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1.  Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization 
BMC Genomics  2015;16(1):82.
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.
PMCID: PMC4335788
RT-PCR; Normalization; ΔCt; Housekeeping genes; Regression
2.  Genome-wide association analysis of anti-TNF drug response in rheumatoid arthritis patients 
Annals of the rheumatic diseases  2012;72(8):1375-1381.
Treatment strategies blocking tumor necrosis factor (anti-TNF) have proven very successful in patients with rheumatoid arthritis (RA). However, a significant subset of patients does not respond for unknown reasons. Currently there are no means of identifying these patients prior to treatment. This study was aimed at identifying genetic factors predicting anti-TNF treatment outcome in patient with RA using a genome-wide association approach.
We conducted a multi-stage, genome-wide association study with a primary analysis of 2,557,253 single nucleotide polymorphisms (SNPs) in 882 RA patients receiving anti-TNF therapy included through the Dutch Rheumatoid Arthritis Monitoring (DREAM) registry and the database of Apotheekzorg. Linear regression analysis of changes in the Disease Activity Score in 28 joints after 14 weeks of treatment was performed using an additive model. Markers with a p<10−3 were selected for replication in 1,821 RA patients from three independent cohorts. Pathway analysis including all SNPs with a p-value < 10−3 was performed using Ingenuity.
Seven hundred seventy two markers demonstrated evidence of association with treatment outcome in the initial stage. Eight genetic loci showed improved p-value in the overall meta-analysis compared to the first stage, three of which (rs1568885, rs1813443 and rs4411591) showed directional consistency over all four studied cohorts. We were unable to replicate markers previously reported to be associated with anti-TNF outcome. Network analysis indicated strong involvement of biological processes underlying inflammatory response and cell morphology.
Using a multi-stage strategy, we have identified 8 genetic loci associated with response to anti-TNF treatment. Further studies are required to validate these findings in additional patient collections.
PMCID: PMC4169706  PMID: 23233654
anti-TNF; gene polymorphism; pharmacogenetics; rheumatoid arthritis; genome-wide association study
3.  Inhibition of Fucosylation Reshapes Inflammatory Macrophages and Suppresses Type II Collagen-Induced Arthritis 
Fucosylation catalyzed by fucosyltransferases (FUTs) is an important post-translational modification involved in a variety of biological processes. The purpose of the current study is to determine the roles of fucosylation in rheumatoid arthritis (RA) and the efficacy of reestablishing the immune homeostasis by using 2-Deoxy-D-galactose (2-D-gal), a fucosylation inhibitor.
Q-PCR was performed to determine the expression of fucosyltransferases (FUTs) in RA and osteoarthritis (OA) synovial tissues and FACS sorted cells from RA synovial fluids. The in vivo inhibitory effect of 2-D-gal was evaluated in a collagen-induced arthritis (CIA) model. The in vitro effects of 2-D-gal on inflammatory macrophage differentiation, cytokine production, antigen uptake, processing, and presenting functions were analyzed.
FUTs that are involved in terminal or sub-terminal, but not core or O-fucosylation, were upregulated in RA, compared to OA synovial tissues. The expression of terminal FUTs was highly positively correlated with that of TNF encoding for tumor necrosis factor α. Terminal FUTs were predominately expressed in M1 macrophages. In vivo, 2-D-gal treatment precluded CIA development with reduced inflammatory macrophages and Th17 in draining lymph nodes, decreased TNF-α, IL-6 and antibodies to type II collagen in the serum. In vitro, 2-D-gal skewed the differentiation of M1 macrophages to IL-10 producing M2 macrophages. Furthermore, 2-D-gal significantly inhibited antigen presenting function of M1 macrophages.
Terminal fucosylation is a novel hallmark of inflammatory macrophages. Inhibition of terminal FUTs reshapes the differentiation and functions M1 macrophages, leading to resolution of inflammation in arthritis.
