The application's broad, long-term objectives include understanding the mechanism of pathophysiology of Werner's syndrome, a syndrome that while caused by a mutation in a single gene, mimics many of the features of normal aging. Although the gene has been identified as a helicase, the distal effects of mutations in this helicase, and how they cause multiple phenotypes of aging, have yet to be clarified. We propose to address the mechanism by which helicase mutations mimic aging by studying global transcript levels using the Affymetrix microarray format, in fibroblasts and lymphoblasts of patients with Werner's Syndrome.
Aim 1 will be a study design of fibroblasts and lymphoblasts from 6 patients with Werner's syndrome, using the multiple techniques we have learned over the last 2 years for separating the mutation-dependent signal from noise, in our other studies. Helicase signal will be separated from noise using these techniques, which include: chip replication, single-chip variability, genetic heterogeneity subtraction, cell autonomous gene expression, and Mean/s.d. comparisons of data quality, small change criteria, and biochemical pathway mapping.
Aim 2 will be to confirm the most important helicase-dependent alterations in gene expression by RT-PCR and Western experiments, and to compare these to our increasing database of microarray data of mitochondrial and oxidative-stress related diseases, to search for similarities and differences.
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