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Abstract

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

Objective

Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. The aim of this study was to examine methyl-CpG–binding protein 2 gene (MECP2) polymorphisms in a large cohort of patients with lupus and control subjects, and to determine the functional consequences of the lupus-associated MECP2 haplotype.

Methods

We genotyped 18 single-nucleotide polymorphisms within MECP2, located on chromosome Xq28, in a large cohort of patients with lupus and control subjects of European descent. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines in female lupus patients with and those without the lupus-associated MECP2 risk haplotype.

Results

We confirmed, replicated, and extended the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of lupus patients and control subjects of European descent (odds ratio 1.35, P = 6.65 × 10−11). MECP2 is a dichotomous transcription regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most (∼81%) were up-regulated. Genes that were up-regulated had significantly more CpG islands in their promoter regions compared with genes that were down-regulated. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms, suggesting that these genes are targets for MECP2 regulation in B cells. Furthermore, at least 13 of the 104 up-regulated genes are regulated by interferon. The disease-risk MECP2 haplotype was associated with increased expression of the MECP2 transcription coactivator CREB1 and decreased expression of the corepressor histone deacetylase 1.

Conclusion

Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.

Systemic lupus erythematosus (SLE) is a chronic, debilitating autoimmune disease associated with significant morbidity and mortality. The disease can affect multiple organs including the brain, kidney, lung, heart, and joints. Lupus is characterized by the production of autoantibodies to a variety of nuclear antigens and by the presence of an autoreactive T cell phenotype in the peripheral blood (1, 2). The pathogenesis of both drug-induced and idiopathic lupus involves a defect in T cell DNA methylation, resulting in overexpression of several methylation-sensitive genes such as ITGAL (CD11a), TNFSF7 (CD70), PRF1 (perforin), and CD40LG (CD40 ligand) (3, 4). Normal CD4+ T cells treated with DNA methylation inhibitors such as 5-azaC overexpress the same methylation-sensitive genes, similar to T cells from lupus patients. T cells treated with DNA methylation inhibitors become autoreactive in vitro; they are capable of spontaneously lysing syngeneic macrophages and inducing autologous B cell activation and immunoglobulin production (5). Furthermore, T cells treated with DNA methylation inhibitors induce a lupus-like disease, with glomerulonephritis and autoantibody production, upon adoptive transfer into mice (6). Interestingly, a CD4+ T cell methylation defect has also been reported in at least 1 murine model of lupus (7).

We have previously reported the genetic association between lupus and common variants within the methyl-CpG–binding protein 2 gene (MECP2) in 2 independent cohorts of lupus patients and controls and identified both risk and protective haplotypes (8). MECP2, located on chromosome Xq28, encodes for a 486-aa protein that binds methylated DNA and is intimately involved in the transcriptional regulation of methylation-sensitive genes.

MECP2 had been largely thought of as a transcription repressor that exerts its effects, at least in part, by recruiting histone deacetylases to promoter sequences of target genes, thereby inducing a transcriptionally inaccessible chromatin configuration (9). Recent evidence, however, indicates that MECP2 is also a transcription activator capable of recruiting the transcription factor CREB1 (10). Indeed, MECP2 acts as a transcription activator in the majority (∼85%) of genes regulated by MECP2 in the murine hypothalamus (10).

In this study, we first confirmed the association of lupus with variants within MECP2 in a large independent cohort of lupus patients and control subjects of European descent. We next determined the expression of the 2 known messenger RNA (mRNA) isoforms of MECP2 in B cell lines from lupus patients with the risk haplotype and those with the protective haplotype. Furthermore, we demonstrated that the MECP2 risk haplotype dictates global changes in B cell gene expression relative to the protective nonrisk haplotype and thereby provides multiple paths toward realization of the phenotype.

PATIENTS AND METHODS

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

Patients and control subjects.

A cohort of 1,418 unrelated lupus patients and 1,876 race-matched control subjects, all of whom were of European descent, were recruited at the Oklahoma Medical Research Foundation as well as at collaborating institutes in the US, the UK, and Sweden. This cohort is independent of the cohort of European descent previously described by our group (8). All patients fulfilled the American College of Rheumatology classification criteria for lupus (11). All protocols were approved by the institutional review boards at the University of Oklahoma Health Sciences Center and the Oklahoma Medical Research Foundation.

