Faculty Bibliography

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4x10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have approximately 33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-kappaB transcription factor. Finally, we develop a high-throughput NF-kappaB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-kappaB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (DeltaDAS) in the etanercept subset of patients (P = 8 x 10(-8)), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3' UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1 x 10(-11) in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better DeltaDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry.

CONTEXT: Large genomic copy number variations have been implicated as strong risk factors for schizophrenia. However, the rarity of these events has created challenges for the identification of further pathogenic loci, and extremely large samples are required to provide convincing replication. OBJECTIVE: To detect novel copy number variations that increase the susceptibility to schizophrenia by using 2 ethnically homogeneous discovery cohorts and replication in large samples. DESIGN: Genetic association study of microarray data. SETTING: Samples of DNA were collected at 9 sites from different countries. PARTICIPANTS: Two discovery cohorts consisted of 790 cases with schizophrenia and schizoaffective disorder and 1347 controls of Ashkenazi Jewish descent and 662 parent-offspring trios from Bulgaria, of which the offspring had schizophrenia or schizoaffective disorder. Replication data sets consisted of 12,398 cases and 17,945 controls. MAIN OUTCOME MEASURES: Statistically increased rate of specific copy number variations in cases vs controls. RESULTS: One novel locus was implicated: a deletion at distal 16p11.2, which does not overlap the proximal 16p11.2 locus previously reported in schizophrenia and autism. Deletions at this locus were found in 13 of 13,850 cases (0.094%) and 3 of 19,954 controls (0.015%) (odds ratio, 6.25 [95% CI, 1.78-21.93]; P = .001, Fisher exact test). CONCLUSIONS: Deletions at distal 16p11.2 have been previously implicated in developmental delay and obesity. The region contains 9 genes, several of which are implicated in neurological diseases, regulation of body weight, and glucose homeostasis. A telomeric extension of the deletion, observed in about half the cases but no controls, potentially implicates an additional 8 genes. Our findings add a new locus to the list of copy number variations that increase the risk for development of schizophrenia.

BACKGROUND: Biobanks have the potential to offer a venue for chronic disease biomarker discovery, which would allow for disease early detection and for identification of carriers of a certain predictor biomarker. To assess the general attitudes towards genetic research and participation in biobanks in the Long Island/Queens area of New York, and what factors would predict a positive view of such research, participants from the NSLIJ hospital system were surveyed. METHODS: Participants were recruited at six hospital centers in the NSLIJ system during the summers of 2009 and again in 2011 (n = 1,041). Those who opted to participate were given a questionnaire containing 22 questions assessing demographics, lifestyle and attitudes towards genetic research. These questions addressed individual participant's beliefs about the importance of genetic research, willingness to participate in genetic research themselves, and their views on informed consent issues. RESULTS: Respondents took a generally positive view of genetic research in general, as well as their own participation in such research. Those with reservations were most likely to cite concerns over the privacy of their medical and genetic information. Those who were married tended to view genetic research as important, while those in the younger age group viewed it as less important. Prior blood donation of respondents was found to be a predictor of their approval for genetic research. Demographic factors were not found to be predictive of personal willingness to participate in genetic research, or of approval for the opt-out approach to consent. CONCLUSIONS: While respondents were generally inclined to approve of genetic research, and those who disapproved did not do so based on an underlying moral objection to such research, there is a disconnect between the belief in the importance of genetic research and the willingness of individuals to participate themselves. This indicates a continued concern for the ways in which genetic materials are safeguarded once they are collected. Also indicated was a general lack of understanding about the various consent processes that go along with genetic research, which should be addressed further to ensure the successful continuation of biobanks.

Mice with a DC-specific deletion of the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp1) exhibit a lupus-like phenotype, secondary to enhanced DC production of IL-6. Here we explored further phenotypic changes in Blimp1-deficient DCs, the molecular mechanism underlying these changes, and their relevance to human disease. Blimp1-deficient DCs exhibited elevated expression of MHC II, and exposure to TLR agonists increased secretion of proinflammatory cytokines. This phenotype reflects enhanced expression of the microRNA let-7c, which is regulated by BLIMP1. Let-7c reciprocally inhibited Blimp1 and also blocked LPS-induced suppressor of cytokine signaling-1 (SOCS1) expression, contributing to the proinflammatory phenotype of Blimp1-deficient DCs. DCs from Blimp1 SLE-risk allele carriers exhibited analogous phenotypic changes, including decreased BLIMP1 expression, increased let-7c expression, and increased expression of proinflammatory cytokines. These results suggest that let-7c regulates DC phenotype and confirm the importance of BLIMP1 in maintaining tolerogenic DCs in both mice and humans.

