Kengo Moriyama

A Novel Homozygous Missense Mutation in the Apo A-I Gene With Apo A-I Deficiency

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 <40% of normal control values. LCAT mass was 550% of normal control. Sequence analysis of polymerase chain reaction-amplified DNA of the probands 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.

Novel mutations in ABCA1 gene in Japanese patients with Tangier disease and familial high density lipoprotein deficiency with coronary heart disease.

Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.

Apolipoprotein E1 Lys-146 → Glu with type III hyperlipoproteinemia

During the screening of samples obtained from 5 individuals with type III hyperlipidemia, we identified a variant of apolipoprotein (apo) E which exhibited a discrepancy in apo E phenotype showing the E3/E1 isoform on isoelectric focusing (IEF) analysis and E3/E3 on gene analysis. Sequence analysis of the DNA of the proband that was amplified by PCR and subcloned, revealed a single substitution of one lysine (AAG) for one glutamic acid (GAG) at position 146, thereby adding two negatively charged units to apo E3. This defect had been described only for apo E1 to date (Mann et al. (1989) Clin. Res. 37, 520A (abstract)). In this case, PCR-mediated site-directed mutagenesis was used to identify the structural alterations forming the abnormal E1 genotype in the probands family. Purified apo E1 Lys-146----Glu showed less than 10% of binding activity to apo B, E receptor on human skin fibroblasts compared with apo E3. This substitution demonstrates that Lys-146 is essential for the binding of apo E to the receptor.

Effects of niceritrol on levels of serum lipids, lipoprotein(a), and fibrinogen in patients with primary hypercholesterolemia

Thirty-three consecutive unselected patients with primary hypercholesterolemia received niceritrol 1.5 g daily for 12 weeks, with the effect of administering divided dose (twice daily (b.i.d.) and three times daily (t.i.d.)) evaluated. The serum concentrations of lipoprotein(a) (Lp(a)), lipids, the major apolipoproteins (apo), cholesteryl ester transfer activity and fibrinogen were determined before and after treatment. The b.i.d. and t.i.d. regimens each significantly reduced the serum levels of total cholesterol and triglyceride. The mean changes in serum lipids and lipoproteins did not differ significantly between the two groups. After 12 weeks of treatment, there was a significant decrease in total plasma cholesterol, triglyceride, low density lipoprotein cholesterol, apo A-II, apo B and fibrinogen and an increase in the high density lipoprotein cholesterol levels. Although the serum level of Lp(a) did not change in every patient, niceritrol significantly reduced the serum Lp(a) level in those with an initially high level of Lp(a) (greater than or equal to 20 mg/dl).

Population frequency of apolipoprotein E5 (Glu3-->Lys) and E7 (Glu244-->Lys, Glu245-->Lys) variants in western Japan.

Apolipoprotein (apo) E modulates the catabolism of chylomicrons and of very low density lipoprotein remnants. It has three major isoforms (apo E2, E3, E4) and some rare variants. To detect the variants of apo E, blood specimens from 1269 Japanese subjects were analyzed using isoelectric focusing with immunoblotting. The E5 and E7 variants were identified by IEF in 2 and 18 subjects, respectively. Both E5 (Glu3→Lys) carriers were confirmed by PCR‐mediated site‐directed mutagenesis, and all E7 (Glu244→Lys, Glu245→Lys) carriers were confirmed by the amplification refractory mutation system. The relative frequencies of the ε5 and ε7 alleles were 0.001 and 0.007, respectively. High concentrations of total cholesterol (>220 mg/dl) were detected in five of the subjects expressing apo E7 and one subject expressing apo E5, and eight subjects heterozygous for apo E7 showed elevated plasma triglyceride concentrations (>150 mg/dl). In 621 healthy subjects, the mean triglyceride concentration in subjects with apo E7/3 appeared to be higher than in those with apo E3/3, but the difference was not statistically significant.

Characterization of a novel variant of apolipoprotein E, E2 Fukuoka (Arg-224 → Gln) in a hyperlipidemic patient with xanthomatosis

A new variant of apolipoprotein (apo) E, designated apo E2 Fukuoka, was identified in a 54-year-old Japanese woman who suffered from hyperlipoproteinemia (total cholesterol 29.7 mmol/l, triglyceride 12.0 mmol/l, when she was 48-year-old) with the presence of xanthoma in the palms, bones, and ocular fundi, and other sites. Foam-cell macrophages were observed in bone marrow specimens. Analysis of apo E phenotype showed the E3/E2 isoform on isoelectric focusing performed on plasma, but the E3/E3 isoform on restriction-fragment-length polymorphism of the apo E gene. This discrepancy indicated that the apo E had an amino-acid substitution outside of amino-acid residues at 112 and 158. Sequence analysis of the patients DNA, which was amplified by PCR and subcloned, revealed a single substitution from arginine (CGG) to glutamine (CAG) at residue 224, thereby adding one negatively charged unit to apo E3. Recombinant apo E2 Fukuoka produced in COS-1 cells showed almost the same binding activity to the LDL receptor on human skin fibroblasts as compared with recombinant apo E3. Recombinant apo E2 Fukuoka showed the same heparin binding ability than recombinant apo E3. Findings indicated that apo E2 Fukuoka was not the primary cause of the hyperlipoproteinemia observed in this case.

Identification of two apolipoprotein variants, A‐I Karatsu (Tyr 100 → His) and A‐I Kurume (His 162 → Gin)

We identified two apolipoprotein (apo) A‐I variants, using isoelectric focusing gel electrophoresis: apo A‐I Karatsu, which had a relative charge of+ 1 compared to normal apo A‐I4, and apo A‐I Kurume, which had a relative charge of ‐1. Direct sequence analysis of the PCR‐amplified DNA from the proband of apo A‐I Karatsu revealed a single substitution of tyrosine (TAC) for histidine (CAC) at position 100. Sequence analysis of apo A‐I Kurume revealed a single substitution of histidine (CAT) for glutamine (CAG) at position 162. Probands of these two mutants and limited family study showed no accelerated atherosclerosis.

Detection of a point mutation in cholesteryl ester transfer protein gene by polymerase chain reaction-mediated site-directed mutagenesis

We describe a method for the rapid and non-radioactive examination of DNA samples for a mutation of cholesteryl ester transfer protein using a polymerase chain reaction-mediated site-directed mutagenesis. CETP deficiencies were studied in 554 Japanese subjects (370 men, 184 women) aged between 18 and 91 (mean 48.3 years). By this method, we detected one homozygote and 3 heterozygotes of the CETP deficiency.