Genotyping.

Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs). Genotyping of 18 single-nucleotide polymorphisms (SNPs) within MECP2 was performed with an Illumina BeadStation 500GX Genetic Analysis System (Illumina, San Diego, CA), using Illumina Infinium II genotyping assays according to the manufacturer's recommendations. These 18 SNPs were selected from the Database of Single-Nucleotide Polymorphisms (online at http://www.ncbi.nlm.nih.gov/projects/SNP/) to cover the entire length of MECP2 and were previously genotyped by our group in 2 independent cohorts of lupus patients and control subjects.

Cell culture and RNA extraction.

Epstein-Barr virus (EBV)–transformed B cell lines from lupus patients were used to study the effect of MECP2 risk and protective haplotypes. B cell lines were prepared from PBMCs isolated from lupus patients by density-gradient centrifugation and then suspended in RPMI 1640 with 10% bovine serum, supplemental glutamine, streptomycin, and penicillin. A small concentration of cyclosporine (1 μg/ml) was added to inhibit T cell suppression of transformed B cell growth. Finally, an aliquot of a fresh culture supernatant from a B95-8 marmoset cell line culture producing infectious EBV was added as the transforming agent. Cell lines grew in a few weeks, were expanded, and were frozen in 90% fetal calf serum (FCS) and 10% DMSO in aliquots of 20 million cells at −70°C. After equilibration at this temperature, the cells were transferred to liquid nitrogen for long-term storage.

EBV-transformed B cell lines from 10 lupus patients homozygous for the MECP2 risk haplotype and 10 lupus patients homozygous for the protective haplotype were thawed into medium, washed, and grown in RPMI 1640 supplemented with 10% FCS, glutamine, streptomycin, and penicillin. Twenty-four hours prior to isolating RNA, all cell lines were washed and grown into fresh medium. RNA was isolated using a combination of TRIzol (Invitrogen, Carlsbad, CA) and RNeasy kits (Qiagen, Valencia, CA). Briefly, 15 × 106 cells were lysed in 1 ml of TRIzol reagent to which 200 μl of chloroform was added, after which the cells were mixed by inversion for 15 seconds and incubated at room temperature for 3 minutes. The lysate was then centrifuged at 14,000 rpm for 15 minutes at 4°C. Ethanol (100%) was added to the supernatant at a ratio of 0.53:1 ethanol:supernatant by volume, the mixture was loaded into the RNeasy column, and RNA isolation was completed following the RNeasy protocol.

Real-time reverse transcription–polymerase chain reaction (RT-PCR).

To measure the levels of MECP2 transcripts (isoform 1 and isoform 2), real-time RT-PCR was performed using the iScript One-Step RT-PCR Kit With SYBR Green (Bio-Rad, Hercules, CA) and the Rotor-Gene 3000 real-time thermocycler (Corbett Research, Mortlake, New South Wales, Australia). RNA was first treated with Turbo DNA-free (Ambion, Austin, TX) to digest any contaminating DNA. A total of 62.5 ng of RNA per reaction was used. The PCR protocol was as follows: 10 minutes at 50°C, 5 minutes at 95°C, 45 cycles of 10 seconds at 95°C and 30 seconds at 55°C. Internal standards prepared by serial dilutions were used to quantify the expression levels of both MECP2 isoforms, CREB1 and HDAC1, followed by normalization to a housekeeping gene (GAPDH or ACTB [β-actin]). The primers used are as follows: for MECP2A (isoform 1), forward 5′-CTGGGATGTTAGGGCTCAGGGA-3′, reverse 5′-AGAGTGGTGGGCTGATGGCT-3′; for MECP2B (isoform 2), forward 5′-AGGCGAGGAGGAGAGACTGGAA-3′, reverse 5′-AGAGTGGTGGGCTGATGGCT-3′; for CREB1, forward 5′-CCAGCAGAGTGGAGATGCAG-3′, reverse 5′-GTTACGGTGGGAGCAGATGAT-3′; for HDAC1, forward 5′-ACCCGGAGGAAAGTCTGTTAC-3′, reverse 5′-GGTAGAGACCATAGTTGAGCAGC-3′; for GAPDH, forward 5′-TGTTGCCATCAATGACCCCTTC-3′, reverse 5′-CTCCACGACGTACTCAGCGC-3′; for ACTB, forward 5′-GCACCACACCTTCTACAATGAGC-3′, reverse 5′-GGATAGCACAGCCTGGATAGCAAC-3′. Real-time RT-PCR, performed as described above, was also used to validate the expression microarray data. The genes examined include CLIC2, IFITM3, IGJ, ITM2B, and TEX15. Primer sequences are available from the corresponding author upon request. All primers were purchased from Integrated DNA Technologies (Coralville, IA).

