Effect of a common mutation (D442G) of the cholesteryl ester transfer protein gene on lipids and lipoproteins in children.

Cholesteryl ester transfer protein (CETP) is thought to regulate plasma HDL. Patients with CETP deficiency caused by mutation of the CETP gene [D442G; a missense mutation (Asp442-->Gly)] have been reported to show high plasma HDL levels. However, there are no data available on children with D442G. To determine the effects of plasma CETP and CETP gene mutation (D442G) on lipids and lipoproteins in children, we screened children by PCR and restriction fragment length polymorphism analysis of the CETP gene. Plasma lipids, apolipoproteins, and CETP mass levels were also determined. In the current study, 22 children with D442G were found (21 heterozygotes and a homozygote). A homozygous child showed high plasma HDL level and very low plasma CETP mass. In heterozygous children, plasma concentrations of HDL cholesterol, apo A-I and apo A-II were not increased, whereas plasma CETP mass was significantly decreased. Plasma CETP mass in heterozygous children was correlated with plasma concentrations of total cholesterol, LDL cholesterol, and apo B. Plasma CETP mass in children without D442G was not correlated with the plasma concentration of any lipid or apolipoprotein. All of these data suggest that the D442G mutation, by itself, might not affect HDL metabolism in children. The CETP mass required for efficient HDL-cholesteryl ester clearance in children may be less than that in older subjects.

Structure of the human acyl-CoA:cholesterol acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia.

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.