Genetic variants associated with susceptibility to idiopathic pulmonary fibrosis in people of European ancestry: a genome-wide association study

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.

Methods: We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.

Findings: 602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18-1·37], p=1·32 × 10^-9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56-3·26], p=1·12 × 10^-66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35-1·54], p=7·81 × 10^-28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).

Interpretation: AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.

Funding: UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

Figure 1

Quality control and sample selection flow chart (A) Stage 1 genome-wide association study. (B) Stage 2 follow-up analyses. *On identification of a pair of individuals who were second-degree relatives or closer, one individual was excluded. IPF=idiopathic pulmonary fibrosis. †One patient had both call rate <98% and sex mismatch.

Figure 2

Manhattan plot for the discovery genome-wide association study of IPF susceptibility The x axis shows chromosomal position and the y axis shows –log₁₀(p value) from the discovery (stage 1) case-control analysis. Green variants are those that reached genome-wide significance in the meta-analysis of stage 1 and 2 results (and any variant in linkage disequilibrium with the lead variant [r²>0·1]). The blue line shows the threshold used for selecting variants for stage 2 (p=5 × 10⁻⁶) and the red line shows genome-wide significance (p=5 × 10⁻⁸). Hollow circles show variants showing an association with genotyping array in the controls, and those that did not show an association with IPF in stage 2 (appendix pp 2 and 6). IPF=idiopathic pulmonary fibrosis.

Figure 3

Comparison of case-control association results and lung eQTL results Region plots for a 2 Mb region on chromosome 15 for the stage 1 case-control GWAS (circles above the x axis) and lung eQTL analysis (squares below the x axis). The x axis shows chromosomal position. The y axis above the x-axis shows the –log₁₀(p value) from the case-control analysis and the y axis below the x axis shows the –log₁₀(p value) for AKAP13 expression from the lung eQTL analysis. The blue dotted line shows the significance threshold (p=5 × 10⁻⁶) used in stage 1 and the red dotted line shows genome-wide significance (p=5 × 10⁻⁸). Boxes at the bottom show gene locations plotted against the same x axis as the case-control and eQTL results, with AKAP13 highlighted in green. Variants are coloured according to linkage disequilibrium with rs62025270 (shown in blue). eQTL=expression quantitative trait locus. GWAS=genome-wide association study.

Figure 4

Lung eQTL results for AKAP13 expression by rs62025270 genotype Three independent datasets are shown. Blue points show normalised residuals of expression of AKAP13 after adjusting for age, sex, and smoking status for each individual by genotype of rs62025270. The box and whiskers show the mean and IQR for each genotype (left-hand y-axis). The grey boxes show the percentage of variance of AKAP13 expression that is explained by rs62025270 (right-hand y-axis). The p value relating the genotype to expression is shown at the top for each sample. Red allele indicates the allele associated with increased IPF susceptibility. eQTL=expression quantitative trait locus.

Figure 5

AKAP13 expression in bronchial mucosa and alveolar cells in patients with IPF and controls Sections of lung tissue from controls show AKAP13 expression in the bronchial mucosa (A) and alveolar cells (B). Sections of lung tissue from patients with IPF show low AKAP13 expression in fibroblastic foci (C), and high expression in the epithelium lining fibrotic alveoli (D) and distal small airways (green arrow; E). (F) Strong staining in lymphoid follicles associated with fibrotic regions in patients with IPF. (G) Section of lung tissue from a patient with IPF showing that areas of lung less affected by fibrosis have high numbers of alveolar cells expressing AKAP13 (green arrows). (H) In tissue from regions of the lung unaffected by fibrosis in patients with IPF, type 1 (green arrows) and type 2 (blue arrow) alveolar epithelial cells primarily express AKAP13 in the parenchyma. (I) AKAP13 mRNA expression in whole lung tissue homogenates from patients with IPF and controls. Each point shows a sample from one person and the line shows the mean fold change (black bars show ± SE) in AKAP13 mRNA in 46 patients with IPF and 51 controls. Relative expression (relative to housekeeping gene) is plotted on a log₂ scale. AKAP13=A-kinase anchoring protein 13. FF=fibroblastic foci. IPF=idiopathic pulmonary fibrosis. LF=lymphoid follicle.