Expression microarray.

After purification, the RNA concentration was determined with a NanoDrop scanning spectrophotometer (Thermo Scientific, Wilmington, DE) and then qualitatively assessed for degradation with 28:18S ribosomal RNA, using a capillary gel electrophoresis system (Agilent 2100 Bionanalyzer; Agilent, Wilmington, DE). Biotinylated amplified RNA was produced from 250 ng of total RNA per sample, using a modification of the Eberwine protocol (16) as described in the Illumina TotalPrep RNA Amplification Kit (Ambion). Briefly, RNA was reverse-transcribed with an oligo(dT) primer containing a T7 promoter. RNA containing biotin-UTP ribonucleotides was amplified by in vitro transcription to generate antisense RNA. This RNA was hybridized overnight at 58°C to human WG-6 version 3 Expression BeadChip microarrays (Illumina). These arrays contain 48,804 50-mer oligonucleotide probes coupled to beads that are mounted on glass slides. Each bead has an ∼20–30-fold redundancy per microarray. Microarrays were washed under high stringency, labeled with streptavidin–Cy3, and scanned with an Illumina BeadStation 500 scanner.

Statistical analysis.

Analysis of genotyping data.

SNPs with a minor allele frequency of ≥5% and a P value for Hardy-Weinberg equilibrium (HWE) of >0.01 were used for further analysis. The genotyping success rate for all SNPs analyzed was ≥97.4%. Allele frequencies were determined in both the patient and control groups, and Pearson's chi-square and P values were calculated to assess differences between the 2 groups. P values for permutation were calculated using Haploview version 4.1 software, to correct for multiple testing (12). Haploview 4.1 was also used to generate a linkage disequilibrium (LD) plot for the analyzed SNPs and to calculate correlation coefficient (r2) values between SNPs. Common haplotypes (those with a frequency of >1%) produced by the disease-associated SNPs were determined, and haplotype frequencies were calculated using Haploview 4.1. Principal components analyses (PCAs) were computed to identify population substratifications in our cohort (13). A total of 64 samples that violated the assumption of sample homogeneity based on the PCA (41 samples from patients and 23 samples from control subjects) were removed prior to data analysis. We then performed genomic control analysis to calculate the inflation factor λ, using 2,218 null SNPs, which produced a value of λ = 1.04, further indicating no evidence for population substratification. The inflation factor is a measure that quantifies the degree to which population stratification increases the chi-square test statistics.

Bioinformatics and statistical analysis of microarray data.

Statistical analysis of microarray data was performed using associative analysis of expression, as previously described (14). CpG islands in the regions 5 kb upstream and 5 kb downstream of the transcription start site of differentially expressed genes were identified algorithmically using Build 36.3 (released March 24, 2008) of the Human Genome Resources database (ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/). CpG islands were defined as a stretch of DNA of at least 200 bp, with a C-plus-G content of at least 50% and an observed:expected CG frequency of at least 0.6. The IRIDESCENT algorithm (15) was used to identify and score the relevance of “objects” (i.e., genes, diseases, phenotypes, small molecules, and ontology categories) that co-occurred with the differentially expressed genes in Medline abstracts. The names and synonyms for these objects can be obtained from publicly available databases including, but not limited to, OMIM (diseases), the Disease Ontology Database (phenotypes), Entrez Gene (genes), CHEMID (chemicals), and Gene Ontology (GO) (GO categories). A shared relationship between a subset of differentially expressed genes and another object in the IRIDESCENT database identifies common processes and associations.

