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The aim of this study is to analyze the effect of apolipoprotein E (apo E) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on serum lipid and homocysteine levels in the general Japanese population.We analyzed the polymorphisms in individuals randomly selected from among participants of Serum Lipid Survey 2000.The frequency of the epsilon2, epsilon3, and epsilon4 alleles of APOE was 4.2, 85.3, and 10.5%, respectively. Individuals with the genotype epsilon4/epsilon4 had the highest total and low-density lipoprotein (LDL) cholesterol levels, while those with epsilon2/epsilon2 had the lowest. Individuals with the epsilon2/epsilon2 and epsilon2/epsilon4 genotypes had higher remnant-like particles (RLP)-cholesterol levels than those with epsilon2epsilon3, epsilon3epsilon3, and epsilon3epsilon4. There was a trend for individuals with the epsilon2/epsilon4 and epsilon2/epsilon2 genotypes to have higher triglyceride levels, although the difference was not significant. The presence of the T allele in a MTHFR polymorphism (C667T) was associated with higher homocysteine levels, which is more prominent in men than in women.Thus in our large-scale analysis we have shown that RLP-cholesterol is better associated with, APOE genotype than triglyceride and the effect of the T allele on MTHFR polymorphism (C667T) homocysteine levels is more prominent in men than in women among Japanese.

Apolipoprotein (apo) J, clusterin, is ubiquitously expressed in many tissues, and is a component of high-density lipoproteins (HDLs). There is experimental evidence that it may be anti-atherogenic through its effects on cholesterol transport, smooth muscle cell proliferation and lipid peroxidation. HDLs containing apo J and apo A-I carry paraoxonase (PON1), which protects low-density lipoproteins from oxidative modification; however, the extent to which apo J affects coronary heart disease (CHD) is not known. We have developed a sandwich ELISA that enables apo J to be assayed in the range of 13-200 microg/mL. Serum apo J was 52.8+/-0.8 microg/mL (mean+/-SEM; range, 36.0-84.3 microg/mL; n=92) in healthy Japanese men, and 49.3+/-0.5 microg/mL (34.5-72.8; n=241) in healthy Japanese women. Multiple regression of these data and results from 67 men with CHD showed that apo J concentration was unrelated to age, sex or body mass index, but was positively related to serum PON1 (p

Background & Aims: The relationship between single nucleotide polymorphisms (SNPs) and clinical outcomes has been intensively studied. We intended to determine SNPs of CYP2C19 and 23S rRNA of Helicobacter pylori by using rapid urease test (RUT)-positive gastric mucosal samples. Methods: One hundred thirty-nine patients with H pylori-positive results based on RUT completed 1-week treatment with lansoprazole 30 mg twice a day, clarithromycin 200 mg 3 times daily, and amoxicillin 500 mg 3 times daily. SNPs from adenine to guanine at positions 2142 and 2143 of 23S rRNA of H pylori (A2142G and A2143G) and SNPs from guanine to adenine at positions 681 in exon 5 (*2) and 636 in exon 4 (*3) of CYP2C19 were determined by the serial invasive signal amplification reaction assay by using DNAs extracted from gastric tissue samples already used for RUT. Minimum inhibitory concentrations of clarithromycin for H pylori were determined by culture test. CYP2C19 genotypes were classified into the rapid metabolizer (*1/*1), intermediate metabolizer (*1/*2 or *1/*3), and poor metabolizer (*2/*2, *2/*3, or *3/*3) groups. Results: H pylori strains with A2142G or A2143G mutation had higher minimum inhibitory concentrations for clarithromycin. Cure rates in rapid, intermediate, and poor metabolizer groups were 57.8% (95% confidence interval, 42.1%–72.4%), 88.2% (78.1%–94.8%), and 92.3% (74.9%–99.1%), respectively (P

