Integrative Genomics Analysis Identifies ACVR1B as a Candidate Causal Gene of Emphysema Distribution

Abstract

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10^-5 in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P < 0.05), with the strongest enrichment observed in CD4⁺, CD8⁺, and regulatory T cells. We selected a putative causal variant, rs7962469, associated with ACVR1B expression in lung tissue for additional functional investigation, and reporter assays confirmed allele-specific regulatory activity for this variant in human bronchial epithelial and Jurkat immune cell lines. ACVR1B expression levels exhibit a nominally significant association with emphysema distribution. EABD-associated loci are preferentially enriched in regulatory elements of multiple cell types, most notably T-cell subsets. Multiple EABD loci colocalize to regulatory elements that are active across multiple tissues and cell types, and functional analyses confirm the presence of an EABD-associated functional variant that regulates ACVR1B expression, indicating that transforming growth factor-β signaling plays a role in the EABD phenotype. Clinical trial registered with www.clinicaltrials.gov (NCT00608764).

Trial registration: ClinicalTrials.gov NCT00608764 NCT00292552.

Keywords: gene; chronic obstructive pulmonary disease; emphysema distribution; integrative genomics; transforming growth factor-β signaling.

Figure 1.

Workflow and summary of the results of the colocalization analysis of emphysema distribution–associated genetic variants with expression quantitative trait loci (eQTL) and epigenomic data. ACVR1B = activin A receptor type 1B; DCBLD1 = discoidin, CUB and LCCL domain containing 1; GWAS = genome-wide association studies; HBE = human bronchial epithelial; IGHV3 = immunoglobulin heavy variable 3; MEI1 = meiotic double-stranded break formation protein 1; PICS = probabilistic identification of causal SNPs.

Figure 2.

Overlap of emphysema apicobasal distribution (EABD) risk variants in multiple tissues and cell types. (A) The majority of EABD loci overlap eQTL regions that are active in more than one tissue, and (B) there is trend toward stronger EABD GWAS association and a greater number of active eQTL tissues at a given locus. (C) The majority of EABD loci also lie within regions of DNase I peaks and (D) enhancer elements that are active in more than one cell type.

Figure 3.

Plot of the cell type enrichment −log₁₀ P value from the Genomic Annotation Shifter (GoShifter) analyses of the overlaps between emphysema distribution–associated genetic variants with DNase I–hypersensitive peaks, DNase I hotspots, enhancer marks, and digital DNase I footprints. The cell types with enrichment P values less than 0.05 are colored red in A and shown in more detail in B. iPS = induced pluripotent stem cell; PMA-I = phorbol 12-myristate 13-acetate plus ionomycin.

Figure 4.

GWAS locus and eQTL plots for (A) the rs7962469 variant near ACVR1B in lung tissue and (B) for rs10152544 near SMAD3 in esophageal mucosa. cMMb = centimorgan per megabase.

Figure 5.

Reporter assays of the rs7962469 variant near the ACVR1B gene in 16HBE cells showing significantly increased expression of the A variant relative to the G variant (P = 0.008) and the empty luciferase vector (pGL4.23) (P = 0.00002). The P value comparing the G variant with pGL4.23 is 0.02. **P < 0.05. Data are from four independent transfections, each performed in triplicate. TK = thymidine kinase.