Whole exome sequencing identifies novel candidate genes that modify chronic obstructive pulmonary disease susceptibility

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible airflow limitation in response to inhalation of noxious stimuli, such as cigarette smoke. However, only 15-20 % smokers manifest COPD, suggesting a role for genetic predisposition. Although genome-wide association studies have identified common genetic variants that are associated with susceptibility to COPD, effect sizes of the identified variants are modest, as is the total heritability accounted for by these variants. In this study, an extreme phenotype exome sequencing study was combined with in vitro modeling to identify COPD candidate genes.

Results: We performed whole exome sequencing of 62 highly susceptible smokers and 30 exceptionally resistant smokers to identify rare variants that may contribute to disease risk or resistance to COPD. This was a cross-sectional case-control study without therapeutic intervention or longitudinal follow-up information. We identified candidate genes based on rare variant analyses and evaluated exonic variants to pinpoint individual genes whose function was computationally established to be significantly different between susceptible and resistant smokers. Top scoring candidate genes from these analyses were further filtered by requiring that each gene be expressed in human bronchial epithelial cells (HBECs). A total of 81 candidate genes were thus selected for in vitro functional testing in cigarette smoke extract (CSE)-exposed HBECs. Using small interfering RNA (siRNA)-mediated gene silencing experiments, we showed that silencing of several candidate genes augmented CSE-induced cytotoxicity in vitro.

Conclusions: Our integrative analysis through both genetic and functional approaches identified two candidate genes (TACC2 and MYO1E) that augment cigarette smoke (CS)-induced cytotoxicity and, potentially, COPD susceptibility.

Fig. 1

Identifying candidate COPD genes through genomic and functional approaches. WES in 62 highly susceptible smokers and 30 exceptionally resistant smokers were conducted to identify exonic variants that may contribute to disease risk or resistance to CS. Top scoring candidate genes from the rare variant and gene set analyses were further filtered by requiring that the gene be expressed in primary HBECs, and 81 candidate genes were selected for in vitro functional testing in CSE-exposed HBECs. Using siRNA-mediated gene silencing experiments, we identified candidate genes whose knockdown augmented CSE-induced cytotoxicity, protected CSE-induced cytotoxicity, or alone-reduced cell viability

Fig. 2

Effects of TACC2 siRNA transfection on CSE-induced cytotoxicity and TACC2 mRNA levels. a Forty-eight hours after transfection with either siRNA targeting TACC2 (TACC2 siRNA) or the scrambled siRNA (scrambled control) as control, HBEC2 cells were incubated in the absence (no CSE) or presence of 2 % CSE (CSE) for 24 h. Cell viability was determined using the MTT assay at 24 h. Data are expressed as mean ± SEM for three independent experiments with triplicated samples (*p < 0.05; **p < 0.01). b Steady-state levels of TACC2 mRNA were measured by RT-PCR and presented as relative fold difference compared with CDKN1B in HBEC2 cells after 48 h with either TACC2 siRNA or scrambled control. Data are expressed as mean ± SEM from two independent experiments with triplicated samples (**p < 0.01). c HBEC2 cells were treated as in a. Cell death was analyzed by Annexin V and propidium iodide (PI) staining 24 h after 2 % CSE exposure. The percentage of Annexin V positive cells/total cell number was expressed as percentage apoptosis. Data are expressed as mean ± SEM for three independent experiments (**p < 0.01). Representative flow cytometry data are shown. d HBEC2 cells were treated as in a. Immunoblot analysis of active caspase-3 was performed 24 h after 2 % CSE exposure. Immunoblotting data are representative of three experiments