A. Effect of the ABCA1 variant on cholesterol efflux in fibroblasts from a proband homozygous for S1731C and a healthy control. Fibroblasts were isolated by taking a biopsy from the forearm of the proband and a healthy individual, plated in 12-well plates and radiolabeled with [3H]-cholesterol for 48 hours. Cholesterol efflux was performed as described in Methods under background diffusion conditions (−22OH −ApoA-I), unstimulated (−22OH +ApoA-I) and stimulated (+22OH −ApoA-I, +22OH +ApoA-I) conditions, with or without ApoA-I. The proband had a significantly reduced ApoA-I-mediated efflux as compared to the control without the variant. Values represent the mean ±S.D. from triplicate wells. Results shown are a representative of three independent experiments. ***P=1.23×10−4 by Student's t-test; and *P=0.0495 using a non-parametric two-sample Wilcoxon rank sum test. 22OH indicates 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; and ApoA-I, apolipoprotein A-I.
B. Elevated concentrations of 17β-estradiol improve cholesterol efflux in the male proband with the ABCA1 S1731C variant. Fibroblasts from a male proband with the ABCA1 S1731C variant and a healthy male control were isolated by taking a biopsy from the forearm of plated in 24-well plates and radiolabeled with [3H]-cholesterol for 24 hours. Cholesterol efflux was performed as described in Methods, with addition of increasing concentrations (2 nM, 20 nM, 50 nM, 0.1µM, 1 µM, 10 µM and 50 µM) of 17β-estradiol while stimulating ABCA1 expression with 22OH/9CR for 17 hours. As in Figure 2A, experiments were done under four conditions (−22OH −ApoA-I, −22OH +ApoA, +22OH −ApoA-I, +22OH +ApoA-I). Efflux results were subsequently adjusted for background basal conditions of passive diffusion. The final stimulated ApoA-I mediated efflux condition is shown. Upon exposure to increasing estradiol concentrations (>20 nM), cholesterol efflux in the S1731C proband significantly increases. Of note, the overall [3H]-cholesterol efflux counts were lesser in magnitude than those observed in given the shorter labelling time period (24 hours). In addition, the difference in efflux between the control and proband was greater than in Figure 2A, given the different basal diffusion of the selected conditions (data not shown), which was now removed from the net ApoAI-mediated efflux. Values represent the mean ±S.D. from triplicate wells. Results shown are representative of three independent experiments. 22OH indicates 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; and ApoA-I, apolipoprotein A-I. ***P=7.2×10−6 (r=0.78) using a non-parametric Spearman trend test for the dose effect on efflux in the S1731C proband; and nsP=0.2 (r=0.25) using a non-parametric Spearman trend test for the dose effect on efflux in the wildtype control.