The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome-wide association studies (GWASs). First, we assessed the contribution of rare coding variants in the 25 genes to the risk of RA in a pooled sequencing study of 500 RA cases and 650 controls of European ancestry. We observed an accumulation of rare nonsynonymous variants exclusive to RA cases in IL2RA and IL2RB (burden test: p = 0.007 and p = 0.018, respectively). Next, we assessed the aggregate contribution of low-frequency and common coding variants to the risk of RA by dense genotyping of the 25 gene loci in 10,609 RA cases and 35,605 controls. We observed a strong enrichment of coding variants with a nominal signal of association with RA (p < 0.05) after adjusting for the best signal of association at the loci (p(enrichment) = 6.4 x 10(-4)). For one locus containing CD2, we found that a missense variant, rs699738 (c.798C>A [p.His266Gln]), and a noncoding variant, rs624988, reside on distinct haplotypes and independently contribute to the risk of RA (p = 4.6 x 10(-6)). Overall, our results indicate that variants (distributed across the allele-frequency spectrum) within the protein-coding portion of a subset of biological candidate genes identified by GWASs contribute to the risk of RA. Further, we have demonstrated that very large sample sizes will be required for comprehensively identifying the independent alleles contributing to the missing heritability of RA.

Suboptimal cellular DNA repair capacity (DRC) has been shown to be associated with enhanced cancer risk, but genetic variants affecting the DRC phenotype have not been comprehensively investigated. In this study, with the available DRC phenotype data, we analyzed correlations between the DRC phenotype and genotypes detected by the Illumina 317K platform in 1,774 individuals of European ancestry from a Texas lung cancer genome-wide association study. The discovery phase was followed by a replication in an independent set of 1,374 cases and controls of European ancestry. We applied a generalized linear model with single nucleotide polymorphisms as predictors and DRC (a continuous variable) as the outcome. Covariates of age, sex, pack-years of smoking, DRC assay-related variables, and case-control status of the study participants were adjusted in the model. We validated that reduced DRC was associated with an increased risk of lung cancer in both independent datasets. Several suggestive loci that contributed to the DRC phenotype were defined in ERCC2/XPD, PHACTR2, and DUSP1. In summary, we determined that DRC is an independent risk factor for lung cancer, and we defined several genetic loci contributing to DRC phenotype.

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.

Recent work has shown that much of the missing heritability of complex traits can be resolved by estimates of heritability explained by all genotyped SNPs. However, it is currently unknown how much heritability is missing due to poor tagging or additional causal variants at known GWAS loci. Here, we use variance components to quantify the heritability explained by all SNPs at known GWAS loci in nine diseases from WTCCC1 and WTCCC2. After accounting for expectation, we observed all SNPs at known GWAS loci to explain 1.29 x more heritability than GWAS-associated SNPs on average (P=3.3 x 10(-)(5)). For some diseases, this increase was individually significant: 2.07 x for Multiple Sclerosis (MS) (P=6.5 x 10(-)(9)) and 1.48 x for Crohn's Disease (CD) (P = 1.3 x 10(-)(3)); all analyses of autoimmune diseases excluded the well-studied MHC region. Additionally, we found that GWAS loci from other related traits also explained significant heritability. The union of all autoimmune disease loci explained 7.15 x more MS heritability than known MS SNPs (P < 1.0 x 10(-)(1)(6) and 2.20 x more CD heritability than known CD SNPs (P = 6.1 x 10(-)(9)), with an analogous increase for all autoimmune diseases analyzed. We also observed significant increases in an analysis of > 20,000 Rheumatoid Arthritis (RA) samples typed on ImmunoChip, with 2.37 x more heritability from all SNPs at GWAS loci (P = 2.3 x 10(-)(6)) and 5.33 x more heritability from all autoimmune disease loci (P < 1 x 10(-)(1)(6) compared to known RA SNPs (including those identified in this cohort). Our methods adjust for LD between SNPs, which can bias standard estimates of heritability from SNPs even if all causal variants are typed. By comparing adjusted estimates, we hypothesize that the genome-wide distribution of causal variants is enriched for low-frequency alleles, but that causal variants at known GWAS loci are skewed towards common alleles. These findings have important ramifications for fine-mapping study design and our understanding of complex disease architecture.