Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening

Abstract

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial kindreds with pulmonary fibrosis. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no previous connection to telomere biology or disease, with five new heterozygous damaging mutations in unrelated cases and none in controls (P = 1.3 × 10(-8)); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more new damaging and missense variants at conserved residues in cases than in controls (P = 1.6 × 10(-6)). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths, and we observed epigenetic inheritance of short telomeres in family members. Together, these genes explain ~7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction.

Figure 1. Q-Q Plot of Observed versus Expected P-values Comparing the Burden of Novel Variants in Protein-Coding Genes in Familial Pulmonary Fibrosis Cases and Controls

Novel variants in European 78 pulmonary fibrosis probands and 2,816 controls were identified and their frequencies compared by Fisher’s exact test. The distribution of observed P-values for each gene was compared to the distribution of expected P-values. (a) Analysis of novel variants that are either damaging or missense at positions that are highly conserved across phylogeny. (b) Analysis of novel damaging variants. The distribution of observed P-values generally follows the expected distribution for damaging plus conserved missense variants, while for damaging mutations only, many P-values are lower than expected due to a paucity of variants. The damaging plus missense set shows two genes (RTEL1 and PARN) with P-values at or near genome-wide significance, while the damaging variant set shows one gene, PARN, with an observed P-value well outside the expected distribution.

Figure 2. Segregation of Heterozygous PARN Mutations in Familial Pulmonary Fibrosis Kindreds and the Location of PARN Alterations in the Different Protein Domains

(a–f) Abridged pedigrees of six kindreds with familial pulmonary fibrosis and PARN mutations. The PARN cDNA mutations and predicted amino acid changes are listed above each family. Individuals with pulmonary fibrosis or an unclassified lung disease are indicated by red and blue symbols, respectively. Unfilled symbols represent individuals with no self-reported lung disease. Arrows denote the probands. Kindreds F349 and F373 were found to be related through a distant ancestor (II.2). Numbers in parentheses indicate individuals for whom no DNA sample is available. The presence or absence of a mutation is indicated by plus or minus signs, respectively. When the mutation was inferred from location in pedigree, the plus sign is in parentheses. The age at the time of blood draw or death is indicated to the upper right of each symbol. (g) Schematic representation of the functional domains of PARN with the position of mutations indicated by the arrows. Conserved protein domains include the CAF1 ribonuclease domain (blue), the R3H domain that binds single stranded nucleic acids (red), and the RNA recognition domain (RRM, green). (h) Clustal alignments of homologous PARN protein sequences from Homo sapiens (human), Macaca mulatta (monkey), Canis familiaris (dog), Bos taurus (cow), Mus musculus (mouse), Rattus norvegicus (rat), Gallus gallus (chicken), Danio rerio (fish).

Figure 3. Segregation of Heterozygous RTEL1 Mutations in Familial Pulmonary Fibrosis Kindreds and the Location of RTEL1 Alterations in the Different Protein Domains

(a–e) Abridged pedigrees of six kindreds with familial pulmonary fibrosis and RTEL1 mutations. The RTEL1 cDNA mutations and predicted amino acid changes are listed above each family. Pedigree information is presented in a similar manner as in Figure 1. (f) Schematic representation of the functional domains of RTEL1 protein with the position of the mutations indicated by the arrows. Conserved protein domains: DEAD2 domain (orange bar), helicase C domain (red), RAD3-related DNA helicase domain (blue), harmonin N-like domian (purple) and the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) domain (green). (g) Clustal alignments of homologous RTEL1 protein sequences from species listed in Figure 2.

Figure 4. PARN and RTEL1 Mutations Are Associated with Short Leukocyte Telomere Lengths

(a) Telomere lengths determined by Southern blot terminal restriction fragment length (TRFL) analysis of nonrelated normal (spouse) controls and PARN probands. The age of each individual is indicated above the blot. Box plots (median, IQR, range) of TRFL (b) and percent short TRFL (< 3 kb) (c) are indicated for the controls and PARN and RTEL1 probands, who have an average age of 64, 64 and 60 years, respectively. (d) Telomere length of genomic DNA isolated from circulating leukocytes from heterozygous PARN and RTEL1 mutation carriers as measured by multiplexed qPCR assay. Telomere lengths are age-adjusted and are expressed as the observed minus the expected length. Each symbol represents a unique individual; the proband for each family is indicated by the red outline. The mean telomere length is indicated by the black bar. The black dotted line shows the median telomere length of an independent control group; approximate age-adjusted prediction bands (percentiles, blue dotted lines) were previously calculated from a linear regression model using the controls. (e) Genomic DNA telomere lengths as measured by the multiplexed qPCR assay of individuals heterozygous for a mutation in TERT, TERC, RTEL1 and PARN are shown as red, orange, green and blue colored circles, respectively. Telomere lengths of 1^st and 2^nd degree relatives of the PARN or RTEL1 mutation carriers who did not inherit the variants are shown as open green and blue circles, respectively. Open black circles represent unrelated normal controls.