RESULTS

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

Association of lupus with polymorphisms within MECP2.

We genotyped 18 SNPs within MECP2 in a large independent cohort of 1,418 lupus patients and 1,876 control subjects of European descent and confirmed the association between SNPs within MECP2 and lupus. PCA was used to detect population substratification and identified “outlier” samples (41 patients and 23 controls) that were excluded from further analysis. Therefore, a total of 1,377 lupus patients (1,293 women and 84 men) and 1,853 control subjects (1,097 women and 756 men) were analyzed.

SNPs with minor allele frequencies of ≥5% and a P value for HWE of >0.01 were included in subsequent analyses. The P value for HWE measures the difference between the observed genotype frequency and the expected genotype frequency, based on the observed allele frequency. A high P value for HWE indicates random mating in a study population. We confirmed the association between lupus and all 8 SNPs within MECP2 that was previously observed in lupus patients and control subjects of European descent and Korean lupus patients and control subjects (8). Indeed, in the new independent cohort, the SNPs with the strongest association, rs3027933, rs1734791, rs1734792, rs1734787, and rs2075596, had odds ratios (ORs) of 1.38, 1.37, 1.37, 1.35, and 1.35, respectively, and P values of 1.50 × 10−5, 1.92 × 10−5, 2.80 × 10−5, 5.22 × 10−5, and 5.66 × 10−5, respectively (Table 1). All 8 lupus-associated SNPs identified were in linkage disequilibrium (LD), with pairwise correlation coefficient values of ≥0.64. The SNPs with the strongest association, mentioned above, were in strong LD, with pairwise correlation coefficient values of ≥0.95, suggesting that they are surrogates for the same genetic effect.

Table 1. Genetic association between SNPs within MECP2 and lupus in an independent cohort of lupus patients and control subjects of European descent*
SNPRisk alleleRisk allele frequencyχ2OR (95% CI)PP for permutationHWE P
Cases, no. (%)Controls, no. (%)
  • *

    Only single-nucleotide polymorphisms (SNPs) with minor allele frequencies of ≥5% were analyzed. OR = odds ratio; 95% CI = 95% confidence interval; HWE = Hardy-Weinberg equilibrium.

rs2075596A446 (16.8)383 (13.0)16.2121.35 (1.17–1.57)5.66 × 10−50.00030.25
rs3027933C464 (17.4)390 (13.2)18.7341.38 (1.19–1.60)1.50 × 10−51.00 × 10−40.28
rs3027935G2476 (93.3)2721 (92.4)1.5811.14 (0.93–1.40)0.20860.53850.76
rs3027939A2447 (94.8)2721 (93.9)1.8281.17 (0.93–1.48)0.17630.47470.99
rs17435T608 (22.8)575 (19.5)9.0051.22 (1.07–1.38)0.00270.01360.79
rs7050901G2542 (95.2)2786 (94.5)1.5341.16 (0.92–1.47)0.21550.55080.98
rs1624766G604 (22.7)576 (19.5)8.41.21 (1.06–1.37)0.00380.01760.96
rs7884370A2523 (94.7)2773 (94.0)1.1981.14 (0.90–1.43)0.27370.64970.75
rs1734787C459 (17.2)393 (13.3)16.3661.35 (1.17–1.56)5.22 × 10−50.00030.40
rs5987201G2529 (94.8)2772 (94.0)1.6551.16 (0.92–1.46)0.19820.51891.00
rs1734791A464 (17.5)395 (13.4)18.2691.37 (1.18–1.59)1.92 × 10−50.00020.24
rs1734792A462 (17.3)392 (13.3)17.5461.37 (1.18–1.58)2.80 × 10−50.00030.31
rs11156611G2530 (94.8)2773 (94.0)1.3871.15 (0.91–1.44)0.2390.58911.00
rs2239464A583 (21.9)541 (18.4)10.8111.25 (1.09–1.42)0.0010.00530.94

Using the 8 SNPs in MECP2 that were associated with lupus in our cohort, we identified 3 haplotypes with a frequency of >1%. Haplotype 1 (ACTGCAAA) is a disease-risk haplotype (OR 1.38, 95% confidence interval [95% CI] 1.19–1.60, P = 2.36 × 10−5), while haplotype 2 (GGAAATCG) is a protective haplotype (OR 0.82, 95% CI 0.72–0.93, P = 0.0022). These data are consistent with and confirm our previously reported findings (8).

