Multiplex PCR Analysis of In vivo-Arising Deletion Mutations in the 'hprt' Geneof Human T-Lymphocytes

摘要

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapidand efficient evaluation of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes for deletions. The hprt clonal assay was used to isolate in-vivo-arising hprt-deficient T-cells from six health males. 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 relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-gamma genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation 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 region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.