Spectrum of point mutations in the coding region of the hypoxanthine- guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes in vivo

The hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in 6-thioguanine (TG) resistant T-lymphocytes is a useful target for the studyof somatic in vivo mutagenesis, since it provides information about a broadspectrum of mutation. Mutations in the hprt coding region were studied in124 TG-resistant T-cell clones from 38 healthy, non- smoking male donorsfrom a previously studied population of bus maintenance workers,fine-mechanics and laboratory personnel. Their mean age was 43 years (range23-64) 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 of hprt cDNA identified 115unique mutations; 76% were simple base substitutions, 10% were +/-1 bpframeshifts, and 10% were small deletions within exons (3-52 bp). Inaddition, two tandem base substitutions and one complex mutation wereobserved. Simple base substitutions were observed at 55 (20%) of 281 sitesknown to be mutable in the hprt coding sequence. The distribution of thesemutations was significantly different than would be expected based upon aPoisson distribution (P < 0.0001), suggesting the existence of'hotspots'. All of the 87 simple base substitutions occurred at knownmutable sites, but eight were substitutions of a kind that have notpreviously been reported at these sites. The most frequently mutated siteswere cDNA positions 197 and 146, with six and five independent mutationsrespectively. Four mutations were observed at position 131, and three eachat positions 143, 208, 508 and 617. Transitions (52%) were slightly morefrequent than tranversions (48%), and mutations at GC base pairs (56%) morecommon than mutations at AT base pairs (44%). GC > AT was the mostcommon type of base pair substitution (37%). The majority of the mutationsat GC base pairs (78%) occurred at sites with G in the non-transcribedstrand. All but one of eight mutations at CpG- sites were of the kindexpected from deamination of methylated cytosine. Deletion of a single basepair (-1 frameshift) was three times more frequent than insertion of asingle bp (+1 frameshift). Almost half (6/13) of the small (3-52 bp)deletions within the coding sequence clustered in the 5' end of exon 2.Short repeats and other sequence motifs that have been associated withreplication error were found in the flanking regions of most of theframeshifts and small deletions. However, several differences in the localsequence context between +/-1 frameshift and deletion mutations were alsonoticed. The present results identify positions 197, 146 and possibly 131as hotspots for base substitution mutations, and confirm previouslyreported hotspots at positions 197, 508 and 617. In addition, the earliernotion of a deletion hotspot in the 5'end of exon 2 was confirmed. Theobservations of these mutational cluster regions in different humanpopulations suggest that they are due to endogeneous mechanisms ofmutagenesis, or to ubiquitous environmental influences. The emergingbackground spectrum of somatic in vivo mutation in the human hprt geneprovides a useful basis for comparisons with radiation or chemicallyinduced mutational spectra, as well as with gene mutations in human tumors.