The Specific Aims of this grant are directed towards understanding the function of human KARP-1 (Ku86 autoantigen related protein-1) as it relates to its role as a candidate gene for DC (dyskeratosis congenita). DC is a rare inherited disorder characterized by a triad of cutaneous hallmarks: abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. The disease presents with significant clinical heterogeneity and patients may also be afflicted with dental, gastrointestinal, neurological, ophthalmic, pulmonary and/or skeletal abnormalities. In particular, tissues that need continual renewal (e.g., epidermal, mucosal and bone marrow lineages) are most affected in DC patients and 70% of all the morbidity associated with DC is due to bone marrow failure. Cells derived from DC patients are characterized by possessing i) short telomeres, ii) reduced expression levels of hTR (human telomeric RNA), iii) reduced activity of telomerase, iv) genomic instability, and v) proliferative dysfunction. Two genes, hTR and DKC1, have been identified, that, when mutated, give rise to DC. Mutation of either hTR or DKC1 accounts for about 50% of all DC patients while the other 50% have mutations in genes that have yet to be identified. Interestingly, hTR and DKC1 produce gene products that are nucleolar in cellular localization. In published reports and in preliminary data provided with this application, we demonstrate that the loss of a single allele of the Ku86:KARP-1 locus in human cells results in precisely the same phenotypes that are observed in DC patients: i) short telomeres, ii) reduced expression levels of hTR, iii) reduced activity of telomerase, iv) genomic instability, and v) proliferative dysfunction. In addition, we also demonstrate that KARP-1 is nucleolar. Thus, in toto, the evidence is compelling that KARP-1 is a candidate gene for DC. Herein, we describe experiments that will determine whether KARP-1 is mutated in DC patients and that will elucidate the role(s) of KARP-1 in telomere maintenance in human cells. The ultimate goal of these studies is to use human somatic cell lines altered in their expression of KARP-1 to understand the molecular mechanisms of telomere dysfunction in human patients.
Our Specific Aims /Goals are: 1. Is the KARP-1 gene mutated and/or is KARP-1 gene expression aberrant in DC patients? 2. Is KARP-1 essential in human somatic cells? 3. Biochemical and molecular characterization of KARP-1 and its interactors.
