Mutation profile at the hprt locus in T-cells of non-smoking males

Sammanfattning: Extensive studies have been done on mutations in several marker genes in order to predict health consequences such as carcinogenesis. However, more information about mutations arising in vivo in somatic cells is needed to understand the relationship between the different factors involved in mutagenesis such as DNA damage, DNA repair capacity and individual susceptibility, and to elucidate the influence of endogenous mechanisms and environmental exposures. To improve the knowledge and understanding of human somatic in vivo mutagenesis, the occur rence and mechanisms of different kinds of mutations were studied in the hypoxanthine phosphoribosyltransferase (hprt) gene in T-lymphocytes of non-smoking males. T-cell cloning in medium containing 6-thioguanine was used to select for mutant clones, and the mutations were further classified and characterized by PCR-based methods and DNA sequencing. Twelve deletions were detected by multiplex-PCR screening of 462 clones from 43 individuals. cDNA was obtained from 327 clones and mutations were identified in 183 of these. Deletion breakpoints were characterized in 16 deletion mutations including 6 mutants from this study population. Most of them seemed to result from a slippage mechanism, causing misalignment of short repeat sequences. Putative secondary structures, possibly acting as intermediates in the deletion formation, were identified in several mutants. A hotspot for deletion breakpoints were identified in the 5' end of exon 2, probably promoted by a 9 nucleotide palindrome and several trinucleotide repeats. Sequence analysis of the cDNA revealed 116 and 58 unique mutations associated with coding and splice errors respectively. Single base substitutions (88) were the most frequent coding mutation, almost equally distributed between transitions (52%) and tranversions (48%). Mutations at GC base pairs (56%) were more common than AT base pairs (44%) w with a majority of the mutations at sites with Gi n the non transcribed strand. Possible hotspots for base substitutions were observed at position 131 and 146 while previously reported hotspots were confirmed at positions 197, 508 and 167. More than half of the small deletions in the coding region clustered in the deletion hotspot region in exon 2. The mutations likely to have caused aberrant splicing were identified in 36 out of 58 mutants characterized with regard to exon skipping. Exons 2-3, 4 and 8 were the most frequently skipped. Most of the splicing errors were caused by base substitutions (28), mainly in the highly conserved dinucleotides in the splice sites. Eight of the splicing mutations occurred in the coding region, and in most of them the predicted change would be a translational stop. This study adds 174 unique in vivo mutations to the human hprt database, induding twelve new mutations and one new mutable site. The total spectrum of in vivo mutations at the hprt locus defined in this study is overall very similar to the types and frequencies of mutations in a previous study of an unrelated population, suggesting that the majority of these mutations are related to endogenous mechanisms and/or by ubiquitous environmental exposures.