Table 2 summarizes the ORs and the Fisher's combined P values for the MECP2 SNPs associated with lupus in the 3 independent cohorts of lupus patients and control subjects that have been studied to date. The MECP2 SNPs with the strongest association were rs1734787, rs1734792, and rs1734791, with Fisher's combined P values of 6.65 × 10−11, 9.67 × 10−11, and 1.52 × 10−10, respectively.

Table 2. Fisher's combined P values for risk alleles in lupus-associated MECP2 SNPs in 3 lupus cohorts*
SNPRisk alleleKorean cohortEuropean descent cohort 1European descent cohort 2Fisher's combined P
  • *

    Values are the odds ratio (95% confidence interval). The Korean cohort (628 lupus patients and 736 control subjects) and cohort 1 (1,080 lupus patients and 1,080 control subjects) were previously described (see ref.8). Cohort 2 (1,377 lupus patients and 1,853 control subjects) is described in the current study. SNP = single-nucleotide polymorphism.

rs2075596A1.49 (1.24–1.80)1.28 (1.09–1.52)1.35 (1.17–1.57)1.57 × 10−9
rs3027933C1.48 (1.23–1.79)1.30 (1.10–1.53)1.38 (1.19–1.60)2.90 × 10−10
rs17435T1.58 (1.31–1.90)1.29 (1.11–1.49)1.22 (1.07–1.38)1.45 × 10−9
rs1624766G1.50 (1.24–1.82)1.28 (1.10–1.48)1.21 (1.06–1.37)3.76 × 10−8
rs1734787C1.55 (1.29–1.87)1.32 (1.12–1.56)1.35 (1.17–1.56)6.65 × 10−11
rs1734791A1.51 (1.25–1.82)1.31 (1.11–1.54)1.37 (1.18–1.59)1.52 × 10−10
rs1734792A1.53 (1.27–1.83)1.31 (1.11–1.54)1.37 (1.18–1.58)9.67 × 10−11
rs2239464A1.51 (1.25–1.82)1.24 (1.07–1.45)1.25 (1.09–1.42)2.92 × 10−8

Expression of MECP2 in lupus patients with and those without the lupus-associated haplotype.

To determine whether the disease-associated polymorphism within the MECP2 locus alters the expression of MECP2, we determined expression of the 2 known MECP2 transcript isoforms (MECP2A and MECP2B) in female lupus patients homozygous for the disease-risk haplotype and in female lupus patients homozygous for the protective haplotype. MECP2A (isoform 1) includes exon 2, where translation is reported to start. The more recently identified transcript variant, MECP2B (isoform 2), lacks exon 2 and has a translation start site in the first exon (17, 18). There was no detectable difference in the level of either transcript variant in lupus patients with the risk haplotype compared with lupus patients with the protective haplotype, as measured by real-time RT-PCR and primers specific for the 2 transcript isoforms (Figure 1A). However, statistical power to detect differences in this experiment was limited by the number of B cell lines available with the risk and protective homozygous MECP2 haplotypes.

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Figure 1. A, Messenger RNA expression levels of MECP2 transcript variants (MECP2A and MECP2B) in B cells from lupus patients homozygous for the MECP2 risk haplotype and those homozygous for the protective haplotype. Bars show the means. B, Number of CpG islands in up-regulated genes (n = 104) compared with down-regulated genes (n = 24) in lupus patients with the disease-associated MECP2 haplotype. Values are the mean and SEM. ∗ = P = 0.04. C, Confirmation of expression microarray data by real-time reverse transcription–polymerase chain reaction, showing the expression of 5 genes differentially expressed in B cells from 5 patients with the MECP2 risk haplotype compared with 6 patients with the MECP2 protective haplotype. Values are the mean and SEM. ∗ = P < 0.05. D and E, Messenger RNA expression levels of CREB1 (D) and HDAC1 (E) in B cells from lupus patients homozygous for the MECP2 risk haplotype and those homozygous for the protective haplotype. Bars show the mean.

