Rare variants in RTEL1 are associated with familial interstitial pneumonia

Abstract

Rationale: Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families.

Objectives: To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis.

Methods: Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds.

Measurements and main results: We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function.

Conclusions: Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis.

Keywords: genetics; idiopathic pulmonary fibrosis; telomere.

Figure 1.

Whole-exome sequencing (WES) analysis pipeline. WES analysis occurred through three phases. In phase 1, raw reads from all samples underwent quality checking, genome mapping, local realignment, and variant calling using Varscan 2.0 to call variants. Following exclusionary quality-control filter, in phase 2 only variants that were found in all affected WES subjects within a family were retained. From among these segregating variants, only those highly likely to affect protein quantity or function (nonsynonymous exonic single-nucleotide variants [i.e., missense or nonsense], insertions and deletions, splice variants) were selected for further analysis. From among these variants, those with minor allele frequency (MAF) greater than 0.001 in the Exome Sequencing Project database (ESPdb) were excluded to yield candidate rare variants (RVs). Sanger requencing was then performed in extended pedigrees encompassing all affected subjects for whom DNA was available to exclude variants that did not segregate fully with disease. These segregating RVs from each family were functionally annotated and aggregated at the gene level across families for further study. GERP = genomic evolutionary rate profiling; INDEL = insertion and deletion; SIFT = Sorting Intolerant from Tolerant; SNV = single-nucleotide variants.

Figure 2.

Pedigrees of familial interstitial pneumonia (FIP) families with RTEL1 rare variants. (A–I) Pedigrees of kindreds with heterozygous RTEL1 rare variants that segregate with FIP are depicted. Roman numerals indicate generation and Arabic numerals indicate subject number within a generation. Diamonds are used to indicate multiple offspring including males and females or at the request of subjects and families to maintain confidentiality. Heterozygous RTEL1 rare variant carriers are denoted as M/+; wild-type RTEL1 subjects are denoted as +/+. In family H, the missense variant is denoted as M, and the 35-base-pair deletion is denoted as Δ. Mutation status and telomere percentile (adjusted for age) are depicted below subjects. Whole-exome sequencing (WES) indicates subjects included in the WES cohort; genotypes for all other subjects were obtained by Sanger sequencing.

Figure 3.

Radiographic and histopathologic changes associated with RTEL1 rare variants. (A, B) Representative high-resolution computed tomography images from affected subjects carrying RTEL1 rare variants show typical usual interstitial pneumonia features. (C, D) Hematoxylin and eosin–stained lung biopsies from RTEL1 rare variant carriers show dense interstitial fibrosis, temporal heterogeneity, and microscopic honeycomb cysts consistent with usual interstitial pneumonia. Original magnification ×40 (C) and ×100 (D).

Figure 4.

RTEL1 rare variants detected by whole-exome sequencing. (A) Modified Sanger sequencing confirmed the RTEL1 rare variants detected by whole-exome sequencing in families A–I. (B) Schematic depiction of the location of RTEL1 rare variants in the 1,219-amino-acid isoform and an alternatively spliced 1,300-amino-acid isoform. PIP = PCNA (proliferating cell nuclear antigen)-interacting protein.

Figure 5.

Carriers of RTEL1 rare variants have short telomeres and increased T-circle formation. (A) Telomere restriction fragment (TRF) is plotted versus age for probands from families in the familial interstitial pneumonia (FIP) registry. All probands in RTEL1-mutation families were less than or equal to 10th percentile for age, similar to what is seen for TERT (n = 18), TERC (n = 2), and DKC1 (n = 1). (B) A 1-D electrophoresis gel from T-circle assay comparing age-matched unaffected married-in control subjects, TERT mutation carriers, and RTEL1 mutation carriers. (C) Quantification of T-circle formation in peripheral blood mononuclear cells, defined as the percentage of T-circles compared with total telomeric DNA in eight unaffected control subjects, nine TERT mutation carriers, and 20 RTEL1 mutation carriers. *P < 0.001 across groups and pairwise RTEL1 carrier versus control and RTEL1 carriers versus TERT carriers.