HIGH-FREQUENCY IN-VIVO LOSS OF HETEROZYGOSITY IS PRIMARILY A CONSEQUENCE OF MITOTIC RECOMBINATION

We have used the adenine phosphoribosyltransferase gene (APRT; 16q24) to investigate the mechanisms of loss of heterozygosity (LOH) in norma l human somatic cells in vivo. APRT-deficient (APRT(-1-), APRT(-/0)) T lymphocytes from the peripheral blood of four obligate APRT heterozyg otes (APRT(+/-)) with characterized germ-line mutations were selected in medium containing 100 mu M 2,6-diaminopurine. A total of 80 2,6-dia minopurine-resistant T-cell clones from 2 of the heterozygotes were an alyzed for this study, The presence or absence of LOH of proximal link ed microsatellite repeat markers was used to divide the clones into tw o groups: (a) those in which LOH was likely due to localized changes i n APRT (e.g., point mutations); and (b) those with LOH at additional l oci, A total of 61 clones (76%) exhibited LOH of linked microsatellite repeat markers at different locations on 16q, which extended from the smallest measured region (<5.5 cM) to the entire 16q arm, The remaini ng 19 clones (24%) had point mutations in APRT or other relatively min or alterations, Ten clones with LOH encompassing different regions of 16q were examined by conventional cytogenetics and by fluorescence in situ hybridization using an APRT cosmid probe, All clones exhibited a normal diploid karyotype, and nine exhibited two copies of APRT, The o ne clone that was hemizygous for APRT had the smallest observed region of LOH in clones from that individual, These results indicate that mi totic recombination and, to a much lesser extent, deletion may be the primary mechanisms for the relatively high frequency of in vivo LOH ob served in normal human T cells, Because LOH leads to the expression of recessive tumor suppressor genes in many cancers, these data have sig nificant implications for the role of LOH in the early stages of tumor development, especially in breast cancer.