Classification of mutations at the human hprt-locus in T-lymphocytes of bus maintenance workers by multiplex-PCR and reverse transcriptase-PCR analysis.

Abstract

Polymerase chain reaction (PCR)-based screening methods were used to classify mutations arising in vivo at the hypoxanthine guanine phosphoribosyl-transferase (hprt) locus in small samples of human T-lymphocyte clones (< 5 x 10(4) cells) from 29 bus maintenance workers exposed to diesel exhaust, and 14 control individuals. All subjects were healthy, non-smoking males. Among 462 T-cell mutants studied by multiplex-PCR of genomic DNA, only 12 (2.6%) deletions were found: three total deletions, five partial exon deletions and four mutants with one or two exons deleted. Point mutations were classified in 323 mutants using reverse transcriptase-PCR amplification: 74 (22.9%) of these had splice site mutations and 241 (74.6%) had coding errors. Splice mutation was more frequent among the garage workers (24.8%) as compared to the controls (19.5%), possibly reflecting a polycyclic aromatic hydrocarbon-specific mutation induction in these workers. Our results also show that both gene deletion and splice mutation at the hprt-locus in T-cells of healthy non-smokers could be less frequent than previously reported.