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Identification of functional consequences of the disease-associated MECP2 haplotype.

MECP2 binds methylated DNA, recruits histone deacetylase or CREB1, and functions as a transcription repressor or activator for genes with CG-rich promoter sequences. Therefore, if the lupus-risk MECP2 haplotype we identified alters the function of MECP2, it is likely to affect the expression of a number of target genes that are regulated by MECP2. To test this hypothesis, we used expression microarrays to investigate the expression patterns of genes in B cell lines from 5 female lupus patients homozygous for the disease-risk haplotype and 6 female lupus patients homozygous for the protective haplotype, all of whom were of European descent.

We identified 128 genes that are differentially expressed as a result of the MECP2 haplotype (see Supplementary Tables 1 and 2, available at the Arthritis & Rheumatism Web site at http://www3.interscience.wiley.com/journal/76509746/home). The majority of differentially expressed genes (n = 104 [∼81%]) were up-regulated (≥1.5 fold) in patients with the risk haplotype compared with patients with the protective haplotype, while 24 genes (∼19%) were down-regulated. Interestingly, the number of CpG islands in the regions 5 kb upstream and 5 kb downstream of the transcription start site was significantly higher in the up-regulated genes compared with the down-regulated genes (t = 2.07, df = 120, P = 0.04) (Figure 1B). Several genes that were up-regulated in patients with the MECP2 risk haplotype are interferon (IFN) regulated. These include BTN3A2, CEBPD, CECR1, IFI6 (G1P3), IFI35, IFITM1, IFITM3, IRF7, ISG20, LY6E, PHGDH, S100A10, and ZBP1. An IFN signature is well documented in PBMCs from lupus patients (19, 20).

We conducted a literature-based analysis of shared commonalities for these genes, as previously described (15), and found that several of these genes are associated with epigenetic mechanisms (Table 3). We confirmed the microarray data by examining the expression of 5 genes (CLIC2, IFITM3, IGJ, ITM2B, and TEX15), 3 of which were up-regulated and 2 of which were down-regulated, using real-time RT-PCR (Figure 1C). We next determined mRNA expression levels of HDAC1 and CREB1 in patients homozygous for the disease-risk haplotype and those homozygous for the protective haplotype. HDAC1 and CREB1 are recruited by MECP2 and function as a transcription corepressor and a transcription coactivator, respectively. We observed that the presence of the lupus-risk MECP2 haplotype is associated with higher expression levels of CREB1 (P = 0.04) and lower expression levels of HDAC1 (P = 0.018) (Figures 1D and E).

Table 3. IRIDESCENT algorithm analysis showing shared relationships with genes that are differentially expressed as a result of the MECP2 haplotype present*
Shared relationshipGenes sharedObserved:expected
  • *

    Relationships were identified in Medline. The observed:expected ratio reflects a statistical enrichment score for the association. The empirically determined mean ± SD ratio for a list of 128 genes is 1.42 ± 0.07.

Histone deacetylase142.48
BDNF132.12
Interferon-inducible134.53
Chromatin structure122.15
CREB1122.03
Hypermethylation112.36
Trichostatin A112.83
CpG methylation93.61
Promoter methylation82.69
Aberrant methylation73.61

DISCUSSION

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

We first replicated the association between SNPs within MECP2 and SLE in a large independent cohort of lupus patients and control subjects of European descent (Table 2). Similarly, using this independent cohort of European descent, we further confirmed the previously identified MECP2 lupus risk haplotype ACTGCAAA and the protective haplotype GGAAATCG.

