SPECTRUM OF POINT MUTATIONS IN THE CODING REGION OF THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE (HPRT) GENE IN HUMAN T-LYMPHOCYTE IN-VIVO

The hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in 6- thioguanine (TG) resistant T-lymphocytes is a useful target for the st udy of somatic in vivo mutagenesis, since it provides information abou t a broad spectrum of mutation. Mutations in the hprt coding region we re studied in 124 TG-resistant T-cell clones from 38 healthy, non-smok ing male donors from a previously studied population of bus maintenanc e workers, fine-mechanics and laboratory personnel. Their mean age was 43 years (range 23-64) and their hprt mutant frequency was 9.3 +/- 5. 2 x 10(-6) (mean +/- SD, range 1.4-22.6 x 10(-6)). Sequence analysis o f hprt cDNA identified 115 unique mutations; 76% were simple base subs titutions, 10% were +/- 1 bp frameshifts, and 10% were small deletions within exons (3-52 bp). In addition, two tandem base substitutions an d one complex mutation were observed. Simple base substitutions were o bserved at 55 (20%) of 281 sites known to be mutable in the hprt codin g sequence. The distribution of these mutations was significantly diff erent than would be expected based upon a Poisson distribution (P < 0. 0001), suggesting the existence of 'hotspots'. All of the 87 simple ba se substitutions occurred at known mutable sites, but eight were subst itutions of a kind that have not previously been reported at these sit es. The most frequently mutated sites were cDNA positions 197 and 146, with six and five independent mutations respectively. Four mutations were observed at position 131, and three each at positions 143, 208, 5 08 and 617. Transitions (52%) were slightly more frequent than tranver sions (48%), and mutations at GC base pairs (56%) more common than mut ations at AT base pairs (44%). GC > AT was the most common type of bas e pair substitution (37%). The majority of the mutations at GC base pa irs (78%) occurred at sites with G in the nontranscribed strand. All b ut one of eight mutations at CpG-sites were of the kind expected from deamination of methylated cytosine. Deletion of a single base pair (-1 frameshift) was three times more frequent than insertion of a single bp (fl frameshift). Almost half (6/13) of the small (3-52 bp) deletion s within the coding sequence clustered in the 5' end of exon 2. Short repeats and other sequence motifs that have been associated with repli cation error were found in the flanking regions of most of the framesh ifts and small deletions. However, several differences in the local se quence context between +/- frameshift and deletion mutations were also noticed. The present results identify positions 197, 146 and possibly 131 as hotspots for base substitution mutations, and confirm previous ly reported hotspots at positions 197, 508 and 617. In addition, the e arlier notion of a deletion hotspot in the 5' end of exon 2 was confir med. The observations of these mutational cluster regions in different human populations suggest that they are due to endogeneous mechanisms of mutagenesis, or to ubiquitous environmental influences. The emergi ng background spectrum of somatic in vivo mutation in the human hprt g ene provides a useful basis for comparisons with radiation or chemical ly induced mutational spectra, as well as with gene mutations in human tumors.