Abstract-Apolipoprotein (apo) A1 plays a central role in the metabolism of HDL. We describe a novel genetic variant of the apoA1 gene identified in a patient with low concentrations of plasma HDL cholesterol. The proband, a 12-year-old Japanese boy, exhibited markedly low levels of both plasma apoA1 and HDL cholesterol. Genomic DNA sequencing of apoA1 genes of the patient showed a compound heterozygosity for an A to C substitution at 27 bp upstream of the transcription start site of 1 apoA1 allele, and a C to T substitution in another allele at residue 84 resulting in aberrant termination. The point mutation at nucleotide position -27 changed ATAAATA of the putative TATA box signal sequence to ATACATA. In addition to this mutation, the patient was heterozygous for a G to A substitution at position -75. Immunoblotting of an isoelectric focusing electrophoresis gel of the proband's plasma showed a trace amount of normal apoA1. No measurable plasma apoA1 and HDL cholesterol in a patient with homozygosity for nonsense mutation at residue 84 has been reported previously. To determine the effects of substitution either at position -27 or -75, plasmids containing the 5Ј-flanking region of the human apoA1 promoter fused to the CAT reporter gene were constructed and transfected in HepG2 cells. A construct with the A to C substitution at position -27 showed 41.

Abstract-A novel variant of apolipoprotein (apo) A-I associated with low high density lipoprotein (HDL) cholesterolemia has been identified in a Japanese family during screening for apoA-I variants by isoelectric focusing (IEF) gel analysis. ApoA-I (Glu23530) Nichinan was caused by a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apoA-I gene. Four subjects in the family were heterozygous carriers for this mutation; the mean plasma concentrations of apoA-I and HDL cholesterol of affected family members were 30% and 32% lower, respectively, than those of unaffected family members. There were no differences in the levels of very low density lipoprotein and low density lipoprotein cholesterol, triglycerides, and other apolipoproteins between the carriers and the noncarrier family members. In the proband, plasma lecithin:cholesterol acyltransferase activity was normal. Functional consequences of the mutation were examined by expressing the mutated and wild-type proapoA-I cDNAs in Escherichia coli. Cholesterol efflux to recombinant proapoA-I Nichinan from mouse peritoneal macrophages loaded with [ 3 H]cholesterol-labeled acetylated low density lipoprotein was decreased by 54% when compared that of normal recombinant proapoA-I. In vivo turnover studies in normal rabbits demonstrated that the recombinant proapoA-I Nichinan was rapidly cleared (22% faster) compared with normal recombinant proapoA-I. We conclude that apoA-I (Glu23530) Nichinan induced a critical structural change in the carboxyl-terminal domain of apoA-I for cellular cholesterol efflux and increased the catabolism of apoA-I, resulting in low HDL cholesterol levels.

Abstract-We analyzed the genetic defect in a 67-year-old Japanese male patient with apolipoprotein (apo) A-I and high density lipoprotein (HDL) deficiencies, corneal opacities, and coronary artery disease. The plasma concentrations of apoA-I and HDL cholesterol were 2.9 to 7.3 mg/dL and 0.08 to 0.19 mmol/L, respectively. The lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate were Ͻ40% of normal control values. LCAT mass was Ϸ50% of normal control. Sequence analysis of polymerase chain reaction-amplified DNA of the proband's apoA-I gene showed a homozygous T-to-A transition resulting in the substitution of Val 156 with Glu (apoA-I Oita). Direct sequencing of samples obtained from other family members showed that the brother was homozygous, whereas the son was a heterozygous carrier of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of normal apoA-I and normal HDL cholesterol levels. In vivo turnover studies in rabbits demonstrated that the variant apoA-I was rapidly cleared from plasma compared with normal human apoA-I. Our data suggest that the Val156Glu substitution is associated with apoA-I and HDL deficiency, partial LCAT deficiency, and corneal opacities and that Val156 of apoA-I may play an important role in apoA-I function. (Arterioscler Thromb Vasc Biol. 1998;18:389-396.)Key Words: HDL deficiency Ⅲ apolipoprotein variant Ⅲ apoA-I Japanese Ⅲ corneal opacities A polipoprotein A-I is composed of 243 amino acid residues that fold into amphipathic helixes, thus forming functional domains. ApoA-I plays a leading role in HDL-mediated cholesterol efflux from peripheral cells and acts as a cofactor for LCAT activation.1 The functional domains of apoA-I have been recently examined by using monoclonal anti-apoA-I antibodies, mutagenized apoA-I, and immunochemical and physicochemical approaches.1,2 In addition, naturally occurring mutations in the apoA-I gene have been structurally and functionally characterized to be the underlying molecular defects of low HDL levels. [3][4][5] These data suggest that the functional domains of apoA-I contain central amphipathic ␣-helixes involving amino acid residues between 137 and 186, which are critical for both activation of LCAT and enhancing cholesterol efflux from peripheral cells.More than 10 apoA-I gene defects of either a deletion or insertion leading to HDL deficiency have been reported. [5][6][7][8][9][10][11][12][13][14][15] However, in subjects with HDL deficiency, CAD is not always present. In addition, Ϸ40 heterozygous structural apoA-I variants with a single amino acid substitution have been characterized.2,16 -21 However, most of these variants do not affect HDL concentration except for nine that are associated with reduced plasma apoA-I and HDL cholesterol levels. [17][18][19][20][21][22][23][24][25] In this article we report a novel, homozygous, missense point mutation in the apoA-I gene that is associated with apoA-I and HDL deficiencies, corneal opacities, and CAD. Methods SubjectsA 67-year-old Japanese man...

Abstract-The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (dϽ1.225 g/mL) at 37°C for 1 hour, 125 I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas 125 I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the ␣-helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [ 3 H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [ 3 H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/rproapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.