Idiopathic pulmonary fibrosis (IPF) is an adult-onset, lethal, scarring lung disease of unknown etiology. Some individuals with IPF have a familial disorder that segregates as a dominant trait with incomplete penetrance. Here we used linkage to map the disease gene in two families to chromosome 5. Sequencing a candidate gene within the interval, TERT, revealed a missense mutation and a frameshift mutation that cosegregated with pulmonary disease in the two families. TERT encodes telomerase reverse transcriptase, which together with the RNA component of telomerase (TERC), is required to maintain telomere integrity. Sequencing the probands of 44 additional unrelated families and 44 sporadic cases of interstitial lung disease revealed five other mutations in TERT. A heterozygous mutation in TERC also was found in one family. Heterozygous carriers of all of the mutations in TERT or TERC had shorter telomeres than age-matched family members without the mutations. Thus, mutations in TERT or TERC that result in telomere shortening over time confer a dramatic increase in susceptibility to adult-onset IPF.genetics ͉ idiopathic pulmonary fibrosis ͉ telomeres ͉ aging

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BackgroundTelomerase is an enzyme that catalyzes the addition of nucleotides on the ends of chromosomes. Rare loss of function mutations in the gene that encodes the protein component of telomerase (TERT) have been described in patients with idiopathic pulmonary fibrosis (IPF). Here we examine the telomere lengths and pulmonary fibrosis phenotype seen in multiple kindreds with heterozygous TERT mutations.Methods and FindingsWe have identified 134 individuals with heterozygous TERT mutations from 21 unrelated families. Available medical records, surgical lung biopsies and radiographs were evaluated retrospectively. Genomic DNA isolated from circulating leukocytes has been used to measure telomere lengths with a quantitative PCR assay. We find that telomere lengths of TERT mutation carriers decrease in an age-dependent manner and show progressive shortening with successive generations of mutation inheritance. Family members without TERT mutations have a shorter mean telomere length than normal, demonstrating epigenetic inheritance of shortened telomere lengths in the absence of an inherited TERT mutation. Pulmonary fibrosis is an age-dependent phenotype not seen in mutation carriers less than 40 years of age but found in 60% of men 60 years or older; its development is associated with environmental exposures including cigarette smoking. A radiographic CT pattern of usual interstitial pneumonia (UIP), which is consistent with a diagnosis of IPF, is seen in 74% of cases and a pathologic pattern of UIP is seen in 86% of surgical lung biopsies. Pulmonary fibrosis associated with TERT mutations is progressive and lethal with a mean survival of 3 years after diagnosis. Overall, TERT mutation carriers demonstrate reduced life expectancy, with a mean age of death of 58 and 67 years for males and females, respectively.ConclusionsA subset of pulmonary fibrosis, like dyskeratosis congenita, bone marrow failure, and liver disease, represents a “telomeropathy” caused by germline mutations in telomerase and characterized by short telomere lengths. Family members within kindreds who do not inherit the TERT mutation have shorter telomere lengths than controls, demonstrating epigenetic inheritance of a shortened parental telomere length set-point.

Idiopathic pulmonary fibrosis (IPF) is a lethal scarring lung disease that affects older adults. Heterozygous rare mutations in the genes encoding telomerase are found in approximately 15% of familial cases. We have used linkage to map another disease-causing gene in a large family with IPF and adenocarcinoma of the lung to a 15.7 Mb region on chromosome 10. We identified a rare missense mutation in a candidate gene, SFTPA2, within the interval encoding surfactant protein A2 (SP-A2). Another rare mutation in SFTPA2 was identified in another family with IPF and lung cancer. Both mutations involve invariant residues in the highly conserved carbohydrate-recognition domain of the protein and are predicted to disrupt protein structure. Recombinant proteins carrying these mutations are retained in the endoplasmic reticulum and are not secreted. These data are consistent with SFTPA2 germline mutations that interfere with protein trafficking and cause familial IPF and lung cancer.

TERT mutation carriers exhibit early preclinical signs of lung fibrosis, bone marrow dysfunction, and premature graying. These clinical features and short telomere lengths characterize patients with germline TERT mutations.

Through the use of donor cells from transgenic rats expressing GFP exclusively in the germline, we have defined culture conditions where male germ cells lose (on STO cells) or maintain (on MSC-1 cells) stem cell activity. A cadre of germ cell transcripts strikingly decrease in relative abundance as a function of testis age or culture time on STO cells, but only a subset of these transcripts (approximately 248) remain elevated when cultured on MSC-1 cells. If specific gene expression regulates stem cell activity, some or all of these transcripts are candidates as such regulators. We establish a spermatogonial stem cell index (SSCI) that reliably predicts relative stem cell activity in rat or mouse testis cell cultures, and through the use of an antibody to a robust signal (Egr3) within the index find intense signals in single or paired cells. As germ cells form longer interconnected chains (incomplete cytokinesis), the Egr3 signal disappears coincident with a loss of stem cell activity. Thus, molecular markers specific for spermatogonial stem cells establish a reliable and rapid means by which to define these cells in culture and alleviate the need for laborious testicular transfers in initial cell culture studies.

A rat line was generated in which genomic integration of a ROSA-EGFP transgene resulted in exclusive expression of EGFP in the germ cells of both sexes. EGFP expression was uniform and robust in cleavage stage embryos beginning at the late 2-cell stage and continuing through blastocyst development where expression became restricted to cells of the inner cell mass. Subsequent analysis showed high EGFP expression exclusively in primordial, embryonic, and adult germ cells. This unique expression pattern makes this EGFP marked locus the first molecular marker of the germline lineage in both sexes in mammals. FISH was used to localize the transgene insertion to chromosome 11q11-q12, proximal to Grik1 and near Ncam2. Analysis of the region did not identify known germ cell-specific genes but did identify 19 ESTs or transcribed loci present in testes, ovary, or pre-implantation libraries from mice or rats. To assess the utility of the transgenic line for germ cell transplantation studies, non-selected, freshly isolated seminiferous tubule cells were transferred to the testis of recipient males. The donor cell population colonized the testis at a surprisingly high efficiency within 30 days following transfer. Since EGFP is a vital marker, the colonization process can be followed in vivo and the extent of colonization quantified. The unique germ cell specific expression of EGFP makes this line of transgenic rats an excellent novel tool to study germ cell origin, development, and differentiation, and to assess the plasticity of adult somatic stem cells to become male germ cells.

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