we observed a higher frequency of heterozygous CT genotype (OR = 1.827; 95% CI 0.91±3.69; p = 0.068) and lower frequency of TT genotype (OR = 0,759; 95% CI: 0.36±1.59; p=0.428) in SLE patients compared to healthy controls. In logistic regression analysis the presence of allele C in the genotype (CT + CC versus TT) was associated with a 1.6 times higher risk of developing systemic lupus erythematosus.
Genotype and allele frequencies of -509C/T polymorphism in TGFB1 which we established in Bulgarian population were comparable to those found in other populations. However, the available data in the literature reveal the existence of ethnic differences in frequencies of the allele variants of this polymorphic marker (Table 5). C allele had the higher representation among Europeans in French [Cambien et al., 1996], German [Wu et al., 2008] and British [Awad et al., 1998] studies with a frequency of 65 to 76 percent, while among healthy individuals from different Asian ethnicities T allele occurs more frequently or almost equally with the C allele [Amirghofran Z, 2009; Zhang et al., 2009; Chung et al.,, 2007]. In Bulgarian population allele C is slightly more common and was found in 57% of healthy subjects and in 59% of cases with SLE.
|Locus||Allele||Frequency (%) among healthy controls|
|Bulgarian studyData from this study||French studyCambien et al., 1996||British studyAwad et al., 1998||German studyWu et al., 2009||Asian studyLu et al., 2004|
Interethnic differences in allele frequencies of -509C/T SNP in TGFB1 in healthy controls.
According to our knowledge there are only a limited number of studies aiming to evaluate the possible role of polymorphisms in the TGF-β1 gene as predisposing factors for SLE, but the results of these studies are contradictory. The polymorphisms in of -509C/T SNP in TGF-β1 have been explored in SLE only by three research teams [Lu et al., 2004; Caserta et al., 2004; Vuong et al., 2010]. In overall, the contradictory results in the literature could be explained by the genetic heterogeneity of SLE in different populations and possible sample stratification. Table 6 summarizes the association studies dealing TGF-β1 gene polymorphisms for SLE susceptibility.
|+915G/C (rs1800471)||No association||203/158||German||Schotte et al., 2003|
|No association||138/182||Taiwanese||Lu et al., 2004|
|-509C/T (rs1800469)||No association||23/32||North America||Caserta et al., 2004|
|Association of +869C allele with SLE; decreased high TGF-β1 producers haplotypes in SLE (codon10 T allele/25 G allele)|
No association with clinical manifestations
|120/102||Colombian||Guarnizo-Zuccardi et al., 2007|
|+869T/C (rs1982073)||No association with susceptibility|
+869TT associated with aseptic necrosis and anti-Ro antibodies
|196/102||Japan||Wang et al., 2007|
intron G/T (rs2241715)
3’-UTR A/G (rs6957)
|No association||272/307||Sweden||Vuong et al., 2010|
|-509C/T (rs1800469)||Decreased frequency of TT|
(OR = 0,759; 95% CI: 0.36±1.59)
allele C (CT+CC vs TT) risk factor for SLE (OR=1.59;
95% CI: 0.83±3.07)
|149/134||Bulgarian||Manolova et al. 2012|
Association of TGF-β1 gene polymorphisms with SLE.
Lu et al. conducted a case-control study involving 134 patients and 182 healthy individuals of Taiwanese origin to evaluate the association of -509C/T SNP in TGFB1 with susceptibility to systemic lupus erythematosus [Lu et al., 2004]. In the same study, authors investigated also association of some others TGFβ1 single nucleotide polymorphisms, including -988C/A, -800G/A, +869T/C (Leu10Pro), and +915 G/C (Arg25Pro) with susceptibility to SLE and shown that none of the TGFβ1 SNPs was strongly associated with SLE in Taiwanese patients. They conclude that these polymorphisms do not represent a genetic predisposition to SLE. In another study, Schotte and colleagues investigated the +915G/C polymorphism at codon25 and did not found any association between this polymorphism and SLE in German population [Schotte et al., 2003]. In addition, authors found no association of major disease manifestations or specific autoantibodies with TGFB1 genotypes or alleles. The authors conclude that +915G/C polymorphism in TGFB1 neither significantly contributes to the disease susceptibility, nor predisposes to clinical and immunological manifestations typical of SLE. Also, there were no significant associations between several SNPs from the TGFβ1 including -509C/T, +869T/C, intronic G/T (rs2241715), and3’-UTR A/G(rs6957) with SLE or with lupus nephritis in Sweden population [Vuong et al., 2010].
In contrast to these data are the results obtained by Guarnizo-Zuccardi et al., 2007 for several cytokine gene polymorphisms in Colombian patients with SLE. They analyze the relation between the +868T/C and the +915G/C SNP in TGFB1 with the development and clinical manifestations of SLE. The authors found a strong association of SLE with the TGF-β1 codon25 C allele, associated with decreased TGF-β1 production and found lower rates of higher-producing GG genotype and a higher frequency of heterozygous genotypes in this polymorphic marker in patients with SLE. As for the +868T/C polymorphism at codon10, Guarnizo-Zuccardi et al., 2007 did not observe association between SLE and codon10 when analyzing independently, but they found a significant association when the haplotypes codon10/20 were evaluated, which could be because of the linkage disequilibrium between the two SNPs. This extended genotypic analysis revealed a lower frequency of high TGF-β1 producers – haplotype 10/25 T/T-G/G in Colombian patients with SLE. Unlike the relationship of +868T/C and +915G/C SNPs of the TGFB1 gene to disease susceptibility, they found no association between clinical features of the disease and the polymorphisms studied. As opposed to this report, Wang et al., 2007 did not find an impact of +869T/C polymorphism in TGF-β1 gene on disease susceptibility in population-based case-control study involving 196 patients with SLE and 106 healthy controls in Japan. However, they found an association between +869T/C TGFB1 polymorphism and several clinical features of SLE. The carriage of TT genotype of +869T/C polymorphism which is associated with a lower serum TGF-β1 level was related to the occurrence of aseptic necrosis and higher incidence of anti-SSA/Ro antibodies in SLE patients. Consistent with the last finding, the children with the TT +869T/C genotype of TGFB1 gene have been reported to be more susceptible to anti-SSA/Ro antibody-associated congenital hear block [Clancy et al., 2003].
Systemic lupus erythematosus is a heterogeneous disease with diverse clinical manifestations that range could be due to genetic factors. In this regard, we also analyzed the effect of the -509C/T polymorphism in TGFB1 the clinical manifestations evolved in the course of the disease [Manolova et al., 2012]. The results of our study demonstrated a weak association of -509C/T polymorphism of TGFB1 with clinical manifestations of SLE. The carriage of the heterozygous genotype was associated with about 2-fold higher risk for the occurrence of hematological manifestations (OR=2.41; 95%CI: 1.10±5.32; p=0.016) and antibodies against dsDNA (OR = 2.0; 95% CI: 0.96±4.2, p = 0.045) in lupus patients, while the CC genotype is a protective factor for these events. Based on our and others data, we could assume the TGFβ1 gene polymorphisms as one of the genetic factors that explain the heterogeneity seen in SLE.