Factors responsible for radiographic severity of rheumatoid arthritis (RA) in African-Americans are poorly understood. We sought to identify genes whose expression in peripheral blood mononuclear cells (PBMCs) is associated with radiographic severity of RA.
We initially performed quantitative real-time (qRT)-PCR for 182 genes based on plausible immune pathways in 40 African-American RA patients with extremes of radiographic damage (low versus high radiographic scores) and disease duration (early versus late) and 20 healthy African-American controls. In the second phase, we analyzed the expression of significantly associated candidate genes (IFNGR2 and its biological partner IFNGR1) with radiographic scores in 576 African-American RA patients and 51 controls not previously analyzed, accounting for autoantibody status and disease duration.
We found significant differences in IFNGR1 expression between RA and controls (P=6 × 10−14) and in IFNGR2 expression between those with erosions vs no erosions (P=0.01) (Wilcoxon sum test). We also found A significant correlations between IFNGR2 expression and radiographic scores (P=0.03 for erosions, P=0.04 for joint space narrowing, and P=0.03 for total radiographic score, zero-inflated negative binomial model) and annualized progression rate (P=0.0024, Spearman correlation).
These findings have important implications with respect to IFNγ for the pathogenesis of RA and may lead to identification of a biomarker for radiographic damage. Additional studies are needed to define cell subsets responsible for the association of IFNγ receptor gene expression with radiographic finding, which downstream mechanisms are involved, and generalizability to other RA populations.
To evaluate African American rheumatoid arthritis HLA-DRB1 genetic risk by three validated allele classification systems, and by amino acid position and residue. To compare the genetic risk between African American and European ancestries.
Four-digit HLA-DRB1 genotyping was performed on 561 autoantibody-positive African American cases and 776 African American controls. Association analysis was performed on Tezenas du Montcel (TdM); de Vries (DV); and Mattey classification system alleles and separately by amino acid position and individual residues.
TdM S2 and S3P alleles were associated with RA (odds ratios (95% CI) 2.8 (2.0, 3.9) and 2.1 (1.7, 2.7), respectively). The DV (P-value=3.2 x 10−12) and Mattey (P-value=6.5 x 10−13) system alleles were both protective in African Americans. Amino acid position 11 (permutation P-value < 0.00001) accounted for nearly all variability explained by HLA-DRB1, although conditional analysis demonstrated that position 57 was also significant (0.01<= permutation P-val <=0.05). The valine and aspartic acid residues at position 11 conferred the highest risk for RA in African Americans.
With some exceptions, the genetic risk conferred by HLA-DRB1 in African Americans is similar to European ancestry at multiple levels: classification system (e.g., TdM), amino acid position (e.g. 11) and residue (Val 11). Unlike that reported from European ancestry, amino acid position 57 was associated with RA in African Americans, but positions 71 and 74 were not. Asp11 (OR = 1 in European ancestry) corresponds to the four digit classical allele, *09:01, also a risk allele for RA in Koreans.
This study investigates the association of CRP single nucleotide polymorphisms (SNPs) with plasma CRP levels and radiographic severity in African Americans with early and established rheumatoid arthritis (RA). Using a cross-sectional case-only design, CRP SNPs were genotyped in two independent sets of African Americans with RA: Consortium for the Longitudinal Evaluation of African Americans with RA (CLEAR 1) and CLEAR 2. Radiographic data and CRP measurements were available in 294 individuals from CLEAR 1 [median (IQR 25-75) disease duration of 1 (0.6-1.6) year] and in 407 persons from CLEAR 2 [median (IQR 25-75) disease duration of 8.9 (3.5 – 17.7) years]. In CLEAR 1, in adjusted models, the minor allele of rs2808630 was associated with total radiographic score [incident rate ratio (IRR) 0.37 (95% CI 0.19-0.74), p value =0.0051]. In CLEAR 2, the minor allele of rs3093062 was associated with increased plasma CRP levels (p value =0.002). For each rs3093062 minor allele, the plasma CRP increased by 1.51 (95% CI 1.15-1.95) mg/dL when all the other covariates remained constant. These findings have important implications for assessment of the risk of joint damage in African Americans with RA.
