Dyskeratosis congenita (DC) is a multisystem disorder caused by defective telomere maintenance. Bone marrow failure, myelodysplastic syndrome, pulmonary fibrosis, solid tumors, and liver disease are life-threatening manifestations of DC. The goal of our research is to understand the molecular genetics of DC, thereby opening avenues for targeted interventions that may be relevant not only to DC but also to other conditions associated with telomere shortening. Mutations in TINF2, which encodes the telomeric shelterin complex component, TIN2, are causes of DC. An outstanding question is why heterozygous and de novo mutations in TINF2 result in extreme telomere shortening and severe, early-onset disease whereas heterozygous mutations in telomerase component genes lead to more severe manifestations of disease and progressively earlier onset disease over generations in kindred's. All DC-associated TINF2 mutations reported to date, whether missense or truncating, cluster in a 34-amino acid segment located centrally within both the short (TIN2S1-354) and long (TIN2L1-451) TIN2 isoforms. Our research focuses on the unstudied TIN2L isoform, which either harbors missense mutations within the DC cluster or is not expressed when truncating mutations are present. Our preliminary data indicate that TIN2L and TIN2S are not equivalent components of shelterin and that the most common DC-associated missense mutation has deleterious effects on TIN2L but not TIN2S. Additionally, we found that TIN2L contains a conserved phosphorylation site that, when mutated, has the same functional effect on TIN2L as mutations within the DC cluster. Lastly, we found that the enhanced interaction between TIN2L and TRF2 requires TRF2-F120, a key residue within the TRF homology (TRFH) domain that serves as a docking site for various DNA-processing and DNA damage response factors. We hypothesize that TIN2L regulates the ability of TRFH domain-interacting proteins to access TRF2 and that altered TIN2L activity and enhanced recruitment of TIN2L to telomeres drives telomere shortening in patients.
In Specific Aim (SA) 1 of this proposal, we will determine the relative impacts of TIN2S and TIN2L on telomere structure and function by generating an allelic series of mutant cell lines and performing complementation tests with patient-derived TINF2 mutant cell lines. We will also determine if TIN2L, compared to TIN2S, differentially interacts with other proteins in addition to TRF2 using a novel immunoprecipitation protocol followed by mass spectrometry sequencing methods. Finally, we will screen genetically uncharacterized patients with very short telomeres for specific mutations in TIN2L. In SA2, we will elucidate the function of TIN2L phosphorylation by identifying its impact on TRF2 binding and clarifying its relationship with the DC cluster through an array of in vitro and in vivo analyses. In SA3, we will determine the functional interplay between TIN2L and TRF2 TRFH domain- interacting proteins and identify how they are altered in TINF2 mutant cell lines. This work is expected to not only shed light on DC pathogenesis but also generate new paradigm shifts in telomere biology.

Public Health Relevance

Dyskeratosis congenita (DC) is a genetic condition that predisposes individuals to a failure of the bone marrow to produce a sufficient numbers of blood cells, lung scarring, cancer and other medical problems. Although it is known that individuals with DC are unable to maintain the ends of their chromosomes, precisely why this occurs in a particularly severe form of DC affecting children is unknown. The goal of our research is to fill this gap through comprehensive genetic and molecular studies on the gene altered in this form of DC, thus leading to better molecular understanding and potentially new directions for effective therapy for patients.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cellular Mechanisms in Aging and Development Study Section (CMAD)
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Qasba, Pankaj
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Baylor College of Medicine
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United States
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