Project Summary The International Registry of Werner Syndrome recruits cases of Werner syndrome (WS) and a range of other segmental progeroid syndromes from around the world with the goal of elucidating underlying mechanisms of accelerated aging. Detailed clinical information, the results of genetic analyses, and biological specimens are made available to a wide range of qualified geroscientists. In this application, we propose to extend our previous studies to include systematic genome-wide searches for the genetic variants responsible for the 78 progeroid cases that we have so far been unable to genetically characterize. An additional important extension of our research agenda is to initiate translational research that can lead to potential therapeutic agents. We will employ a combination of next generation sequencings, array CGH, and Sanger sequencing, followed by confirmatory Western analysis and quantitative PCR. These approaches have successfully identified novel pathogenic variants of WRN (a DNA helicase), LMNA (a component of nuclear structure), POLD1 (DNA polymerase delta), SPRTN (recruiter of DNA polymerase), ERCC4 (nucleotide excision repair), CTC1 (telomere replication), MDM2 (an inhibitor of P53) and SAMHD1 (regulation of dNTP pools). These loci highlight major roles for genome instability, now widely accepted as one of the hallmarks of aging. We also made progress in the identification of disease mutations that suggest other mechanisms of accelerated aging, such as BSCL2 (lipid droplet formation) and SMAD4 (intracellular signaling of a component of SASP, TGFβ). As indicated above, we will pursue translational research with the potential for the development of ameliorative therapies for our progeroid patients. Based on our previous studies, our collaborator, Dr. Yokote Koutaro at the Japanese Werner Consortium, is evaluating the efficacy of an NAD intermediate and an mTOR inhibitor, metformin, in WS patients. We shall begin independent studies of the effects of suppressors of chronic inflammation, namely Janus kinase (JAK) inhibitors, in cultures from WS patients and controls. This effort was motivated by our findings that SMAD4 mutant fibroblasts exhibited increased accumulation of DNA damage and that WRN mutant fibroblasts showed elevated SMAD4 expressions and dramatically higher levels of SASP compared to control cells, suggesting that a synergy of persistent DNA damage and inflammation may be one of the common key mechanisms leading to the accelerated aging. Concordant experiments will explore the effects of these novel therapeutic targets with high throughput screening of cell cultures from patients with other progeroid syndromes. An initial small scale experiment involving siRNA screening has revealed that siRNAs and drugs that alter the intranuclear dNTP concentration are able to modulate the cellular disease phenotypes of POLD1 mutants. Larger scale siRNA screening will be employed to identify additional novel relevant ta...