Identification of Functional Variants in the FAM13A Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Locus by Massively Parallel Reporter Assays

Abstract

Rationale: The identification of causal variants responsible for disease associations from genome-wide association studies (GWASs) facilitates functional understanding of the biological mechanisms by which those genetic variants influence disease susceptibility.

Objective: We aim to identify causal variants in or near the FAM13A (family with sequence similarity member 13A) GWAS locus associated with chronic obstructive pulmonary disease (COPD).

Methods: We used an integrated approach featuring conditional genetic analysis, massively parallel reporter assays (MPRAs), traditional reporter assays, chromatin conformation capture assays, and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing to characterize COPD-associated regulatory variants in the FAM13A region in human bronchial epithelial cell lines.

Measurements and main results: Conditional genetic association suggests the presence of two independent COPD association signals in FAM13A. MPRAs identified 45 regulatory variants within FAM13A, among which six variants were prioritized for further investigation. Three COPD-associated variants demonstrated significant allele-specific activity in reporter assays. One of three variants, rs2013701, was tested in the endogenous genomic context by CRISPR-based genome editing that confirmed its allele-specific effects on FAM13A expression and on cell proliferation, providing functional characterization for this COPD-associated variant.

Conclusions: The human GWAS association near FAM13A may contain independent association signals. MPRAs identified multiple functional variants in this region, including rs2013701, a putative COPD-causing variant with allele-specific regulatory activity.

Keywords: CRISPR editing; chromosome conformation capture; functional genomics; human bronchial epithelial cells; reporter assay.

Figure 1.

Overview of massively parallel reporter assay (MPRA) design. (A) Schematic illustration to indicate the design of oligonucleotides. (B) Experimental procedures of MPRA screening. TSS = transcription start site.

Figure 2.

Massively parallel reporter assay (MPRA) significant oligonucleotides with allele-specific effects shown by genomic position. Oligonucleotides representing 606 SNPs near and within FAM13A were tested in MPRA experiments with either a strong (A) or weak (B) promoter. The y-axis represents the P values for allele-specific expression from the MPRA experiment, and the x-axis represents the genomic position. Each dot represents one oligonucleotide sequence in the array. Different colors represent the relative correlation (linkage disequilibrium) with previously published GWAS SNP rs4416442 (marked in purple) as indicated by r². The blue line shows the estimated recombination rate at each genomic location from HapMap data. FAM13A = family with sequence similarity member 13A; minP = minimal promoter; SV40 = simian virus 40 (stronger promoter).

Figure 3.

Allelic effects of significant massively parallel reporter assay (MPRA) SNPs with respect to promoter, flanking sequence, and oligonucleotide orientation. (A) Allelic gene regulatory effects are highly correlated for the same SNP within identical oligonucleotide sequences connected with either a weak (minimal promoter) or strong promoter (simian virus 40). The MPRA effect corresponds to the Hodges-Lehmann estimate of the difference between the input/output ratios comparing two SNP alleles. (B and C) Of the 23 SNPs that had significant MPRA results in more than one oligonucleotide context (orientation or position), 11 SNPs that were significant in one orientation showed consistent MPRA effects across different SNP positions within the oligonucleotide. (B) Twelve SNPs that were significant in both orientations had divergent effects, that is, opposite allelic effects in the forward and reverse orientations. (C) Each dot represents one significant oligonucleotide. Only significant MPRA results are displayed. Black arrows indicate SNPs that were chosen for reporter assay validation in Figure 5. Squares indicate forward orientation; triangles indicate reverse orientation. minP = minimal promoter; SV40 = simian virus 40.

Figure 4.

Local chronic obstructive pulmonary disease (COPD) association plots near FAM13A (family with sequence similarity member 13A) for subjects of European descent only. (A) The unconditioned association peak at FAM13A. (B) Local association conditioned on the lead secondary association variant rs147089648. (C) Local association conditioned on rs4416442. (D) Conditioning on both rs4416442 and rs147089648 completely attenuates the association peak. Analyses were performed in COPD cases (n = 5,772) and smoking controls (n = 3,944) of European descent from five cohorts.

Figure 5.

Reporter assay in 16HBE cells testing the allele-specific enhancer activity of massively parallel reporter assay SNPs. Results are given as ratios of firefly to Renilla luciferase activity. Mean ± SEM is shown from four to seven independent experiments with triplicate measurements in each repeat. ***P < 0.001, unpaired Student t test.

Figure 6.

Expression levels of FAM13A (family with sequence similarity member 13A) and cellular proliferation assays in clustered regularly interspaced short palindromic repeats (CRISPR)-edited cell clones targeting rs2013701. (A) Expression levels of FAM13A were measured in 16HBE cell lines. After CRISPR editing, 16HBE cells homozygous for the G allele were altered to the homozygous T genotype of rs2013701. Means ± SEM are shown from two to three individual CRISPR single clones with three repeats for each line. (B) Cell growth curve in 16HBE CRISPR lines. Results from four single colonies with genotype GG or TT at rs2013701 are shown. Means ± SD are shown from triplicate wells. One representative experiment from four independent experiments is shown. *P < 0.05, unpaired Student’s t test.