MULTIPLEX PCR ANALYSIS OF IN-VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine gu anine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. Th e hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 x 10(-6). Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The rel atively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand confo rmation polymorphism (SSCP) analysis of rearranged T-cell receptor (TC R)-gamma genes. Eighteen the 22 partial hprt gene deletion mutants wer e determined to be of independent origin based on a unique hprt mutati on or SSCP-TCR -gamma pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this re gion of the hprt gene is prone to deletion. The small deletions effect ing exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) woul d not have been detected by conventional Southern blot analysis and ma y represent a new, previously unrecognized class of mutations. The rea dy isolation of such intragenic deletions will allow the characterizat ion of breakpoint junctions and may provide insights into the importan t processes of DNA breakage and rejoining. (C) 1994 Wiley-Liss, Inc.