Although it is well established that radiation is a mutagen, more information is needed on the consequences of radiation exposure for specific genetic loci in somatic cells of man, and of the variables that may affect these consequences. Central to evaluation of these consequences is the ability to measure genetic damage. Assessment of a variety of types of genetic damage is needed to ensure that both the quantity and character of damage are ascertained. The objective of this project is to evaluate the biodosimetric information that analysis of mutations of the hypoxanthine phosphoribosyltransferase (HPRT) gene of human peripheral blood lymphocytes can provide for individuals that were exposed to radiation as a consequence of the Chernobyl nuclear power accident. The frequency of mutation will be assessed by the established in vitro limiting dilution assay of thioguanine-resistant, HPRT-deficient T lymphocytes. The molecular nature of mutations present will be assessed using genomic DNA of mutant clones. Gross alterations of the HPRT gene will be detected by Southern analysis of restriction enzyme digested genomic DNA. The extent of deletion of sequences that flank the HPRT gene on the X chromosome will also be determined. Smaller deletion and insertion events that affect the exons of the HPRT gene will be detected with a multiplex polymerase chain reaction (PCR) analysis; PCR analysis will also confirm the identity of exons affected by gross deletions. These molecular analyses will be applied to multiple mutants per individual for selected subsets of the population to study both interindividual differences in mutation spectra and correlations of the molecular nature of mutation with mutation frequency and exposure history. The molecular analyses will also be applied in a cross sectional mode to one mutant of each member of a larger population to assess whether bias is introduced by studying smaller groups of individuals in detail. The stability over time of the frequency and molecular nature of damage at this genetic locus in lymphocytes will be determined for subsets of the population. By studying individuals who received different doses of radiation following the Chernobyl accident, the relationship between exposure history and mutational responses at the HPRT gene will be determined. In combination with the biodosimetry data acquired from the other projects of this Program, these results will advance efforts to assess human risk for somatic and germinal mutation following exposure to mutagenic agents.
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