The T-helper (Th)1 immune reaction is essential for the eradication of hepatitis C virus (HCV) during interferon (IFN) therapy in patients with chronic hepatitis C. Osteopontin is a cytokine crucial for the initiation of the Th1 response. Recently, we identified four single-nucleotide polymorphisms (SNPs) in the promoter region of the osteopontin gene (OPN), at nucleotide (nt) -155, -443, -616, and -1748, and suggested that the SNP at nt -443 was a marker reflecting hepatitis activity in patients with HCV. Therefore, we examined the possibility that SNPs in OPN were also markers predicting the therapeutic efficacy of IFN in patients with chronic hepatitis C. Blood was collected from 77 patients with chronic hepatitis C who had received either IFN monotherapy or IFN-ribavirin combination therapy (IFN-based therapies). SNPs in OPN, MxA, MBL, and LMP7 were analyzed by Invader assay. Promoter SNPs of OPN at nt -155, -616, and -1748 showed linkage disequilibrium at 100% to each other. Sustained virological response (SVR) was observed in 58% of all patients. The SVR rate was higher in patients with the G/G or G/A alleles in the OPN promoter SNP at nt -1748 than in those with A/A (85% vs 45%; P < 0.05). The SVR rate was also higher in patients with T/T at nt -443 than in those with C/C or C/T (86% vs 47%; P < 0.05). Such differences were particularly evident in patients with HCV genotype 1b who had a pretreatment viral load greater than 100 KIU/ml. All the patients who had G/G or G/A at nt -1748 and T/T at nt -443 obtained an SVR. On the other hand, there was no relationship between the efficacy of IFN-based therapies and SNPs in MxA, MBL, and LMP7, which had been shown to have association with the response to IFN monotherapies. SNPs in the promoter region of OPN may be useful as a marker to predict the efficacy of IFN-based therapies in patients with chronic hepatitis C, and further investigation regarding their real significance is warranted in a large series of patients.

We studied the association of six common polymorphisms of four genes related to lipid metabolism with serum lipid levels. We selected single-nucleotide polymorphisms (SNPs) in the genes for cholesteryl ester transfer protein (CETP), lipoprotein lipase (LPL), hepatic lipase (LIPC), and apolipoprotein CIII (APOC3), and studied 2267 individuals randomly selected from the participants of Serum Lipid Survey 2000. There was a significant association of CETP polymorphism (D442G, Int14 +1 G --A, and TaqIB), LPL polymorphism (S447X), and LIPC polymorphism (-514 --CT) with HDL-cholesterol levels. We also found a significant association of LPL polymorphism (S447X) and APOC3 polymorphism (SstI) with triglyceride levels. This is the largest database showing the association of common genetic variants in lipid metabolism with serum lipid levels in the general Japanese population. Further study is necessary to elucidate the role of these gene polymorphisms in cardiovascular events.

Background: A lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor of oxidized LDL in endothelial cells. The expression of LOX-1 was shown to be upregulated in atherosclerotic lesions. Recently, LOX-1 gene polymorphism (G501C) was reported to be associated with myocardial infarction (MI). Hypothesis: Our study was undertaken to elucidate the association between this polymorphism and coronary artery disease (CAD). Methods: We evaluated LOX-1 gene polymorphism using Invader assay in 586 patients undergoing coronary angiography. Results: Study patients were categorized into three groups: normal/minimal stenosis (≤25%) (n = 128); mild stenosis (26-50%) (n = 39); and significant stenosis (> 50%) (n = 419). Of the 419 patients with significant stenosis, 163 had single-vessel, 165 had double-vessel, and 91 had triple-vessel disease. Myocardial infarction was present in 171 patients. The frequency of LOX-1 gene variants (C/C or C/G) was lower in patients with significant than in those with normal/minimal stenosis (36 vs. 49%, p < 0.01). The frequency of LOX-1 gene variants did not differ between patients with and without MI (34 vs. 37%). However, a stepwise decrease in the frequency of such variants was found depending on the severity of CAD: 49% in normal/minimal stenosis, 41% in mild stenosis, 39% in single-vessel, 35% in double-vessel, and 32% in triple-vessel disease. Multivariate analysis demonstrated LOX-1 gene variants to be inversely associated with the presence of significant stenosis (odds ratio = 0.61; 95% confidence interval = 0.41-0.92). Conclusions: The LOX-1 gene variants at 501 were found to be inversely associated with the severity of CAD. This polymorphism may be modifying the severity of CAD.

We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.

Purpose: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader® assay was applied. Methods: To determine the optimum condition for the Invader® assay, mtDNAs were assayed in various amounts of total DNA in 1–4-h incubations at 63°C. To evaluate the suitability of the Invader® assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader® assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader® assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. Results: In 1–8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader® assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader® assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader® assay were consistent with those by the PCR-SSCP analysis. Conclusions: Invader® assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.