C reactive protein; genetics; rheumatoid arthritis; joint damage; radiography
Methotrexate (MTX) has emerged as first-line therapy for early moderate to severe rheumatoid arthritis (RA), but individual variation in treatment response remains unexplained. We tested the associations between 863 known pharmacogenetic variants and MTX response in 471 TEAR Trial participants with early RA. Efficacy and toxicity were modeled using multiple regression, adjusted for demographic and clinical covariates. Penalized regression models were used to test joint associations of markers and/or covariates with the outcomes. The strongest genetic associations with efficacy were in CHST11 (five markers with P <0.003), encoding carbohydrate (chondroitin 4) sulfotransferase 11. Top markers associated with MTX toxicity were in the cytochrome p450 genes CYP20A1 and CYP39A1, solute carrier genes SLC22A2 and SLC7A7, and the mitochondrial aldehyde dehydrogenase gene ALDH2. The selected markers explained a consistently higher proportion of variation in toxicity than efficacy. These findings could inform future development of personalized therapeutic approaches.
Methotrexate; rheumatoid arthritis; pharmacogenetics
To examine the degree to which shared risk factors explain the relationship of periodontitis (PD) with rheumatoid arthritis (RA) and to examine associations of PD and Porphyomonas gingivalis (Pg) with disease features.
RA cases (N=287) and controls (N=330) underwent a standardized periodontal examination. HLA-DRB1 status was imputed using SNPs from the extended MHC. Circulating anti-Pg antibody was measured using ELISA and subgingival plaque was assessed for the presence of Pg using PCR. Associations of PD with RA were examined using multivariable regression.
PD was more common in RA (35%, p = 0.022) and aCCP positive RA (n=240; 37%; p = 0.006) vs. controls (26%). There were no RA-control differences in anti-Pg or the frequency of Pg positivity by PCR. Anti-Pg antibody showed weak but statistically significant associations with both anti-CCP (r=0.14, p=0.022) and RF (r=0.19, p=0.001). PD was associated with increased swollen joint counts (p=0.004), DAS-28-CRP (p=0.045), total Sharp scores (p=0.015), aCCP (p=0.011), and RF (p<0.001). Select anti-citrullinated peptide antibody (ACPA; including antibody to citrullinated filaggrin) were higher in patients with subgingival Pg and higher anti-Pg antibody levels irrespective of smoking. Associations of PD with established seropositive RA were independent of all covariates examined including evidence of Pg infection.
Both PD and Pg appear to shape RA-related autoreactivity in RA. In addition, PD demonstrates an independent relationship with established seropositive RA.
rheumatoid arthritis; periodontitis; Porphyromonas gingivalis; Prevotella intermedia; Fusobacterium nucleatum; anti-citrullinated protein antibody; disease severity
Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard for quantifying relative gene expression. Normalization of RT-qPCR data is commonly achieved by subtracting the Ct values of the internal reference genes from the Ct values of the target genes to obtain ΔCt. ΔCt values are then used to derive ΔΔCt when compared to a control group or to conduct further statistical analysis.
We examined two rheumatoid arthritis RT-qPCR low density array datasets and found that this normalization method introduces substantial bias due to differences in PCR amplification efficiency among genes. This bias results in undesirable correlations between target genes and reference genes, which affect the estimation of fold changes and the tests for differentially expressed genes. Similar biases were also found in multiple public mRNA and miRNA RT-qPCR array datasets we analysed. We propose to regress the Ct values of the target genes onto those of the reference genes to obtain regression coefficients, which are then used to adjust the reference gene Ct values before calculating ΔCt.
The per-gene regression method effectively removes the ΔCt bias. This method can be applied to both low density RT-qPCR arrays and individual RT-qPCR assays.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1274-1) contains supplementary material, which is available to authorized users.
