The aim of this research is to explore the molecular mechanisms of somatic gene mutations in the pediatric population, and to examine the implications of these in vivo events on human development, tumorigenesis, secondary malignancies, and the monitoring of autoimmune diseases. In Phase I, the focus is to determine the somatic mutant frequency in the hprt gene of normal human pediatric subjects from birth to late adolescents. Analysis of the molecular rearrangements in hprt negative clones, specifically the frequency of V(D)J recombinase mediated events resulting in the deletion of exons 2 and 3 will be performed. In Phase II, the aim is to examine hprt somatic mutations in children prior to, during, and after the completion of chemotherapy and/or radiation treatment for a malignant process. The frequency of thioguanine resistant T-cell clones will be determined and compared with normal subjects. Folic acid levels will be correlated with mutant frequency and a master bank set up for long term follow up of these patients for determining if the frequency and types of somatic gene rearrangements can be used as markers for predicting secondary malignancies. The second hypothesis in Phase II will examine the treatment of pre, and new onset diabetic children with azathioprine utilizing the principle of surrogate selection to isolate hprt negative T-cell clones. The frequency of thioguanine resistant T-cell lymphocyte clones will be determined prior to, during and after the completion of azathioprine treatment. Analysis of molecular rearrangements as well as the clonality of these hprt negative clones will be determined. The techniques to be utilized include: 1) T-cell cloning assay, selecting for thioguanine resistant clones. 2) Southern blot analysis of mutant and wildtype clones to determine TCR clonality and structural changes at the hprt locus. 3) Multiplex PCR analysis of hprt gene for the detection of deletions and point mutations. 4) DNA sequence analysis of hprt mutant clones to determine V(D)J recombinase mediated events. These investigations should answer questions on the importance of somatic mutations in developing healthy children and those children with cancer. It will also provide the foundation for devising a novel approach to monitoring azathioprine treatment of Type I diabetes.
