The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Randomized Controlled Trial Substudy of Cell-specific Mechanisms of Janus Kinase 1 Inhibition With Upadacitinib in the Crohn's Disease Intestinal Mucosa: Analysis From the CELEST Study.

BACKGROUND: Janus kinase (JAK) inhibition shows promise for treatment of patients with moderate to severe Crohn's disease. We aimed to provide mechanistic insights into the JAK1-selective inhibitor upadacitinib through a transcriptomics substudy on biopsies from patients with Crohn's disease from CELEST. METHODS: Seventy-four patients consented to this optional substudy. Ileal and colonic biopsies were collected during endoscopy at screening and week 12 or 16. RNA isolated from 226 samples was analyzed by RNAseq, with additional qPCR analysis. Additional biopsies from patients with Crohn's disease receiving anti-tumor necrosis factor (anti-TNF; n = 34) and healthy controls (n = 10) were used for qPCR. Single-cell RNAseq public profiles were used to evaluate treatment effects on specific cellular subsets, associations with endoscopic improvement, and indirect comparisons with the anti-TNF-treated cohort. RESULTS: In involved areas of mucosa with endoscopic remission after upadacitinib treatment, 1156 and 76 protein-coding genes were significantly regulated (false discovery rate < 0.05) at week 12/16 in colonic and ileal biopsies, respectively (60 overlapped), compared with baseline. Upadacitinib did not significantly affect transcriptomes of noninvolved intestinal areas. CELEST patients (mostly anti-TNF-refractory) showed baseline differences in gene expression compared with a separate cohort of biologic-naïve patients. Notably, upadacitinib reversed overexpression of inflammatory fibroblast and interferon-γ effector signature markers. CONCLUSIONS: Upadacitinib modulates inflammatory pathways in mucosal lesions of patients with anti-TNF-refractory Crohn's disease, including inflammatory fibroblast and interferon-γ-expressing cytotoxic T cell compartments. This substudy is the first to describe the molecular response to JAK1 inhibition in inflammatory bowel disease and differential effects relative to anti-TNF treatment. (Clinical trial identifier: NCT02365649).

Estimating colocalization probability from limited summary statistics.

BACKGROUND: Colocalization is a statistical method used in genetics to determine whether the same variant is causal for multiple phenotypes, for example, complex traits and gene expression. It provides stronger mechanistic evidence than shared significance, which can be produced through separate causal variants in linkage disequilibrium. Current colocalization methods require full summary statistics for both traits, limiting their use with the majority of reported GWAS associations (e.g. GWAS Catalog). We propose a new approximation to the popular coloc method that can be applied when limited summary statistics are available. Our method (POint EstiMation of Colocalization, POEMColoc) imputes missing summary statistics for one or both traits using LD structure in a reference panel, and performs colocalization using the imputed summary statistics. RESULTS: We evaluate the performance of POEMColoc using real (UK Biobank phenotypes and GTEx eQTL) and simulated datasets. We show good correlation between posterior probabilities of colocalization computed from imputed and observed datasets and similar accuracy in simulation. We evaluate scenarios that might reduce performance and show that multiple independent causal variants in a region and imputation from a limited subset of typed variants have a larger effect while mismatched ancestry in the reference panel has a modest effect. Further, we find that POEMColoc is a better approximation of coloc when the imputed association statistics are from a well powered study (e.g., relatively larger sample size or effect size). Applying POEMColoc to estimate colocalization of GWAS Catalog entries and GTEx eQTL, we find evidence for colocalization of 150,000 trait-gene-tissue triplets. CONCLUSIONS: We find that colocalization analysis performed with full summary statistics can be closely approximated when only the summary statistics of the top SNP are available for one or both traits. When applied to the full GWAS Catalog and GTEx eQTL, we find that colocalized trait-gene pairs are enriched in tissues relevant to disease etiology and for matches to approved drug mechanisms. POEMColoc R package is available at https://github.com/AbbVie-ComputationalGenomics/POEMColoc .

A Genetic Variant in the BCL2 Gene Associates with Adalimumab Response in Hidradenitis Suppurativa Clinical Trials and Regulates Expression of BCL2.

Hidradenitis suppurativa is a chronic skin disease with a significant genetic component and prevalence from 0.5% to 4%. Adalimumab is the only treatment approved by either the European Medicines Agency or the U.S. Food and Drug Administration for the management of moderate to severe hidradenitis suppurativa. To identify genetic variants associated with adalimumab response, we performed a genome-wide association study (GWAS) from the most extensive two phase 3 hidradenitis suppurativa clinical trials (PIONEER I and II) to date. Through direct genotyping and imputation, we tested almost 7 million genetic variants with minor allele frequency > 5% and identified one single linkage disequilibrium block, located in the intron of the BCL2 gene, which reached genome-wide significance (lead single-nucleotide polymorphism, rs59532114; P = 2.35E-08). Bioinformatic analysis and functional genomics experiments suggested a correlation of the most strongly associated single-nucleotide polymorphism minor allele with increased BCL2 gene and protein expressions in hair follicle tissues. In reciprocal knockdown experiments, we found that BCL2 is down-regulated by TNF inhibition. These results highlight a pathway that involves BCL2 in response to adalimumab. Further work is required to determine how this pathway influences adalimumab effectiveness in patients with hidradenitis suppurativa.

