PCSK6 and Survival in Idiopathic Pulmonary Fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P < 5 × 10^-5) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (P < 5 × 10^-8). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance. Measurements and Main Results: After quality controls, 1,481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of PCSK6 (proprotein convertase subtilisin/kexin type 6) reaching genome-wide significance (hazard ratio, 4.11 [95% confidence interval, 2.54-6.67]; P = 9.45 × 10^-9). PCSK6 protein was highly expressed in IPF lung parenchyma. PCSK6 lung staining intensity, peripheral blood gene expression, and plasma concentration were associated with reduced TFS. Conclusions: We identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein plays a potentially important role in IPF progression.

Keywords: IPF; PCSK6 protein; genome-wide association study; genomics; survival.

Figure 1.

Manhattan plot of stage I gene variants associated with idiopathic pulmonary fibrosis survival. Each dot represents a gene variant, arranged on the x-axis by chromosome. Variants falling above the blue line, which corresponds to P < 5 × 10⁻⁵, are considered to reach nominal significance, whereas those falling above the red line, which corresponds to P < 5 × 10⁻⁸, are considered to reach genome-wide significance. All variants crossing the nominal significance threshold were advanced for stage II testing. Chr = chromosome; DAZAP1 = deleted in azoospermia-associated protein 1; PCSK6 = proprotein convertase subtilisin/kexin type 6; RNU6 = U6 small nuclear RNA; SUCLG1 = succinate-CoA ligase GDP/ADP-forming subunit α; UBE2Q2 = ubiquitin-conjugating enzyme E2Q family member 2.

Figure 2.

(A–F) PCSK6 immunohistochemistry showed increased cytoplasmic PCSK6 (proprotein convertase subtilisin/kexin type 6) expression in ciliated epithelial cells (A) and alveolar epithelial cells (B and C) compared with normal lung control sections (D–F). (G and H) Parallel idiopathic pulmonary fibrosis sections confirm increased PCSK6 staining (G) compared with control section (H). (I) Human kidney positive control is provided for reference. Scale bars, 20 μm (A–F) and 50 μm (G–I).

Figure 3.

(A) Comparison of relative PCSK6 (proprotein convertase subtilisin/kexin type 6) staining intensity between idiopathic pulmonary fibrosis (IPF) cases and non-IPF control subjects demonstrated significantly higher median intensity in IPF lungs (P < 0.001). (B) Survival was lower among IPF cases with PCSK6 staining intensity above the median. NL = non-IPF control subjects.

Figure 4.

Relationship between proprotein convertase subtilisin/kexin type 6 and clinically relevant idiopathic pulmonary fibrosis endpoints. (A and B) Higher peripheral blood gene expression (A) and circulating plasma protein concentration (B) are associated with reduced transplantation-free survival. CI = confidence interval; COMET = Correlating Outcomes with Biochemical Markers to Estimate Time-Progression in IPF.