Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes
cholesterol esterification in mammalian cells. Two
isoforms of ACAT have been reported to date (ACAT-1 and ACAT-2). ACAT-1 is ubiquitously expressed in tissues except the intestine. In contrast, ACAT-2 is expressed mainly in the intestine in humans. To investigate the relationship between ACAT-2 and
dyslipidemia, we determined the structure of the human ACAT-2 gene and then studied the relationship between mutations of the ACAT-2 gene and
dyslipidemia. To isolate human ACAT-2 genomic
DNA, we designed primers based on the human ACAT-2
cDNA sequence: forward primer 5'-ACACCTCGATCTTGGTCCTGCCATA-3' and reverse primer 5'-GGAATGCAGACAGGGAGTCCT-3'. Using these primers, a human P1-derived artificial chromosome (PAC) library was screened by PCR-based procedures. Isolated PAC clones were completely digested with BamHI and subcloned into plasmid vector. Subclones that contained exons were screened by dot-blot hybridization using partial ACAT-2
cDNA fragments. The coding region of the ACAT-2 gene was encoded in 15 exons from 51 to 265 base pairs on a 21 kilobase span of genomic
DNA. The exonic sequences coincided completely with that of ACAT-2
cDNA, and each exon-intron junction conserved splicing consensus sequences. Next, 187 (91 dyslipidemic and 96 normolipidemic) subjects were screened by PCR single-strand conformational polymorphism analysis of the ACAT-2 gene. Three mutations were identified by
DNA sequencing: two missense mutations (E14G in exon 1 and T254I in exon 7) and a point mutation in intron 7 (-35G-->A). Mutations in exon 1 and intron 7 were not associated with plasma concentrations of
lipids and
apolipoproteins (apo). However, plasma
apoC-III levels in T254I heterozygotes were significantly higher than those in subjects without mutation. Plasma
triglyceride (TG) levels in T254I heterozygotes were similar to those in subjects without mutation. Although further studies are needed, our data suggest that ACAT-2 may contribute to
apoC-III gene expression and the assembly of
apoC-III and TG, possibly in the intestine.