To study the functional consequences of MECP2 polymorphisms on lupus susceptibility, we used transformed B cell lines from lupus patients homozygous for the MECP2 risk haplotype and lupus patients homozygous for the MECP2 protective haplotype. This approach has the advantage of removing any potential confounding effects of environmental factors or medication exposures among lupus patients. We observed no difference in the steady-state mRNA levels of the 2 known MECP2 transcript variants between lupus patients with the risk and protective haplotypes. MECP2 binds to methylated CG dinucleotides in promoter sequences of methylation-sensitive genes and functions as a key transcription repressor, in part by recruiting histone deacetylases, thereby altering chromatin configuration to a transcriptionally inaccessible form (9, 21).

Surprisingly, recent evidence suggests that MECP2 is a key transcription activator that associates with the transcription factor CREB1 in promoter sequences of MECP2-activated genes (10). Indeed, MECP2 functions as a transcription activator in the majority of genes dysregulated in the hypothalamus of MECP2-transgenic and Mecp2-null mice (10). MECP2 target genes are tissue specific, perhaps related to the relative abundance of the various corepressors or coactivators that facilitate the effects of MECP2. Given the dichotomous effects of MECP2 on gene expression, we determined the functional consequences of the lupus-risk MECP2 haplotype compared with the lupus-protective MECP2 haplotype in B cell lines from lupus patients, using expression microarrays.

We identified 128 genes that were differentially expressed as a result of the MECP2 haplotype carried. Interestingly, the majority of the differentially expressed genes (∼81%) were up-regulated in lupus patients homozygous for the risk haplotype. Approximately 85% of genes regulated by MECP2 in the hypothalamus were overexpressed in MECP2-transgenic mice and underexpressed in Mecp2-null mice (10). If this relationship remains true in human B cells, then the lupus-associated MECP2 polymorphism is a surrogate for a gain of MECP2 function. This hypothesis will more readily explain the predominance of lupus in women, who have a copy of MECP2 on each of the 2 X chromosomes coupled with reactivation of the normally inactive X chromosome due to defective DNA methylation that has been described in lupus patients (22).

Genes that are up-regulated in patients homozygous for the risk haplotype contained significantly more CpG islands in their promoter regions compared with down-regulated genes (P = 0.04) (Figure 1B). This is consistent with a gain of MECP2 function as a result of the MECP2 risk haplotype, because genes that are activated by MECP2 were reported to contain more CpG islands compared with genes repressed by MECP2 (10).

The expression of CREB1 was increased in patients with the MECP2 risk haplotype as compared with patients with the protective haplotype. In contrast, the expression of HDAC1, which is an important MECP2 transcription corepressor, was decreased. This predicts that the MECP2 disease-risk haplotype induces an overall overexpression of MECP2-regulated genes, consistent with the results of our expression microarray experiment.

GO analysis revealed several interesting features in the group of genes that are differentially expressed as a result of MECP2 haplotypes. A number of genes up-regulated in B cell lines carrying the risk haplotype are IFN regulated. This is particularly interesting, because up-regulation of IFN-regulated genes in the PBMCs of lupus patients is well established and is linked to disease activity and production of anti–double-stranded DNA antibodies (19, 23, 24). Of note, both IFNγ and IFNβ are known to be regulated by epigenetic mechanisms (25, 26), suggesting that epigenetic dysregulation of IFN genes is a plausible functional consequence of MECP2 polymorphism in lupus patients.

In a mouse model with an inducible ERK signaling defect resulting in reduced DNA methyltransferase 1 expression and abnormal expression of methylation-sensitive genes, differential expression of IFN-regulated genes has also been reported (27). Furthermore, stimulated T cells from female mice with a truncated form of MECP2 (Mecp2308/308) demonstrate significant overexpression of IFNγ compared with T cells from wild-type mice (Sawalha AH, et al: unpublished observations).

