Chest
Original Research: Diffuse Lung DiseaseExome Sequencing Identifies Mutant TINF2 in a Family With Pulmonary Fibrosis
Section snippets
Human Subjects
Subjects were recruited to a Johns Hopkins study aimed at understanding the genetics and natural history of telomere-mediated disease.10, 11 The study was approved by the Johns Hopkins Medicine Institutional Review Board, NA_33072, and all the participants gave written informed consent.
Exome Sequencing
Exome sequencing was performed using the SureSelect XT All Exome V4 kit and was sequenced on the Illumina HiSEquation 2000 platform as described.4 Variants were called using the Genome Analysis Tool Kit (GATK) and were annotated using ANNOVAR (http://www.openbioinformatics.org/annovar/).4 Unique variants in telomere genes that were not found in the 1000 Genome Project Database and the Exome Variant Server were prioritized for additional studies.
Telomere Length and DNA Sequencing
Telomere length was measured on peripheral blood mononuclear cells by flow cytometry and fluorescence in situ hybridization.12 TERT and TR sequencing was performed as outlined previously.13 TINF2 exon 6 was sequenced using the following primer sets: TINF2.E6F 5′-CCTGGAGACAATATGGTGTGG-3′ and TINF2.E6R 5′-AGGCTGTTGATCCAATCCTG-3′ (834 bp product). Because DNA derived from formalin-fixed, paraffin-embedded tissues is fragmented, we used two primer sets to genotype the two variants: TINF2.E6.1F
Haplotype and Clonality Studies
Genomic DNA was extracted from peripheral blood using a Puregene kit (QIAGEN) and from paraffin-embedded lung tissue using standard protocols. Variants were confirmed by Sanger sequencing, and the haplotype of adjacent variants was determined by thymidine and adenosine cloning polymerase chain reaction (PCR) products into a pCR4-TOPO vector according to the manufacturer's protocol (Life Technologies) and by the sequencing of individual clones.
TIN2 Expression Studies
We measured TINF2 mRNA levels in lymphoblastoid lines generated from the proband as well as from healthy control subjects, as described previously,3 using the following quantitative real-time PCR primers: TINF2.E3F: GATTTTGGAGGCACAGGAAA and TINF2.E5R: CTGCATCCAACTCAGCACAT. To test the in vivo stability of mutant TIN2 proteins, we cloned the TINF2 genomic locus (inclusive of introns and exons) into a pCDNA5/FRT/TO vector (Life Technologies). An N-terminal Myc tag was introduced immediately after
Exome Sequencing Identifies TINF2 Mutations in a Proband With FPF
The proband presented with IPF at 49 years of age and died at age 50 (Figs 1A, 1B). She did not have premature graying, blood count abnormalities, or any mucocutaneous features of dyskeratosis congenita. Her brother died of IPF at age 44 years. Both the proband and her brother had a documented history of infertility that did not respond to reproductive assistance. The family history was notable for two maternal uncles who died of cryptogenic liver disease in their 30s (Fig 1A). Lymphocyte
Haplotype Studies Suggest Acquired Mosaicism of the TINF2 Genotype in the Blood
To determine the phase of the mutations, we amplified and cloned the TINF2 region encompassing the variants and quantified the proportion of each clone by Sanger sequencing. Among the clones sequenced from blood-derived DNA (n = 56), the wild-type allele was in 34%. The Δ15 and Thr284Arg mutations were detected in cis in 45% of the clones. The remaining clones contained either the Δ15 (17%) or Thr284Arg (4%) alone. The clonal heterogeneity suggested two compatible possibilities. The first was
Segregation Studies Support Mutant TINF2 Segregates With IPF Phenotype
To test for segregation in the family, we sequenced genomic DNA from the proband's father and sister, who had no telomere syndrome features and normal telomere lengths, and found no mutations (Figs 1A, 1C, 1D). These findings, along with the maternal history of liver disease, implicated the patient's mother as a likely obligate carrier of the Thr284Arg mutation (Fig 1A).
TINF2 Deletion Disrupts Protein Expression
We examined the functional consequences of the mutations on endogenous TIN2 expression by measuring the mRNA levels in cells derived from the proband and found a reduction by quantitative real-time PCR (37% compared with 100 ± 21%, n = 4 control subjects). This was consistent with the Δ15-Thr284Arg mutation causing nonsense-mediated decay. To directly examine whether the Δ15 mutation affected the expression of the missense mutation, we cloned the TINF2 genomic sequence under the regulation of a
TINF2 Mutations Are a Rare Cause of FPF
We had previously screened 73 probands with FPF for TINF2 mutations in exon 6.6 In this study, we screened 40 additional probands with FPF and found no mutations. These data suggest that TINF2 mutations are a rare cause of FPF and explain the genetic risk in approximately 1% of cases.
A History of Infertility May Precede the Onset of IPF in Telomere Syndrome Cases
In both the proband and her brother, infertility preceded the onset of IPF, and we tested whether this pattern was recurrent in other telomere syndrome cases. We reviewed the history of 45 consecutively evaluated patients with pulmonary fibrosis with telomerase mutations in a Johns Hopkins-based study and documented a history of referral for reproductive assistance in five cases (11%, three male and two female, TERT n = 3, TR n = 2). In all these cases, the infertility evaluations preceded the
Discussion
Here, we report mutations in TINF2 in association with FPF. Our observations add to a growing body of literature underscoring the intimate connection between telomere dysfunction and IPF risk. The proband and her affected brother with IPF had no features of classic dyskeratosis congenita and no bone marrow failure, the predominant phenotypes heretofore linked to TINF2 mutations that usually manifest before the age of 10 years.15, 16, 17 As such, our exome findings are notable and indicate that
Conclusions
The genetic diagnosis of telomere-mediated pulmonary fibrosis has important implications for treatment. In the lung transplant setting, patients with pulmonary fibrosis are prone to otherwise rare complications caused by immunosuppressive medications that include excessive transfusion support for cytopenias, renal failure, and GI bleeding.33 The link between TINF2 and FPF, therefore, has implications for genetic evaluation as well as for patient care.
Acknowledgments
Author contributions: M. A. is guarantor of the manuscript and takes responsibility for the integrity of the data and the accuracy of the data analysis. J. K. A., S. E. S., and M. A. designed the experiments and interpreted the data; J. K. A., S. E. S., C. L. W., M. H., V. S. H., and M. A. performed experiments; and M. A. wrote the manuscript.
Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations
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Dr Alder and Ms Stanley contributed equally to this work.
FUNDING/SUPPORT: This work was supported by the National Institutes of Health (NIH) [Grant RO1 CA160433] and by the Commonwealth Foundation (to Dr Armanios). Dr Alder received support from the NIH [Grant R00 HL113105], and Ms Stanley received support from the NIH [Grant T32 GM007309].
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