ACTIVATION STATUS OF THE X-CHROMOSOME IN HUMAN MICRONUCLEATED LYMPHOCYTES

The frequency of X chromosome aneuploidy in human female peripheral bl ood lymphocytes has been reported by several investigators to be signi ficantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsib le for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive a nd not the active X. Blood samples were obtained from two unrelated fe males, 36 and 33 years of age, each with a different X; 9 reciprocal t ranslocation. In each, the normal X chromosome is inactive and the tra nslocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 ye ar old, while 9,500 binucleated cells were scored from the 33 year old . The slides were first labeled and the kinetochore status of each mic ronucleus was determined. This was followed by simultaneous hybridizat ion with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronuclea ted cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2% ) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) cont ained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inact ive X chromosome is preferentially included in the micronuclei, and su ggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon.