Exome sequencing identifies mutant TINF2 in a family with pulmonary fibrosis

Abstract

Background: Short telomeres are a common defect in idiopathic pulmonary fibrosis, yet mutations in the telomerase genes account for only a subset of these cases.

Methods: We identified a family with pulmonary fibrosis, idiopathic infertility, and short telomeres.

Results: Exome sequencing of blood-derived DNA revealed two mutations in the telomere-binding protein TINF2. The first was a 15-base-pair deletion encompassing the exon 6 splice acceptor site, and the second was a missense mutation, Thr284Arg. Haplotype analysis indicated both variants fell on the same allele. However, lung-derived DNA showed predominantly the Thr284Arg allele, indicating that the deletion seen in the blood was acquired and may have a protective advantage because it diminished expression of the missense mutation. This mosaicism may represent functional reversion in telomere syndromes similar to that described for Fanconi anemia. No mutations were identified in over 40 uncharacterized pulmonary fibrosis probands suggesting that mutant TINF2 accounts for a small subset of familial cases. However, similar to affected individuals in this family, we identified a history of male and female infertility preceding the onset of pulmonary fibrosis in 11% of TERT and TR mutation carriers (five of 45).

Conclusions: Our findings identify TINF2 as a mutant telomere gene in familial pulmonary fibrosis and suggest that infertility may precede the presentation of pulmonary fibrosis in a small subset of adults with telomere syndromes.

Figure 1 –

Clinical features of family with pulmonary fibrosis. A, Pedigree of index case with summary of clinical history. The proband is delineated by the arrow; □ = male family members; ○ = female family members. The black shade refers to individuals with clear telomere phenotypes and gray indicates probable obligate carrier of a TINF2 mutation. TINF2 genotypes are denoted adjacent to the individuals sequenced. B, Lung window images from a chest CT scan from the proband showing peripheral honeycombing predominantly in the lung bases, typical of IPF. The UIP histology was subsequently confirmed on lung biopsy. C, Lymphocyte telomere length of affected and unaffected individuals from A are plotted relative to age-matched control subjects. D, Granulocyte telomere length relative to age-matched control subjects. Nomograms and percentiles were based on data from 192 control subjects. The pedigree identifiers refer to individuals in A. C/G = missense mutation; Δ15 = deletion at the intron 5-exon 6 junction; IPF = idiopathic pulmonary fibrosis; UIP = usual interstitial pneumonia; WT = wild-type reference sequence.

Figure 2 –

TINF2 mutations studies in a proband with pulmonary fibrosis. A, Schema of TINF2 genomic locus showing position of mutations detected by exome sequencing. The exon 6 hotspot is highlighted in red. B, Chromatogram traces of sequence data from the proband derived from blood and lung genomic DNA. C, Percentage of the polymerase chain reaction-amplified products derived from clonal analysis in the blood and lung.

Figure 3 –

Consequences of mutations on TIN2 protein stability and proposed model of functional consequences of TINF2 mutations. A, Schema of TINF2 at its genomic locus inserted in FRT sites in HeLa cells. B, Immunoblot of Myc-tagged TIN2 expressed from a single promoter shows its two isoforms, TIN2L and TIN2S as described in Kaminker and Campisi Cell Cycle 2009. A third band, possibly representing a third TIN2 isoform, is also seen at an intermediate size between TIN2L and TIN2S. In contrast to the wild-type, and the two missense variants, Δ15-Thr284Arg abolishes the expression of TIN2L and creates low levels of a smaller degenerate protein product below the expected size for TIN2S. C, Six known familial pulmonary fibrosis genes and their frequency estimates. D, Schema of model summarizing the putative functional consequences of the TINF2 mutations within the context of genetic and functional studies delineated. CMV = cytomegalovirus; FRT = flippase recognition target; TetO = Tet operator; TetR = Tet repressor; TIN2L = long-form isoform; TIN2S = short-form isoform. See Figure 1 legend for expansion of other abbreviations.