RT-PCR; Normalization; ΔCt; Housekeeping genes; Regression
The aim of this study was to identify genetic variants associated with rheumatoid arthritis (RA) risk in black South Africans. Black South African RA patients (n = 263) were compared with healthy controls (n = 374). Genotyping was performed using the Immunochip, and four-digit high-resolution human leukocyte antigen (HLA) typing was performed by DNA sequencing of exon 2. Standard quality control measures were implemented on the data. The strongest associations were in the intergenic region between the HLA-DRB1 and HLA-DQA1 loci. After conditioning on HLA-DRB1 alleles, the effect in the rest of the extended major histocompatibility (MHC) diminished. Non-HLA single nucleotide polymorphisms (SNPs) in the intergenic regions LOC389203|RBPJ, LOC100131131|IL1R1, KIAA1919|REV3L, LOC643749|TRAF3IP2, and SNPs in the intron and untranslated regions (UTR) of IRF1 and the intronic region of ICOS and KIAA1542 showed association with RA (p < 5 × 10−5). Of the SNPs previously associated with RA in Caucasians, one SNP, rs874040, locating to the intergenic region LOC389203|RBPJ was replicated in this study. None of the variants in the PTPN22 gene was significantly associated. The seropositive subgroups showed similar results to the overall cohort. The effects observed across the HLA region are most likely due to HLA-DRB1, and secondary effects in the extended MHC cannot be detected. Seven non-HLA loci are associated with RA in black South Africans. Similar to Caucasians, the intergenic region between LOC38920 and RBPJ is associated with RA in this population. The strong association of the R620W variant of the PTPN22 gene with RA in Caucasians was not replicated since this variant was monomorphic in our study, but other SNP variants of the PTPN22 gene were also not associated with RA in black South Africans, suggesting that this locus does not play a major role in RA in this population.
The Disease Activity Score based on 28 joints (DAS28) has been increasingly used in clinical practice and research studies of rheumatoid arthritis (RA). Studies have reported discordance between DAS28 based on erythrocyte sedimentation rate (ESR) versus C-reactive protein (CRP) in RA patients. However such comparison is lacking in African-Americans with RA.
This analysis included participants from the Consortium for the Longitudinal Evaluation of African Americans with Early Rheumatoid Arthritis (CLEAR) Registry which enrolls self-declared African-Americans with RA. Using tender and swollen joint counts separate ESR-based and CRP-based DAS28 scores (DAS28-ESR3 and DAS28-CRP3) were calculated, as were DAS28-ESR4 and DAS28-CRP4, which included the patient’s assessment of disease activity. The scores were compared using paired t-test, simple agreement and kappa, correlation coefficient and Bland-Altman plots.
Of the 233 included participants, 85% were women, mean age at enrollment was 52.6 years, and median disease duration at enrollment was 21 months. Mean DAS28-ESR3 was significantly higher than DAS28-CRP3 (4.8 vs. 3.9; p<0.001). Similarly, mean DAS28-ESR4 was significantly higher than DAS28-CRP4 (4.7 vs. 3.9; p<0.001). ESR-based DAS28 remained higher than CRP-based DAS28 even when stratified by age, sex, and disease duration. Overall agreement was not high between DAS28-ESR3 and DAS28-CRP3 (50%) or between DAS28-ESR4 and DAS28-CRP4 (59%). DAS28-CRP3 underestimated disease activity in 47% of the participants relative to DAS28-ESR3 and DAS28-CRP4 in 40% of the participants relative to DAS28-ESR4.
There was significant discordance between the ESR-based and CRP-based DAS28 which could impact clinical treatment decisions in African-Americans with RA.
DAS28; Rheumatoid Arthritis; African-Americans
Gene expression profiling may be used to stratify patients by disease severity to test the hypothesis that variable disease outcome has a genetic component. In order to define unique expression signatures in African American rheumatoid arthritis (RA) patients with severe erosive disease, we undertook a gene expression study using samples of RNA from peripheral blood mononuclear cells (PBMCs). RNA from baseline PBMC samples of 96 African American RA patients with early RA (<2 years disease duration) was hybridized to cDNA probes of the Illumina Human HT-V3 expression array. Expression analyses were performed using the ca. 25,000 cDNA probes, and then expression levels were compared to the total number of erosions in radiographs of the hands and feet at baseline and 36 months. Using a false discovery rate cutoff of Q = 0.30, 1,138 genes at baseline and 680 genes at 36 months significantly correlated with total erosions. No evidence of a signal differentiating disease progression, or change in erosion scores between baseline and 36 months, was found. Further analyses demonstrated that the differential gene expression signature was localized to the patients with the most erosive disease (>10 erosions). Ingenuity Pathway Analysis demonstrated that genes with fold change greater than 1.5 implicated immune pathways such as CTLA signaling in cytotoxic T lymphocytes. These results demonstrate that CLEAR patients with early RA having the most severe erosive disease, as compared to more mild cases (<10 erosions), may be characterized by a set of differentially expressed genes that represent biological pathways with relevance to autoimmune disease.
