An Exome Sequencing Study to Assess the Role of Rare Genetic Variation in Pulmonary Fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is an increasingly recognized, often fatal lung disease of unknown etiology.

Objectives: The aim of this study was to use whole-exome sequencing to improve understanding of the genetic architecture of pulmonary fibrosis.

Methods: We performed a case-control exome-wide collapsing analysis including 262 unrelated individuals with pulmonary fibrosis clinically classified as IPF according to American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association guidelines (81.3%), usual interstitial pneumonia secondary to autoimmune conditions (11.5%), or fibrosing nonspecific interstitial pneumonia (7.2%). The majority (87%) of case subjects reported no family history of pulmonary fibrosis.

Measurements and main results: We searched 18,668 protein-coding genes for an excess of rare deleterious genetic variation using whole-exome sequence data from 262 case subjects with pulmonary fibrosis and 4,141 control subjects drawn from among a set of individuals of European ancestry. Comparing genetic variation across 18,668 protein-coding genes, we found a study-wide significant (P < 4.5 × 10^-7) case enrichment of qualifying variants in TERT, RTEL1, and PARN. A model qualifying ultrarare, deleterious, nonsynonymous variants implicated TERT and RTEL1, and a model specifically qualifying loss-of-function variants implicated RTEL1 and PARN. A subanalysis of 186 case subjects with sporadic IPF confirmed TERT, RTEL1, and PARN as study-wide significant contributors to sporadic IPF. Collectively, 11.3% of case subjects with sporadic IPF carried a qualifying variant in one of these three genes compared with the 0.3% carrier rate observed among control subjects (odds ratio, 47.7; 95% confidence interval, 21.5-111.6; P = 5.5 × 10^-22).

Conclusions: We identified TERT, RTEL1, and PARN-three telomere-related genes previously implicated in familial pulmonary fibrosis-as significant contributors to sporadic IPF. These results support the idea that telomere dysfunction is involved in IPF pathogenesis.

Keywords: collapsing analysis; exome sequencing; genetics; interstitial lung disease; pulmonary fibrosis.

Figure 1.

Quantile–quantile plot of pulmonary fibrosis collapsing analyses. Results are shown for the analysis of 262 pulmonary fibrosis case and 4,141 control subjects. (A) A total of 15,393 genes had at least one case or control carrier. Qualifying variants have a minor allele frequency less than 0.05% in the test cohort and are absent among external reference cohorts. Variants are annotated as loss-of-function, in-frame indel, or missense predicted to be “probably damaging” by Polymorphism Phenotyping version 2 (HumDiv). Two genes, TERT and RTEL1, achieved study-wide significance (adjusted α = [0.05/(6 × 18668)] = 4.46 × 10⁻⁷). (B) A total of 10,710 genes had at least one loss-of-function case or control carrier. Qualifying variants are variants with a population minor allele frequency less than or equal to 0.1% and are annotated as loss-of-function single-nucleotide variants or indels. PARN and RTEL1 achieved study-wide significance (P < 4.46 × 10⁻⁷).

Figure 2.

Distribution of case and control qualifying variants for TERT, RTEL1, and PARN from the primary and loss-of-function model analyses. Case and control variants are plotted on the canonical transcript of the corresponding gene. Frameshift (F) and nonsense (N) variants are filled in red. Missense (M) and in-frame indel (C) variants are filled in blue. Case variants are shown with red lines, and control variants are shown with blue lines. Dashed lines indicate a qualifying variant found in both case and control subjects. Variants reported below the protein line indicate canonical splice (S) variants. Known conserved domains from the Conserved Domains Database are listed below the gene names. UTR = untranslated region.

Figure 3.

Enrichment levels of different qualifying variant categories among the three study-wide significant pulmonary fibrosis genes. We compared enrichment of qualifying variants across increasing minor allele frequency (MAF) and varying in silico effects to assess which variation type was most attributable to pulmonary fibrosis disease risk. (A) Six paired comparisons were done to a single background variation estimate: the percentage of exome-wide ultrarare autosomal synonymous variants that belong to the case subjects (8,279 of 141,449; Ps = 0.0583). (B) Forest plot derived from a multivariate logistic regression assessing the relative contribution of different qualifying variant categories. All categories are mutually exclusive (see Methods section in online supplement). The loss-of-function (LoF) category represents LoF variants with an MAF less than or equal to 0.1%. The LoF category is prematurely truncated for illustrative purposes; however, the effect size and 95% confidence interval (CI) are provided. The missense probably damaging ultrarare category includes variants predicted to be “probably damaging” by Polymorphism Phenotyping version 2 with an MAF less than 0.05% in the test population and absent in Exome Variant Server (EVS) and Exome Aggregation Consortium (ExAC release 0.3.1). The missense probably damaging MAF less than or equal to 0.1% represents variants not in the ultrarare category and that are predicted to be “probably damaging” with a MAF less than or equal to 0.1% across the test population, EVS, and ExAC cohorts. The missense “possibly/benign” category includes missense variants predicted to be “possibly damaging” or “benign” with a MAF less than or equal to 0.1% across the test population, EVS, and ExAC cohorts (including ultrarare possibly/benign missense variants). The “neutral” category represents ultrarare putatively neutral (synonymous) variants across the three pulmonary fibrosis genes (TERT, RTEL1, and PARN). N/A = not applicable; OR = odds ratio.