PMCID: PMC4146698  PMID: 24838610
4.  Genetics of rheumatoid arthritis contributes to biology and drug discovery 
Okada, Yukinori | Wu, Di | Trynka, Gosia | Raj, Towfique | Terao, Chikashi | Ikari, Katsunori | Kochi, Yuta | Ohmura, Koichiro | Suzuki, Akari | Yoshida, Shinji | Graham, Robert R. | Manoharan, Arun | Ortmann, Ward | Bhangale, Tushar | Denny, Joshua C. | Carroll, Robert J. | Eyler, Anne E. | Greenberg, Jeffrey D. | Kremer, Joel M. | Pappas, Dimitrios A. | Jiang, Lei | Yin, Jian | Ye, Lingying | Su, Ding-Feng | Yang, Jian | Xie, Gang | Keystone, Ed | Westra, Harm-Jan | Esko, Tõnu | Metspalu, Andres | Zhou, Xuezhong | Gupta, Namrata | Mirel, Daniel | Stahl, Eli A. | Diogo, Dorothée | Cui, Jing | Liao, Katherine | Guo, Michael H. | Myouzen, Keiko | Kawaguchi, Takahisa | Coenen, Marieke J.H. | van Riel, Piet L.C.M. | van de Laar, Mart A.F.J. | Guchelaar, Henk-Jan | Huizinga, Tom W.J. | Dieudé, Philippe | Mariette, Xavier | Bridges, S. Louis | Zhernakova, Alexandra | Toes, Rene E.M. | Tak, Paul P. | Miceli-Richard, Corinne | Bang, So-Young | Lee, Hye-Soon | Martin, Javier | Gonzalez-Gay, Miguel A. | Rodriguez-Rodriguez, Luis | Rantapää-Dahlqvist, Solbritt | Ärlestig, Lisbeth | Choi, Hyon K. | Kamatani, Yoichiro | Galan, Pilar | Lathrop, Mark | Eyre, Steve | Bowes, John | Barton, Anne | de Vries, Niek | Moreland, Larry W. | Criswell, Lindsey A. | Karlson, Elizabeth W. | Taniguchi, Atsuo | Yamada, Ryo | Kubo, Michiaki | Liu, Jun S. | Bae, Sang-Cheol | Worthington, Jane | Padyukov, Leonid | Klareskog, Lars | Gregersen, Peter K. | Raychaudhuri, Soumya | Stranger, Barbara E. | De Jager, Philip L. | Franke, Lude | Visscher, Peter M. | Brown, Matthew A. | Yamanaka, Hisashi | Mimori, Tsuneyo | Takahashi, Atsushi | Xu, Huji | Behrens, Timothy W. | Siminovitch, Katherine A. | Momohara, Shigeki | Matsuda, Fumihiko | Yamamoto, Kazuhiko | Plenge, Robert M.
Nature  2013;506(7488):376-381.
A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.
PMCID: PMC3944098  PMID: 24390342
5.  CASPASE-12 and rheumatoid arthritis in African-Americans 
Immunogenetics  2014;66(4):281-285.
CASPASE-12 (CASP12) has a down-regulatory function during infection, and thus may protect against inflammatory disease. We investigated the distribution of CASP12 alleles (#rs497116) in African-Americans (AA) with rheumatoid arthritis (RA). CASP12 alleles were genotyped in 953 RA patients and 342 controls. Statistical analyses comparing genotype groups were performed using Kruskal-Wallis non-parametric ANOVA with Mann-Whitney U tests and chi-square tests. There was no significant difference in the overall distribution of CASP12 genotypes within AA with RA, but CASP12 homozygous patients had lower baseline joint narrowing scores. CASP12 homozygosity appears to be a subtle protective factor for some aspects of RA in AA patients.
PMCID: PMC4139147  PMID: 24515649
6.  Immunochip Identifies Novel, and Replicates Known, Genetic Risk Loci for Rheumatoid Arthritis in Black South Africans 
Molecular Medicine  2014;20(1):341-349.
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.
PMCID: PMC4153842  PMID: 25014791
7.  Genetic Variants Associated with Methotrexate Efficacy and Toxicity in Early Rheumatoid Arthritis: Results from the Treatment of Early Aggressive Rheumatoid Arthritis Trial 
The pharmacogenomics journal  2013;14(1):48-53.
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.
