The major objectives of this proposal are to study the mechanism and biological consequences of age-specific in vivo somatic genetic events in children. The hypothesis is that somatic mutations arising during fetal development and early childhood are more reflective of genetic changes with disease potential and environmental exposures than are mutations arising later in life. This hypothesis will be tested by three specific aims: 1) To complete studies comparing somatic mutational events that occur during fetal development and childhood with those that occur in adults; 2) To compare the prevalence of """"""""illegitimate"""""""" V(D)J recombinase mediated mutations in a reporter gene, hprt, and a gene of disease significance, p53, and, 3) To determine the frequencies, spectra, and persistence of individual hprt mutations in newborns and children exposed to known genotoxic agents, and to correlate these molecular events with subsequent diseases. The frequency of somatic mutational events will be determined by the hprt T-cell cloning assay. Genetic analysis of hprt and p53 mutations will be performed using several methods: multiplex PCR, RT-PCR, IPCR, Southern blotting and DNA sequencing. These methods have previously been used for detailed molecular characterization of mutational events for inferences regarding genetic mechanisms and genotoxic monitoring. The research plan is designed to study the association/prediction between somatic mutations and specific diseases by determining: 1) whether the background frequency and mutational spectra of in vivo somatic mutations at the hprt locus in children are age-specific, reflecting unique biologic differences at the molecular level when compared to adults. These studies will provide information on the age dependent nature of mutational events in children and provide the database required for comparison studies in children with specific clinical diseases and genotoxic exposures; 2) if V(D)J recombinase mediated rearrangements captured at the hprt locus occur in a clinically specific tumor suppressor gene, p53. These studies may provide insights into the clinical effects of spontaneous age-specific mutagenic mechanism occurring during early human development; and 3) if somatic mutations induced by environmental exposures (cigarette smoke and chemotherapy) in children are predictive of subsequent somatic disease. The testing of potential mutagenic exposures in children is important since somatic mutational events during this period could have significant clinical consequences as well as long term effects as an adult. Results obtained from these studies will provide fundamental insights regarding the clinical relevance of age-specific, spontaneous and environmentally induced somatic mutations in children.
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