Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease that is strongly linked to deficits in telomere maintenance. Telomeres are protective non-coding DNA end caps at the ends of eukaryotic chromosomes. The telomere sequence, TTAGGG, is conserved in all vertebrates and is bound by a protective protein complex termed shelterin in a sequence-dependent manner. Displacement of the shelterin complex leads to a DNA damage response, resulting in chromosomal fusions and cell cycle arrest. With each round of cell division, telomere ends shorten due to a combination of the end replication problem and genomic stress. To counterbalance this shortening, cells utilize the enzyme telomerase. Telomerase is composed of a protein component telomerase reverse transcriptase (TERT) and an RNA template, the telomerase RNA component (TR). Mutations in IPF are often linked to genes required for telomere maintenance, such as mutations in TERT, TERC (TR), RTEL1, DKC1, and related genes. TR is 451 base pairs and contains an 11 base pair template responsible for binding the telomeric overhang and acting as a template for the reverse transcription of six new bases. We have identified a patient with IPF that is heterozygous for a mutation in the TR template region which is predicted to code for a variant telomere sequence. Remarkably, whole genome sequencing from the patient demonstrated that the telomeres display a significant portion of variant telomere sequences. Strict conservation of the telomere sequence throughout 400 million years of evolution suggests that variation in the telomere sequence would not be tolerated due to blocking shelterin binding. The nature of this unexpected find will be examined in this proposal. We hypothesize that this variant telomerase template, and subsequent variant telomere addition, may cause cellular dysfunction through two independent mechanisms; through telomere shortening and/or a loss of shelterin binding. These mechanisms will be explored in the following aims. Aim 1 will examine how the addition of a variant telomere sequence affects the enzymatic activity of telomerase and resulting telomere length in cells. Aim 2 will examine the effect that the variant sequence has on genomic stability, including shelterin binding and the DNA damage response in cells. Completion of this proposal will give us new insights into how telomeric sequence degeneration can affect genomic stability and telomere maintenance.