PMCID: PMC3701736  PMID: 23545897
Methotrexate; rheumatoid arthritis; pharmacogenetics
10.  Changes in Lipoproteins Associated with Treatment with Methotrexate or Combination Therapy in Early Rheumatoid Arthritis: Results from the TEAR Trial 
Arthritis and rheumatism  2013;65(6):1430-1438.
To study changes in lipid profiles at 24 weeks among early rheumatoid arthritis (RA) patients participating in the Treatment of Early Rheumatoid Arthritis (TEAR) Trial randomized to initiate methotrexate plus etanercept (MTX+ETA), triple therapy (TT) [MTX plus sulfasalazine plus hydroxychloroquine] or aggressively-titrated MTX monotherapy.
The TEAR biorepository study had 459 participating patients. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured in serum plasma at 0 and 24 weeks.
At 24 weeks, there were statistically significant mean increases in cholesterol levels in the MTX + ETA, TT, and MTX monotherapy arms, the observed increases were 31.4, 28.7 and 30 mg/dL in LDL-C; 19.3, 22.3 and 20.6 mg/dL in HDL-C and 56.8, 53 and 57.3 mg/dL values in TC (p < 0.001 all compared to baseline). There was a statistically significant decrease in TC/HDL-C ratio at 24 weeks in all 3 treatment groups from baseline. There was no difference in any lipid changes between the 3 treatment arms. After multivariable adjustment, change in C-reactive protein was associated with change in LDL-C (p=0.03), HDL-C (p=0.09), and TC (p=0.01), but disease activity score in 28-joints was not. Baseline glucocorticoid use was associated with changes in HDL-C (p=0.03) and TC (p=0.02).
Levels of TC, LDL-C, and HDL-C increased equivalently shortly after initiation of MTX + ETA, TT and MTX monotherapy among early RA patients with active disease participating in a clinical trial. The clinical relevance of short term changes in traditional lipids on cardiovascular outcomes remains to be determined.
PMCID: PMC3672346  PMID: 23460074
rheumatoid arthritis; etanercept; methotrexate; cholesterol; lipoprotein; cardiovascular
12.  A Randomized Comparative Effectiveness Study of Oral Triple Therapy versus Etanercept plus Methotrexate in Early, Aggressive Rheumatoid Arthritis 
Arthritis and rheumatism  2012;64(9):2824-2835.
To assess if it is better to intensively treat all early RA patients with drug combinations or reserve this for those who do not appropriately respond to methotrexate monotherapy and assess if the combination therapy of methotrexate plus etanercept is superior to the combination of methotrexate plus sulfasalazine plus hydroxychloroquine.
The TEAR study is a 2-year, randomized, double-blind trial. Using a 2×2 factorial design, participants were randomized to one of four treatment arms: immediate combination therapy of methotrexate plus etanercept; or oral triple therapy (methotrexate plus sulfasalazine plus hydroxychloroquine); or initial methotrexate monotherapy with a step-up to one of the combination therapies (all arms included matching placebos). The primary outcome was an observed-group analysis of DAS28-ESR scores from weeks 48 to 102.
At the week 24 step-up period, those receiving immediate combination therapy (etanercept plus methotrexate; or triple therapy) demonstrated greater reduction in DAS28-ESR compared to those on initial methotrexate monotherapy (DAS28-ESR: 3.6 vs. 4.6, p<0.0001), with no differences between regimens of combination therapy. For weeks 48 through 102, participants randomized to step-up arms had a DAS28-ESR clinical response that was not different than those who received initial combination therapy, regardless of the treatment arm (3.2 vs. 3.2, p=0.75). There was no significant difference in DAS28-ESR between participants receiving oral triple therapy versus combination methotrexate plus etanercept (3.1 vs. 3.2, p=0.42). By week 102, there was a small, statistically significant difference in change in radiographic measurements from baseline between methotrexate plus etanercept compared to oral triple therapy (0.64 vs. 1.69, p= 0.047). The absolute difference at week 102 was small.
