The accumulation of somatic mutations has been proposed to play an important role in the human aging process. Precisely quantifying this increase in genetic damage, however, has been a major technical challenge because of the extremely low baseline rate of mutation. To date, the best evidence that unrepaired replication errors accumulate with age comes from cell type-specific selection systems in culture or from transgenic mouse models. Direct measurement of mutation frequency in most human tissues has been precluded by a lack of sufficiently sensitive and generalizable biochemical tools. Our group has recently developed a restriction endonuclease-based method for mutation detection known as Random Mutation Capture (RMC), which has the ability to identify a single base change among more than 108 wild type sequences. My preliminary data establish the feasibility of using this approach to assay mutation frequency at the multi-copy human ribosomal DNA (rDNA) locus. I propose to use this assay to determine how the prevalence of single base substitutions and larger deletions changes in different tissue types over the human lifespan. ? ? In Specific Aim 1, I will adapt the RMC method to interrogate a site in the multi-copy 18s ribosomal RNA gene to overcome sample-size limitations inherent to the very low mutation frequency of normal human tissues.
In Specific Aim 2, I will use this improved assay to compare the prevalence and, spectrum of single base substitutions in different tissues obtained from 24 individuals ranging eighty years in age.
In Specific Aim 3, I will use these tissue samples and a form of the assay that is specific for detection of deletions and rearrangements, to determine how the load of such genetic alterations develops with time. ? ? The aging phenotype is highly diverse in how it manifests across different individuals with respect physiological rate of progression and mode of disability and disease induced. It is possible that a similar diversity in the quantity of genetic damage that accrues over the course of a lifetime exists to explain this variation and might ultimately have utility as a life-year-independent marker of molecular aging to predict clinically relevant outcomes such as disease risk or lifespan. ? ? ?
| Schmitt, Michael W; Kennedy, Scott R; Salk, Jesse J et al. (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 109:14508-13 |
| Salk, Jesse J; Horwitz, Marshall S (2010) Passenger mutations as a marker of clonal cell lineages in emerging neoplasia. Semin Cancer Biol 20:294-303 |
| Loh, Ern; Salk, Jesse J; Loeb, Lawrence A (2010) Optimization of DNA polymerase mutation rates during bacterial evolution. Proc Natl Acad Sci U S A 107:1154-9 |
| Salk, Jesse J; Fox, Edward J; Loeb, Lawrence A (2010) Mutational heterogeneity in human cancers: origin and consequences. Annu Rev Pathol 5:51-75 |
| Salk, Jesse J; Salipante, Stephen J; Risques, Rosa Ana et al. (2009) Clonal expansions in ulcerative colitis identify patients with neoplasia. Proc Natl Acad Sci U S A 106:20871-6 |
| Fox, Edward J; Salk, Jesse J; Loeb, Lawrence A (2009) Cancer genome sequencing--an interim analysis. Cancer Res 69:4948-50 |