Identification of HLA-DRB1 association to adalimumab immunogenicity.

Anti-drug antibody formation occurs with most biological agents across disease states, but the mechanism by which they are formed is unknown. The formation of anti-drug antibodies to adalimumab (AAA) may decrease its therapeutic effects in some patients. HLA alleles have been reported to be associated with autoantibody formation against interferons and other TNF inhibitors, but not adalimumab. We analyzed samples from 634 subjects with either rheumatoid arthritis (RA) or hidradenitis suppurativa (HS): 37 subjects (17 RA and 20 HS) developed AAA (AAA+) during adalimumab treatment and 597 subjects (348 RA, 249 HS) did not develop AAA (AAA-) during the clinical trials. Using next-generation sequencing-based HLA typing, we identified three protective HLA alleles (HLA-DQB1*05, HLA-DRB1*01,and HLA-DRB1*07) that were less prevalent in AAA+ than AAA-subjects (ORs: 0.4, 0.25 and 0.28, respectively; and P values: 0.012, 0.012 and 0.018, respectively) and two risk HLA alleles (HLA-DRB1*03 and HLA-DRB1*011) that were more abundant in AAA+ than AAA-subjects (ORs: 2.52, and 2.64, respectively; and P values: 0.006 and 0.019). Similar to the finding of Billiet et al. who found that carriage of the HLA-DRB1*03 allele was more prevalent in those with anti-infliximab antibodies (OR = 3.6, p = 0.002, 95% CI: [1.5,8.6]).), we found HLA-DRB1*03 allele was also more prevalent in anti-adalimumab positive (OR = 2.52, p = 0.006, 95% CI: [1.37,4.63]). The results suggest that specific HLA alleles may play a key role in developing AAAs in RA and HS patients treated with adalimumab.

The expression of RUNDC3B is associated with promoter methylation in lymphoid malignancies.

DNA methylation is an epigenetic modification that plays an important role in the regulation of gene expression. The function of RUNDC3B has yet to be determined, although its dysregulated expression has been associated with malignant potential of both breast and lung carcinoma. To elucidate the potential of using DNA methylation in RUNDC3B as a biomarker in lymphoid malignancies, the methylation status of six regions spanning the CpG island in the promoter region of RUNDC3B was determined in cancer cell lines. Lymphoid malignancies were found to have more prominent methylation and did not express RUNDC3B compared with myeloid malignancies and solid tumours, supporting the potential use of DNA methylation in this region as a biomarker for lymphoid malignancies. RUNDC3B contains a RUN domain in its N-terminal region that mediates interaction with Rap2, an important component of the mitogen-activated protein kinase (MAPK) cascade, which regulates cellular proliferation and differentiation. The protein sequence of RUNDC3B also contains characteristic binding sites for MAPK intermediates. Therefore, it is possible that RUNDC3B serves as a mediator between Rap2 and the MAPK signalling cascade. Three genes with MAPK-inducible expression were downregulated in a methylated leukaemia cell line (HSPA5, Jun and Fos). Jun and Fos combine to form the activating protein 1 transcription factor, and loss of this factor is associated with the dysregulation of genes involved in differentiation and proliferation. We hypothesize that the loss of RUNDC3B secondary to aberrant hypermethylation of the early growth response 3 transcription factor binding site results in dysregulated MAPK signalling and carcinogenesis in lymphoid malignancies. © 2015 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.

MedlinePlus-based health information prescriptions: a comparison of email vs paper delivery.

BACKGROUND: The internet can provide evidence-based patient education to overcome time constraints of busy ambulatory practices. Health information prescriptions (HIPs) can be effectively integrated into clinic workflow, but compliance to visit health information sites such as MedlinePlus is limited. OBJECTIVE: Compare the efficacy of paper (pHIP) and email (eHIP) links to deliver HIPs; evaluate patient satisfaction with the HIP process and MedlinePlus information; assess reasons for noncompliance to HIPs. METHOD: Of 948 patients approached at two internal medicine clinics affiliated with an academic medical centre, 592 gave informed consent after meeting the inclusion criteria. In this randomised controlled trial, subjects were randomised to receive pHIP or eHIP for accessing an intermediate website that provided up to five MedlinePlus links for physician-selected HIP conditions. Patients accessing the intermediate website were surveyed by email to assess satisfaction with the health information. Survey non-responders were contacted by telephone to determine the reasons for no response. RESULTS: One hundred and eighty-one patients accessed the website, with significantly more 'filling' eHIP than pHIP (38% vs 23%; P < 0.001). Most (82%) survey respondents found the website information useful, with 77% favouring email for future HIPs delivery. Lack of time, forgot, lost instructions or changed mind were reasons given for not accessing the websites. CONCLUSIONS: Delivery of MedlinePlus-based HIPs in clinic is more effective using email prescriptions than paper. Satisfaction with the HIP information was high, but overall response was low and deserves further investigation to improve compliance and related outcomes.

Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells.

We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2.