Functional studies of a Tangier Disease mutation

Low levels of high density lipoprotein (HDL) are a well established, independent risk factor for the development of atherosclerosis. The ABCA1 (ATP-binding cassette A1) protein is a key regulator of HDL levels as it promotes efflux of cholesterol and phospholipids to circulating lipid-poor apoA-I to form mature HDL particles. Mutations in the ABCA1 gene can cause hypoalphalipoproteinaemia or Tangier Disease (TD) due to a lack of circulating HDL. A New Zealand individual with TD was previously identified and reported as homozygous for a novel R1068H mutation in the ABCA1 gene. The 42 year old female presented with virtually no circulating HDL and peripheral neuropathy, but no sign of atherosclerosis. Interestingly, the TD proband carried two different ABCA1 promoter haplotypes, indicating that her maternal and paternal ABCA1 alleles may be under differential regulatory control. Analysis of the R1068H mutation was carried out to determine impact of the mutation on ABCA1 function. In silico analysis of the mutation suggested that it was ‘probably damaging’, and modeling of the mutation based on a bacterial homologue confirmed the proximity of residue 1068 to the first nucleotide binding domain of ABCA1. Cholesterol efflux assays in HEK293 cells transfected with ABCA1 expression vectors showed that the R1068H mutation severely impaired cholesterol efflux, supporting the in silico analysis. Confocal microscopy of ABCA1 in HEK293 cells suggested that the R1068H mutation lead to reduced plasma membrane localisation of the protein. Primary skin fibroblast cultures were established from R1068H family members to perform further ABCA1 functional analyses. Cholesterol efflux assays performed in TD fibroblasts displayed very little cholesterol efflux potential. Interestingly, fibroblasts of R1068H carriers also showed a marked reduction in cholesterol efflux (< 40% of wildtype). The two ABCA1 promoter haplotypes present in the TD proband were investigated for their relative promoter activities. The maternal haplotype carried the minor alleles for 5 SNPs and an insertion/deletion, and showed decreased promoter activity compared to the paternal haplotype in both RAW264.7 and HepG2 cells. However, in RAW264.7 cells the maternal haplotype was upregulated to a greater extent by LXR and RXR agonists, and showed higher promoter activity than the paternal promoter haplotype. ABCA1 expression was examined at the mRNA and protein level in all primary fibroblast cell lines. mRNA expression was very variable between cell lines, and did not appear to correlate with ABCA1 promoter genotype, R1068H genotype, cholesterol efflux level or HDL level. Protein expression was examined by Western blot and was not significantly different between individual family members. Allele-specific ABCA1 expression was examined in R1068H heterozygotes to measure the relative contribution of each allele to overall expression. The mutant 1068H allele was shown to contribute only a small amount of total ABCA1 expression in paternal carriers (20 – 37%) and surprisingly, no 1068H expression at all was detected from the maternal ABCA1 allele. Further investigation revealed that the maternal ABCA1 allele does not contain a copy of the R1068H mutation, and instead harbours a large genomic deletion within the ABCA1 gene. This deletion was not detectable by sequencing of coding regions of the ABCA1 gene which had been the previous approach used to genotype the TD proband, and which incorrectly assigned homozygosity for 1068H. The R1068H mutation leads to a dysfunctional ABCA1 protein which has reduced cell membrane localisation and impaired cholesterol efflux capability. The mutation does not affect expression levels of either ABCA1 mRNA or protein. ABCA1 promoter SNPs do cause changes in the promoter activity of the ABCA1 promoter and respond differently to known ABCA1 agonists, but do not cause changes in ABCA1 expression levels. This suggests that complex regulatory and compensatory systems are in place with regards to ABCA1 regulation