Genome-wide gene expression; Sharp/van der Heijde; Pathway analysis; CLEAR; ABCoN
Rapidly predicting future outcomes based upon short-term clinical response would be helpful to optimize RA management in early disease.
To derive and validate a clinical prediction rule to predict low disease activity (LDA) at 1 year among patients participating in the Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) trial escalating RA therapy by adding either etanercept (E) or sulfasalazine + hydroxychloroquine [triple therapy (TT)] after 6 months of methotrexate (MTX) therapy.
Eligible subjects included in the derivation cohort (used for model building, n=186) were participants with moderate or higher disease activity (DAS28ESR>3.2) despite 24 weeks of MTX monotherapy who added either etanercept or sulfasalazine+hydroxychloroquine. Clinical characteristics measured within the next 12 weeks were used to predict LDA 1 year later using multivariable logistic regression. Validation was performed in the cohort of TEAR patients randomized to initially receive either MTX+E or TT.
The derivation cohort yielded three prediction models of varying complexity that included age, DAS28 at various time points, body mass index, and ESR (AUROC up to 0.83). Accuracy of the prediction models ranged between 80 and 95% in both derivation and validation cohorts, depending on the complexity of the model and the cutpoints chosen for response and non-response. Approximately 80% of patients could be predicted to be responders or non-responders at week 12.
Clinical data collected early after starting or escalating DMARD/biologic treatment could accurately predict LDA at 1 year in early RA patients. For patients predicted to be non-responders, treatment could be changed at 12 weeks to optimize outcomes.
rheumatoid arthritis; prediction; anti-TNF; triple therapy
We previously reported an analysis of single nucleotide polymorphisms (SNPs) in three validated European rheumatoid arthritis (RA) susceptibility loci, TAGAP, TNFAIP3, and CCR6 in African-Americans with RA. Unexpectedly, the disease-associated alleles were different in African-Americans than in Europeans. In an effort to better define their contribution, we performed additional SNP genotyping in these genes.
Seven SNPs were genotyped in 446 African Americans with RA and 733 African American controls. Differences in minor allele frequency between cases and controls were analyzed after controlling for global proportion of European admixture, and pairwise linkage disequilibrium (LD) was estimated among the SNPs.
Three SNPs were significantly associated with RA: TNFAIP3 rs719149 A allele (OR (95% CI) 1.22 (1.03–1.44) (p =0.02); TAGAP rs1738074 G allele OR 0.75 (0.63–0.89), (p =0.0012); and TAGAP rs4709267 G allele 0.74 (0.60–0.91), (p =0.004). Pairwise LD between the TAGAP SNPs was low (R2=0.034). The haplotype containing minor alleles for both TAGAP SNPs was uncommon (4.5%). After conditional analysis on each TAGAP SNP, its counterpart remained significantly associated with RA (rs1738074 for rs4709267 p=0.00001; rs4709267 for rs1738074 p=0.00005), suggesting independent effects.
SNPs in regulatory regions of TAGAP and an intronic SNP (TNFAIP3) are potential susceptibility loci in African Americans. Pairwise LD, haplotype analysis, and SNP conditioning analysis suggest that these two SNPs in TAGAP are independent susceptibility alleles. Additional fine mapping of this gene and functional genomic studies of these SNPs should provide additional insight into the role of these genes in RA.
Large-scale genetic association studies have identified over 20 rheumatoid arthritis (RA) risk alleles among individuals of European ancestry. The influence of these risk alleles has not been comprehensively studied in African-Americans. We therefore sought to examine whether these validated RA risk alleles are associated with RA in an African-American population.