We used the IRIDESCENT algorithm (28, 29) to search the Medline database for relationships in the literature with the list of the genes differentially expressed as a result of MECP2 haplotypes. Several interesting significant relationships with epigenetic-related mechanisms were identified (Table 3). For example, among the up-regulated genes in patients with the risk haplotype, TMS1 (target of methylation-induced silencing 1; PYCARD; ASC) is a proapoptotic gene that is methylation sensitive and is epigenetically silenced in some cancers (30) and was recently shown to affect the innate inflammatory response (31). The results of our current study suggest that it is also sensitive to MECP2, either directly or indirectly. The vimentin and p18 genes (VIM and CDKN2C, respectively), which have been shown to be hypermethylated in some cancers (32, 33), were also up-regulated. The expression of ITGAL (CD11a), an integrin molecule, is known to be regulated by DNA methylation (34). ITGAL is hypomethylated and overexpressed in lupus T cells, and its overexpression is associated with T cell autoreactivity in lupus patients (2, 35). ITGAL expression was also up-regulated in B cells from lupus patients with the MECP2 risk haplotype.

Among the down-regulated genes was the proto-oncogene MYC (c-myc), which is known to affect DNA methylation and histone modifications (36, 37) and has previously been implicated in autoimmunity and SLE (38, 39). SMARCA2 is a member of the chromatin remodeling family (SWI/SNF) of genes that regulate transcription by altering chromatin structure and was recently reported to be up-regulated in the immunodeficiency–centromeric instability–facial anomalies syndrome, which is known to result from a mutation in the DNA-methylating enzyme DNMT3B (40). PEG10 (paternally expressed gene 10), an imprinted gene (41), was also down-regulated in lupus patients with the risk haplotype.

We observed a strong relationship in the literature between the differentially expressed genes, as a consequence of the MECP2 haplotype carried, and epigenetic mechanisms including DNA methylation and histone modification. This provides further evidence for a role of the identified MECP2 haplotypes in epigenetic dysregulation and supports the fact that the differentially expressed genes reflect target genes for MECP2 that are altered as a result of the lupus-associated MECP2 polymorphism. Of interest, this literature search identified a relationship between a set of our differentially expressed genes and CREB1. CREB1 is a known transcription factor that has recently been identified as a key player in MECP2-induced transcription activation (10). Furthermore, we identify a relationship in the literature with brain-derived neurotrophic factor, which is the first mammalian neuronal target gene for MECP2 identified and is thought to play a pathogenic role in patients with Rett Syndrome–associated MECP2 mutations (42).

In conclusion, our data replicate and further confirm the genetic association of polymorphism within the MECP2 gene and lupus. We identified several target genes that are dysregulated in B cells from lupus patients with the MECP2 lupus-risk haplotype. Importantly, the MECP2 risk haplotype is associated with increased expression of a number of IFN-regulated genes and may play a role in the IFN signature observed in lupus patients. Furthermore, the MECP2 target genes identified in B cells from lupus patients can potentially uncover various aspects in the pathogenesis of the disease and help provide new therapeutic targets for lupus.

AUTHOR CONTRIBUTIONS

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

Dr. Sawalha 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 design. Webb, Kaufman, Kimberly, Harley, Sawalha.

Acquisition of data. Webb, Jeffries, Kaufman, Frank, Merrill, Ramsey-Goldman, Petri, Reveille, Alarcón, Vilá, James, Moser, Gaffney, Gilkeson, Harley, Sawalha.

Analysis and interpretation of data. Wren, Kaufman, Tang, Frank, Harley, Sawalha.

Manuscript preparation. Webb, Wren, Merrill, Reveille, Alarcón-Riquelme, James, Harley, Sawalha.

Statistical analysis. Wren, Kelly, Kaufman, Tang, Sawalha.

Collection/provision of samples/biologic material. Merrill, Kimberly, Edberg, Alarcón-Riquelme, Vyse.

Acknowledgements

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

We thank Dr. Peter Gregersen for providing control DNA samples, and the Lupus Family Registry and Repository at the Oklahoma Medical Research Foundation for recruiting patients with lupus and providing the clinical materials 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
  9. Supporting Information

Supporting Information

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

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ART_24360_sm_supplTable1-2.doc561KSupplementary Table 1. Genes upregulated (≥1.5 fold) in lupus patients homozygous for the lupus-associated MECP2 risk haplotype as compared to lupus patients homozygous for the MECP2 protective haplotype; Supplementary Table 2. Genes downregulated (≥1.5 fold) in lupus patients homozygous for the lupus-associated MECP2 risk haplotype as compared to lupus patients homozygous for the MECP2 protective haplotype

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