Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) risk has a strong genetic component. Studies have implicated variations at several loci, including TERT, surfactant genes, and a single nucleotide polymorphism at chr11p15 (rs35705950) in the intergenic region between TOLLIP and MUC5B. Patients with IPF who have risk alleles at rs35705950 have longer survival from the time of IPF diagnosis than do patients homozygous for the non-risk allele, whereas patients with shorter telomeres have shorter survival times. We aimed to assess whether rare protein-altering variants in genes regulating telomere length are enriched in patients with IPF homozygous for the non-risk alleles at rs35705950.

Methods: Between Nov 1, 2014, and Nov 1, 2016, we assessed blood samples from patients aged 40 years or older and of European ancestry with sporadic IPF from three international phase 3 clinical trials (INSPIRE, CAPACITY, ASCEND), one phase 2 study (RIFF), and US-based observational studies (Vanderbilt Clinical Interstitial Lung Disease Registry and the UCSF Interstitial Lung Disease Clinic registry cohorts) at the Broad Institute (Cambridge, MA, USA) and Human Longevity (San Diego, CA, USA). We also assessed blood samples from non-IPF controls in several clinical trials. We did whole-genome sequencing to assess telomere length and identify rare protein-altering variants, stratified by rs35705950 genotype. We also assessed rare functional variation in TERT exons and compared telomere length and disease progression across genotypes.

Findings: We assessed samples from 1510 patients with IPF and 1874 non-IPF controls. 30 (3%) of 1046 patients with an rs35705950 risk allele had a rare protein-altering variant in TERT compared with 34 (7%) of 464 non-risk allele carriers (odds ratio 0·40 [95% CI 0·24-0·66], p=0·00039). Subsequent analyses identified enrichment of rare protein-altering variants in PARN and RTEL1, and rare variation in TERC in patients with IPF compared with controls. We expanded our study population to provide a more accurate estimation of rare variant frequency in these four loci, and to calculate telomere length. The proportion of patients with at least one rare variant in TERT, PARN, TERC, or RTEL1 was higher in patients with IPF than in controls (149 [9%] of 1739 patients vs 205 [2%] of 8645 controls, p=2·44 × 10^-8). Patients with IPF who had a variant in any of the four identified telomerase component genes had telomeres that were 3·69-16·10% shorter than patients without a variant in any of the four genes and had an earlier mean age of disease onset than patients without one or more variants (65·1 years [SD 7·8] vs 67·1 years [7·9], p=0·004). In the placebo arms of clinical trials, shorter telomeres were significantly associated with faster disease progression (1·7% predicted forced vital capacity per kb per year, p=0·002). Pirfenidone had treatment benefit regardless of telomere length (p=4·24 × 10^-8 for telomere length lower than the median, p=0·0044 for telomere length greater than the median).

Interpretation: Rare protein-altering variants in TERT, PARN, TERC, and RTEL1 are enriched in patients with IPF compared with controls, and, in the case of TERT, particularly in individuals without a risk allele at the rs35705950 locus. This suggests that multiple genetic factors contribute to sporadic IPF, which might implicate distinct mechanisms of pathogenesis and disease progression.

Funding: Genentech, National Institutes of Health, Francis Family Foundation, Pulmonary Fibrosis Foundation, Nina Ireland Program for Lung Health, US Department of Veterans Affairs.

Figure 1.

Gene diagram of TERT and rare variant location. SNPs are identified by amino acid change and position and colored red if the MAF is ≤ 0.001, blue if the MAF > 0.001. DEL indicates a frameshift affecting >1 amino acids.

Figure 2.

Gene diagram of TERC and rare variant location. SNPs are identified by nucleic acid position and base change and colored red if the MAF is < 0.001, blue if the MAF > 0.001.

Figure 3.

Gene diagram of RTEL1 and rare variant location. SNPs are identified by amino acid change and position and colored red if the MAF is < 0.001, blue if the MAF > 0.001. DEL indicates a frameshift affecting >1 amino acids. “.” indicates variation affecting a predicted splice site.

Figure 4.

Gene diagram of PARN and rare variant location. SNPs are identified by amino acid change and position and colored red if the MAF is < 0.001, blue if the MAF > 0.001. DEL indicates a frameshift affecting >1 amino acids. “.” indicates variation affecting a predicted splice site. Exons have 200 bp added both up and downstream to enhance visibility.

Figure 5.

Rates of telomere complex gene rare protein-altering variants in clinical trial patients. Each patient was assessed for the presence of a missense/nonsense variant in TERT, or RTEL1 or any variant in TERC. The fraction of patients in each trial that have at least one such variant are plotted. The size of each point is proportional to the number of patients in that trial.

Figure 6.

Telomere lengths of clinical trial patients. Mean telomere lengths as measured by TelomereHunter analysis of whole genome sequencing of blood samples from patients in various clinical trials. Age vs. telomere length by disease. Each line is a least squares fit to all patients from a given primary disease indication. Shaded areas are 95% confidence interval estimates for the linear fit to the true population.

Figure 7.

Telomere lengths of clinical trials patients by TERT common variant (rs2736100) genotype and rare variant status. "A" is the risk allele for rs2736100, while "C" is the protective allele. "Wildtype" here refers to patients with a TERT coding sequence containing no missense mutations, while "TERT RV" indicates patients with at least one missense mutation. Boxes indicate the median and quartile values, while the whiskers extend to the most distant value no further than 1.5 times the interquartile range from the quartiles.

Figure 8.

Profile of mean percent decline over time of forced vital capacity (FVC) in the ASCEND and CAPACITY phase III clinical trials of pirfenidone for IPF. Patients in the ASCEND and CAPACITY trials were stratified by whether they received pirfenidone treatment (dashed lines) or a placebo control treatment (solid lines), and by whether they had peripheral blood telomeres that were longer (blue lines) or shorter (red lines) than the median length of the cohort. Error bars are Standard Error of the Mean. Figure A represents telomere length stratified by the median, Figure B is by MUC5B promoter variant status (orange denotes patients positive for the MUC5B risk-allele; green denotes patients negative for the MUC5B risk allele).