27 candidate SNPs were genotyped in 556 autoantibody-positive African-Americans with RA and 791 healthy African-American controls. Odds ratios (OR) and 95% confidence intervals (CI) for each SNP were compared to previously published ORs of RA patients of European ancestry. We then calculated a composite Genetic Risk Score (GRS) for each individual based on the sum of all risk alleles.
There was overlap in the OR and 95% CI between the European and African-American populations in 24 of the 27 candidate SNPs. Conversely, 3 of the 27 SNPs (CCR6 rs3093023, TAGAP rs394581, TNFAIP3 rs6920220) demonstrated an OR in the opposite direction from those reported in RA patients of European ancestry. The GRS analysis indicated a small but highly significant probability that African-American cases were enriched for the European RA risk alleles relative to controls (p=0.00005).
The majority of RA risk alleles previously validated among European ancestry RA patients showed similar ORs in our population of African-Americans with RA. Furthermore, the aggregate GRS supports the hypothesis that these SNPs are risk alleles for RA in the African-American population. Future large-scale genetic studies are needed to validate these risk alleles and identify novel risk alleles for RA in African-Americans.
Canonical analysis measures nonlinear selection on latent axes from a rotation of the gamma matrix (γ) of quadratic and correlation selection gradients. Here we document that the conventional method of testing eigenvalues (double regression) under the null hypothesis of no nonlinear selection is incorrect. Through simulation we demonstrate that under the null the expectation of some eigenvalues from canonical analysis will be nonzero, which leads to unacceptably high type 1 error rates. Using a two trait example, we prove that the expectations for both eigenvalues depend on the sampling variability of the estimates in γ. An appropriate test is to slightly modify the double regression method by calculating permutation p-values for the ordered eigenvalues, which maintains correct type 1 error rates. Using simulated data of nonlinear selection on male guppy ornamentation, we show that the statistical power to detect curvature with canonical analysis is higher compared to relying on the estimates from γ alone. We provide a simple R script for permutation testing of the eigenvalues in order to distinguish curvature in the selection surface induced by nonlinear selection from curvature induced by random processes.
Nonlinear Selection; Phenotypic Selection; Selection Surface; Fitness Surface; Stabilizing Selection
We proposed hierarchical Poisson and binomial models for mapping multiple interacting quantitative trait loci (QTL) for count traits in experimental crosses. We applied our methods to two counted reproductive traits, live fetuses (LF) and dead fetuses (DF) at 17 days gestation, in a F2 female mouse population. We treated observed number of corpora lutea (ovulation rate) as the baseline and the total trials in our Poisson and binomial models, respectively. We detected more than 10 QTL for LF and DF, most having epistatic and pleiotropic effects. The epistatic effects were larger, involved more QTL, and explained a larger proportion of phenotypic variance than the main effects. Our analyses revealed a complex network of multiple interacting QTL for the reproductive traits, and increase our understanding of the genetic architecture of reproductive characters. The proposed statistical models and methods provide valuable tools for detecting multiple interacting QTL for complex count phenotypes.
Count phenotypes; Generalized linear models; Epistasis; Quantitative trait loci; Reproduction
Notwithstanding the well established association of HLA-DRB1 shared epitope alleles, interest remains in identifying additional Major Histocompatibility Complex (MHC) region variants associated with rheumatoid arthritis (RA). We used a panel of 1,201 haplotype tagging single nucleotide polymorphisms (SNPs) designed for African Americans to find genetic variants associated with RA in a 3.8 Mb region encompassing the MHC. Conditioning on seven covariates including HLA-DRB1 risk alleles and population structure, we identified a SNP in HLA-DOA (rs9276977) significantly associated with RA; minor allele frequency (MAF) 0.27 in cases versus 0.21 in controls: OR(±95% CI) = 2.86 (1.61, 5.31). Genotyping of rs9276977 in an independent sample of African-American RA patients and controls did not replicate the association (MAF 0.28 in cases versus 0.27 in controls). This study points to the potential association of a SNP in the HLA-DOA gene with RA in African-Americans, but also underscores the importance of replication of findings in larger patient cohorts.
genetic association; MHC; HLA-DOA; African American; HLA-DRB1
A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD+ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.