Background and aims. Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV. Methods. Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30 IU/L (low-activity group), between 30 and 80 IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80 IU/L irrespective of hepatoprotective treatment (high-activity group). Results. There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt −155, −443, −616, and −1748) were detected in the promoter region of OPN. Among them, the SNP at nt −443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D′ and r2 greater than 0.937 to each other without correlation to disease activity. Conclusions. OPN promoter region SNP at nt −433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.

Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins. Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, CETP is considered to be a key protein in reverse cholesterol transport (RCT), a protective system against atherosclerosis. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with marked hyperalphalipoproteinemia (HALP). Genetic CETP deficiency is the most important and common cause of HALP in the Japanese. Ten mutations of the CETP gene have been demonstrated as causes of HALP, including two common mutations: an intron 14 splicing defect (Int14 + 1 G --> A) and an exon 15 missense mutation (D442G). The subjects with CETP deficiency show a variety of abnormalities in the concentration, composition, and function of both HDL and low density lipoprotein (LDL). CETP deficiency is considered a physiological state of impaired RCT, which may possibly lead to the development of atherosclerosis despite high HDL cholesterol levels. However, the pathophysiological significance of CETP in terms of atherosclerosis has been controversial. Epidemiological studies in Japanese-Americans living in Hawaii and Japanese in the Omagari area, where HALP subjects with an intron 14 splicing defect of the CETP gene are markedly frequent, have shown a relatively increased incidence of coronary atherosclerosis in CETP deficiency. On the other hand, the TaqIB polymorphism-B2 allele with low CETP mass and increased HDL cholesterol has been related to a decreased risk for coronary heart disease (CHD) in many studies, including the Framingham Offspring Study. The current review focused on the characterization of the Japanese subjects with CETP deficiency, including our recent findings.

Polymorphisms in beta-cell transcription factor genes, Ala45Thr in the NeuroD1 gene and Arg121Trp in the Pax4 gene, have been reported. To clarify the role of these mutations in the pathogenesis of late-onset diabetes, we examined the insulin secretion and sensitivity in diabetic patients carrying the homozygous mutation in the NeuroD1 gene or Pax4 gene. We screened for the polymorphisms in NeuroD1 and Pax4 genes in 296 late-onset diabetic patients and 177 unrelated control subjects over 60 years of age. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by direct sequencing. Acute insulin secretion was evaluated using a 2-compartment model analysis of C-peptide kinetics after intravenous glucose load (CS1). Insulin sensitivity was estimated by the insulin-modified minimal model analysis (Si). Four diabetic patients carried the homozygous mutation (Thr/Thr) in the NeuroD1 gene and 3 patients carried the homozygous mutation (Trp/Trp) in the Pax4 gene, while both homozygous mutations were not detected in the control subjects. In patients A, B, C, and D with homozygous mutations in NeuroD1, CS1 (normal range, 6.8 to 18.5 ng/mL/min) was 0.508, 1.481, 1.223, and 1.584 ng/mL/min, respectively, and Si (normal range, 2.6 to 7.6 x 10(-4)/min/[microU/mL]) was 0.727, 3.31, 3.79, and 0.00 x 10(-4)/min/(microU/mL), respectively. In patients X, Y, and Z with homozygous mutation in Pax4, CS was 0.418, 0.208, and 1.279 ng/mL/min, respectively, and Si was 1.11, 2.88, and 0.00 x 10(-4)/min/(microU/mL), respectively. Since acute insulin secretion in response to glucose was markedly impaired and insulin resistance was varied in the patients carrying the homozygous mutations in the NeuroD1 and Pax4 genes, the mutations are ones of the factors involved in the beta-cell dysfunction and do not relate to the insulin resistance. These homozygous mutations appear to play a part in the pathogenesis of beta-cell defect in about 2.5% of Japanese patients with late-onset diabetes.

