PMC

ABCA1 and LPL variants co-segregate with low HDL-C in the multigenerational French Canadian low HDL-C family with 75 (35 males and 40 females) genotyped family members. All of the affected subjects who have HDL-C<the 5^th age-sex specific population percentile comprise risk alleles for either one of the two variants or both, except in one separate branch the low HDL-C traits appears to be inherited from the affected spouse’s side (indicated in yellow). The subjects with both variants have a lower HDL-C than the subjects with only one variant. The subjects whose samples were exome sequenced are indicated by an arrow.

Sex-dependent effect of ABCA1 variants. Figure 3 shows the age-sex specific population HDL-C percentiles by ABCA1 genotypes and sex in 200 French Canadian family members from 11 French Canadian families with different ABCA1 mutations (DelED1893, G616V, K776N, N1800H, Q2210H, R1851X, R2084X, R909X and S1731C). MUT indicates carriers of a mutation in the ABCA1 gene and WT stands for wild-type genotype (i.e. non-carriers). Q2 stands for the median quartile and IQR for interquartile range (Q3-Q1) of HDL-C percentiles. The distributions of the age-sex percentiles are similar between genders in the WT genotype group whereas in the MUT genotype group the distribution is more restricted to the lower tail in males than in females. This difference describes the significant result of the genotype-sex interaction analysis. It should be noted that the displayed age-sex percentiles are not adjusted for relatedness whereas the family relation was taken into account in the genotype-sex interaction analysis performed using SOLAR.

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.