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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HD035309-03
Application #
6138812
Study Section
Special Emphasis Panel (ZRG4-ALTX-4 (02))
Program Officer
Patel, Appasaheb1 R
Project Start
1998-01-08
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
3
Fiscal Year
2000
Total Cost
$107,052
Indirect Cost
Name
University of Vermont & St Agric College
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Murray, Janet M; Messier, Terri; Rivers, Jami et al. (2012) VDJ recombinase-mediated TCR ? locus gene usage and coding joint processing in peripheral T cells during perinatal and pediatric development. J Immunol 189:2356-64
Finette, Barry A (2006) Analysis of mutagenic V(D)J recombinase mediated mutations at the HPRT locus as an in vivo model for studying rearrangements with leukemogenic potential in children. DNA Repair (Amst) 5:1049-64
Kendall, Heather E; Vacek, Pamela M; Rivers, Jami L et al. (2006) Analysis of genetic alterations and clonal proliferation in children treated for acute lymphocytic leukemia. Cancer Res 66:8455-61
Murray, Janet M; O'Neill, J Patrick; Messier, Terri et al. (2006) V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development. J Immunol 177:5393-404
Vacek, Pamela M; Messier, Terri; Rivers, Jami et al. (2005) Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites. Environ Mol Mutagen 45:339-45
Rice, Sederick C; Vacek, Pamela; Homans, Alan H et al. (2004) Genotoxicity of therapeutic intervention in children with acute lymphocytic leukemia. Cancer Res 64:4464-71
Kendall, Heather E; Vacek, Pamela M; Finette, Barry A (2004) Analysis of microsatellite instability in children treated for acute lymphocytic leukemia with elevated HPRT mutant frequencies. Mutagenesis 19:409-12
Rice, Sederick C; Vacek, Pamela M; Homans, Alan H et al. (2003) Comparative analysis of HPRT mutant frequency in children with cancer. Environ Mol Mutagen 42:44-9
Finette, Barry A; Kendall, Heather; Vacek, Pamela M (2002) Mutational spectral analysis at the HPRT locus in healthy children. Mutat Res 505:27-41
Yoshioka, M; O'Neill, J P; Vacek, P M et al. (2001) Gestational age and gender-specific in utero V(D)J recombinase-mediated deletions. Cancer Res 61:3432-8

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