There were no differences in the mean DAS28-ESR during weeks 48-102 between participants randomized to methotrexate plus etanercept or triple therapy, regardless of whether they received immediate combination treatment or step-up from methotrexate monotherapy. At 24 months, immediate combination treatment with either strategy was more effective than methotrexate monotherapy prior to step-up. Initial use of methotrexate monotherapy with the addition of sulfasalazine plus hydroxychloroquine; or etanercept, if necessary after 6 months, is a reasonable therapeutic strategy for early RA. The combination of etanercept plus methotrexate resulted in a statistically significant, but clinically small, radiographic benefit over oral triple therapy.
PMCID: PMC4036119  PMID: 22508468
The Journal of rheumatology  2013;40(9):1498-1504.
To investigate the associations of alcohol consumption and radiographic disease progression in African Americans with recently diagnosed rheumatoid arthritis (RA).
RA patients included in the study were participants in the Consortium for the Longitudinal Evaluation of African Americans with Early Rheumatoid Arthritis (CLEAR) registry. Patients were categorized based on self-reported alcohol consumption; those consuming < 15 beverages per month versus those with ≥ 15 per month. Association of radiographic disease progression over a one to three year period of observation with alcohol consumption was evaluated using multivariate generalized estimating equations.
There were 166 patients included in the study, 39% reported that they had never consumed alcohol. Of the 61% who had consumed alcohol, 73% reported that they on average consumed less than 15 alcoholic beverages per month and 27% reported consuming ≥ 15 per month. In multivariate analysis, consumption of ≥ 15 alcoholic beverages per month was associated with an increased risk of radiographic disease progression (p = 0.017). There was no evidence of a relationship in those consuming < 15 beverages per month (p = 0.802).
There appears to be a dose-dependent relationship between alcohol use and radiographic disease progression in RA. Individuals who consume 15 or more alcoholic beverages per month may have accelerated rates of radiographic joint damage than with lower levels of consumption.
PMCID: PMC4026220  PMID: 23772080
Rheumatoid Arthritis; alcohol consumption; disease severity; disease activity
15.  Comparison of the Disease Activity Score using Erythrocyte Sedimentation Rate and C-reactive Protein in African-Americans with Rheumatoid Arthritis 
The Journal of rheumatology  2013;40(11):1812-1822.
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.
PMCID: PMC3987124  PMID: 23950187
DAS28; Rheumatoid Arthritis; African-Americans
16.  Complementary and Alternative Medicine Use in African Americans With Rheumatoid Arthritis 
Arthritis care & research  2014;66(2):180-189.
Racial/ethnic differences with regard to complementary and alternative medicine (CAM) use have been reported in the US. However, specific details of CAM use by African Americans with rheumatoid arthritis (RA) are lacking.
Data were collected from African Americans with RA enrolled in a multicenter registry regarding the use of CAM, including food supplements, topical applications, activities, and alternative care providers. Factors associated with CAM use by sex and disease duration were assessed using t-test, Wilcoxon’s rank sum test, chi-square test, and logistic regression analyses.
Of the 855 participants, 85% were women and mean age at enrollment was 54 years. Overall, ever using any of the CAM treatments, activities, and providers was 95%, 98%, and 51%, respectively (median of 3 for number of treatments, median of 5 for activities, and median of 1 for providers). Those with longer disease duration (>2 years) were significantly more likely (odds ratio >2.0, P < 0.05) to use raisins soaked in vodka/gin, to take fish oils, or to drink alcoholic beverages for RA treatment than those with early disease. As compared to men, women were significantly (P < 0.05) more likely to pray/attend church, write in a journal, and use biofeedback, but were less likely to smoke tobacco or topically apply household oils for treatment of RA.
CAM use was highly prevalent in this cohort, even in individuals with early disease. Health care providers need to be aware of CAM use as some treatments may potentially have interactions with conventional medicines. This could be important within this cohort of African Americans, where racial disparities are known to affect access to conventional care.
PMCID: PMC3977347  PMID: 23983105
17.  Gene-body mass index interactions are associated with methotrexate toxicity in rheumatoid arthritis 
Annals of the rheumatic diseases  2013;73(4):785-786.
