Analysis of gene-dosage effects on the expression of CD18 by trisomy 21 lymphoblastoid cell-lines using a statistical model to fit flow cytometry profiles.

It is not clear whether Down syndrome, the phenotypic expression of constitutional trisomy for chromosome 21 (T21), is the result of generalised disruption of homeostasis resulting from genetic imbalance, or the over-expression of specific genes on chromosome 21. In order to understand the effect of gene dosage more clearly, we have analysed the predicted and actual levels of expression of the leucocyte integrin beta subunit CD 18 on the surface of T21 leucocytes. Previous studies showed that CD18 expression by T21 lymphoid cell lines (LCL) is greater than on normal LCL. We have now developed a computer model that compares the observed and predicted CD18 flow cytometric profiles for trisomy 21 LCL. Three parameters (alpha, beta and gamma) have been defined that measure different aspects of gene dosage. Using the computer model to calculate these parameters, we have carried out a series of paired comparisons between normal and T21 LCL. The results show that, in some T21 LCL, increased CD18 expression is proportional to the existing gene dosage, in another set the effect is additive, whereas in others there is a combination of proportional and additive effects. The results suggest that gene regulation can exert pleiotropic effects on gene-dosage, and is consistent with a model in which gene dosage itself is the cause of disrupted homeostasis.

Sequential flow cytometric analysis of cell-cycle related changes in LFA-1 (CD18/CD11a) expression by trisomy 21 (Down's syndrome) lymphoblastoid cells.

Lymphocyte function associated antigen 1 (LFA-1) is a heterodimeric leucocyte adhesion molecule comprising non-covalently associated 95 kD, CD18 and 180 kD, CD11a subunits. Lymphoblastoid cell-lines (LCL) derived from persons with Down's syndrome (Trisomy 21) exhibit increased expression of LFA-1 compared with normal LCL. Although this is probably due to a gene-dosage related increase in the synthesis of CD18, cell-cycle differences between Trisomy 21 (T21) and normal LCL could also influence LFA-1 expression. We have therefore analysed expression of CD18 on G1 and G2M cells using sequential flow cytometry. T21 and normal LCL were co-stained with the DNA-binding vital dye HO342 (Hoechst 33342), and with a CD18 monoclonal antibody. The LCL were first sorted on the basis of HO342 staining into G1 and G2M populations, and these fractions then analysed for CD18 expression. Irrespective of the stage of the cell-cycle, expression of CD18 was increased on T21 compared with normal LCL. Although more CD18 was detected on both T21 and normal G2M compared with G1 cells, the relative density of CD18 in G2M was less than in G1 because G2M cells were larger.