PMCID: PMC3970399  PMID: 24291656
Gene polymorphism; methotrexate; rheumatoid arthritis
18.  Integration of Sequence Data from a Consanguineous Family with Genetic Data from an Outbred Population Identifies PLB1 as a Candidate Rheumatoid Arthritis Risk Gene 
PLoS ONE  2014;9(2):e87645.
Integrating genetic data from families with highly penetrant forms of disease together with genetic data from outbred populations represents a promising strategy to uncover the complete frequency spectrum of risk alleles for complex traits such as rheumatoid arthritis (RA). Here, we demonstrate that rare, low-frequency and common alleles at one gene locus, phospholipase B1 (PLB1), might contribute to risk of RA in a 4-generation consanguineous pedigree (Middle Eastern ancestry) and also in unrelated individuals from the general population (European ancestry). Through identity-by-descent (IBD) mapping and whole-exome sequencing, we identified a non-synonymous c.2263G>C (p.G755R) mutation at the PLB1 gene on 2q23, which significantly co-segregated with RA in family members with a dominant mode of inheritance (P = 0.009). We further evaluated PLB1 variants and risk of RA using a GWAS meta-analysis of 8,875 RA cases and 29,367 controls of European ancestry. We identified significant contributions of two independent non-coding variants near PLB1 with risk of RA (rs116018341 [MAF = 0.042] and rs116541814 [MAF = 0.021], combined P = 3.2×10−6). Finally, we performed deep exon sequencing of PLB1 in 1,088 RA cases and 1,088 controls (European ancestry), and identified suggestive dispersion of rare protein-coding variant frequencies between cases and controls (P = 0.049 for C-alpha test and P = 0.055 for SKAT). Together, these data suggest that PLB1 is a candidate risk gene for RA. Future studies to characterize the full spectrum of genetic risk in the PLB1 genetic locus are warranted.
PMCID: PMC3919745  PMID: 24520335
19.  Gene expression patterns in peripheral blood cells associated with radiographic severity in African Americans with early rheumatoid arthritis 
Rheumatology international  2012;33(1):129-137.
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.
PMCID: PMC3769702  PMID: 22238028
Genome-wide gene expression; Sharp/van der Heijde; Pathway analysis; CLEAR; ABCoN
20.  Serum cotinine as a biomarker of tobacco exposure is not associated with treatment response in early rheumatoid arthritis 
Arthritis care & research  2012;64(12):10.1002/acr.21758.
Cigarette smoking has emerged as a risk factor for development of rheumatoid arthritis (RA). Recent studies have suggested that cigarette smoking may lead to lower treatment response rates with methotrexate (MTX) and some biologic agents in RA. Knowledge of whether tobacco exposure reduces treatment efficacy is important as smoking could represent a modifiable factor in optimizing RA treatment.
Study participants included patients with early RA (<3 years duration) enrolled in the Treatment of Early Aggressive RA (TEAR) trial, a randomized, blinded, placebo-controlled clinical trial (RCT) comparing early intensive therapy (MTX + etanercept or MTX + hydroxychloroquine + sulfasalazine [triple therapy]) versus initial treatment with MTX with step-up to MTX + etanercept or to triple therapy if still active at 24 weeks. Serum cotinine was measured using a commercially available ELISA at baseline and 48 weeks with detectable concentrations at both visits serving as indicator of smoking status. Mean Disease Activity Score (DAS-28) was compared by smoking status, adjusting for baseline disease activity.
Of 412 subjects included in the analysis, 293 (71%) were categorized as ‘non-smokers’ and 119 (29%) as ‘current smokers’. There were no differences in the mean DAS-28 between 48 and 102 weeks based on smoking status for the overall group (p=0.881) or by specific treatment assignment.
Among patients enrolled in a large RCT of early RA with poor prognostic factors, smoking status did not impact treatment responses for those receiving early combination or initial MTX with step-up therapy at 24 weeks if still active.
PMCID: PMC3467327  PMID: 22730343
21.  Measurement of Erythrocyte Methotrexate Polyglutamate Levels: Ready for Clinical Use in Rheumatoid Arthritis? 
Current rheumatology reports  2010;12(5):342-347.
Methotrexate (MTX) is one of the most commonly prescribed and most effective drugs for the treatment of rheumatoid arthritis (RA). Given the partial response of many patients and the side effect profile of the drug, there is considerable interest in identification of biomarkers to guide MTX therapy in RA. Upon entering cells, MTX is polyglutamated. Measuring methotrexate polyglutamates (MTX PGs) levels in circulating red blood cells (RBC) has been proposed as an objective measure that can help to optimize MTX therapy in RA. There is conflicting data with regard to the clinical utility of measurement of MTX PGs measurements as a predictor of the efficacy or toxicity of low-dose MTX effects in RA. Should large, randomized clinical trials of this assay show consistent, reproducible, long-term correlations between MTX PG levels and efficacy and toxicity, this test could become a prominent tool for clinicians to optimize the use of MTX in RA.
PMCID: PMC3769795  PMID: 20665136
Rheumatoid Arthritis; Methotrexate; Polyglutamates
23.  Pharmacogenetics of Etanercept in Rheumatoid Arthritis 
Pharmacogenomics  2008;9(8):1011-1015.
Etanercept is one of several TNF inhibitors approved for rheumatoid arthritis (RA) and a variety of other immune-mediated inflammatory conditions. Given the plethora of drugs approved for RA and the wide variations in cost and treatment response, markers of efficacy would be very useful. Several candidate genes, including HLA-DRB1 alleles and those encoding TNF, TNF receptors, and Fc receptors, have been examined for a role in the response to treatment with etanercept. In this review, we discuss pharmacogenetic studies of etanercept in RA and other diseases, and comment on the future of such analyses to advance the goal of personalized medicine in RA.
PMCID: PMC3746504  PMID: 18681777
24.  The HLA–DRB1 Shared Epitope Is Associated With Susceptibility to Rheumatoid Arthritis in African Americans Through European Genetic Admixture 
Arthritis and rheumatism  2008;58(2):349-358.
To determine whether shared epitope (SE)–containing HLA–DRB1 alleles are associated with rheumatoid arthritis (RA) in African Americans and whether their presence is associated with higher degrees of global (genome-wide) genetic admixture from the European population.
In this multicenter cohort study, African Americans with early RA and matched control subjects were analyzed. In addition to measurement of serum anti–cyclic citrullinated peptide (anti-CCP) antibodies and HLA–DRB1 genotyping, a panel of >1,200 ancestry-informative markers was analyzed in patients with RA and control subjects, to estimate the proportion of European ancestry.
The frequency of SE-containing HLA–DRB1 alleles was 25.2% in African American patients with RA versus 13.6% in control subjects (P = 0.00005). Of 321 patients with RA, 42.1% had at least 1 SE-containing allele, compared with 25.3% of 166 control subjects (P = 0.0004). The mean estimated percent European ancestry was associated with SE-containing HLA–DRB1 alleles in African Americans, regardless of disease status (RA or control). As reported in RA patients of European ancestry, there was a significant association of the SE with the presence of the anti-CCP antibody: 86 (48.9%) of 176 patients with anti-CCP antibody–positive RA had at least 1 SE allele, compared with 36 (32.7%) of 110 patients with anti-CCP antibody–negative RA (P = 0.01, by chi-square test).
HLA–DRB1 alleles containing the SE are strongly associated with susceptibility to RA in African Americans. The absolute contribution is less than that reported in RA among populations of European ancestry, in which ~50–70% of patients have at least 1 SE allele. As in Europeans with RA, the SE association was strongest in the subset of African American patients with anti-CCP antibodies. The finding of a higher degree of European ancestry among African Americans with SE alleles suggests that a genetic risk factor for RA was introduced into the African American population through admixture, thus making these individuals more susceptible to subsequent environmental or unknown factors that trigger the disease.
PMCID: PMC3726059  PMID: 18240241
25.  Clinical Response within 12 Weeks as a Predictor of Future Low Disease Activity in Early RA Patients: Results from the TEAR Trial 
The Journal of rheumatology  2013;40(5):572-578.
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.
PMCID: PMC3694569  PMID: 23588939
rheumatoid arthritis; prediction; anti-TNF; triple therapy

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