In the course of investigations of familial coronary artery disease in Hokkaido, the northland of Japan, we identified 13 families affected by familial hypercholesterolemia. Among them, we identified eight novel mutations of the low-density lipoprotein (LDL) receptor gene, four of which caused frameshifts: (1) a 7-bp deletion at nucleotide (nt) 578–584 (codon 172–174, exon 4); (2) a 14-bp insertion at 682 nt (codon 207–208, exon 4); (3) a 49-bp deletion at nt 943–991 (codon 294–310, exon 7); and (4) a one-base insertion of C to a stretch of C3 at nucleotides 1687–1689 or codon 542. The others included (5) a T-to-C transition at nt 1072 causing substitution of Cys for Arg at codon 337 (C337R, exon 8); (6) a splice-site G-to-T substitution in intron 11; (7) a splice-site G-to-C substitution in intron 11; and (8) a G-to-T transition at nt 1731 causing substitution of Trp for Cys at codon 556 (W556C, exon 12). To disclose the functional consequences of novel mutations, we characterized each of these mutations by two assays in peripheral lymphocytes, i.e., uptake of fluorescently labeled LDL by LDL receptors, and measurement of cell surface-bound LDL receptor protein using specific monoclonal antibody against LDL receptor.

Background: Plasma phospholipid transfer protein (PLTP) plays a central role in the remodeling of HDLs. Reliable and accurate methods for assaying PLTP concentration are required.Methods: A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625–15.0 ng/assay (1.2–30.0 mg/L). Intra- and interassay CVs were Results: PLTP concentrations were 12.0 ± 3.0 mg/L (mean ± SD; range, 4.9–20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P Conclusion: This new ELISA will be of value for further studies of PLTP in health and disease.

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxida- tive modification of low density lipoproteins. We have de- veloped a sensitive sandwich enzyme-linked immunosor- bent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 6 1.3 m g/mL (mean 6 SEM; n 5 87). Serum PON concentrations in relation to the PON 192 genetic poly- morphism were: 69.5 6 2.9 m g/mL in the QQ genotype; 63.0 6 1.9 m g/mL in the QR genotype; and 52.8 6 1.7 m g/mL in the RR genotype. Concentrations were signifi- cantly lower in the RR than in the QQ genotype ( P , 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 6 0.40 vs. 2.56 6 0.05 nmol/min/ m g, P , 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was posi- tively associated ( P , 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Sub- jects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 6 2.3 m g/mL; n 5 35, P , 0.01). This association was independent of the position 192 ge- notype. Our new ELISA should be of value for epidemi- ologic and clinical studies of serum PON concentration. — Kujiraoka, T., T. Oka, M. Ishihara, T. Egashira, T. Fujioka, E. Saito, S. Saito, N. E. Miller, and H. Hattori. A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration. J. Lipid Res. 2000. 41: 1358- 1363.

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an in- herent protease activity. After incubation of HDL 3 in the presence of purified plasma PLTP, the d , 1.25 g/ml parti- cles (fusion particles) contained intact 28.2 kDa apoA-I while the d . 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleav- age of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typ- ically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lag- phase was necessary for the development of the cleavage activity during incubation at 37 8 C. The specific apoA-I cleav- age activity of different PLTP preparations varied between 0.4-0.8 m g apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the C- terminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleav- age occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N- terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Re- combinant PLTP produced in CHO cells or using the bacu- lovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated pro- teolytic cleavage of apoA-I might affect plasma HDL metab- olism by generating a novel kinetic compartment of apoA- I with an increased turnover rate. —Jauhiainen, M., J. Huuskonen, M. Baumann, J. Metso, T. Oka, T. Egashira, H. Hattori, V. M. Olkkonen, and C. Ehnholm. Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apo- lipoprotein A-I. J. Lipid Res. 1999. 40: 654-664.

Background Interleukin (IL)-12 is thought to play an important role in the development of atherosclerosis and recently, polymorphism of the 3'-untranslated region of the IL-12 p40 gene (A1188C) was reported to be associated with diabetes and multiple sclerosis. However, the association between this genetic polymorphism and coronary artery disease (CAD) has not been studied. Methods and Results The frequency of this polymorphism was investigated in 555 patients undergoing coronary angiography: 395 had CAD, of whom 161 also had a myocardial infarction (MI). With regard to the IL-12 p40 polymorphism, 125 had the A/A, 268 had the A/C, and 162 had the C/C genotype. The prevalence of CAD did not differ among the groups (71%, 73%, and 69%, respectively; p= NS). The prevalence of MI was also similar among the groups (28%, 27%, and 33%, respectively; p= NS). Moreover, the number of >50% stenotic vessels, >50% stenotic segments, and ≤50% stenotic segments did not differ among the 3 groups. Conclusions Polymorphism of IL-12 p40 gene was not found to be associated with the presence or severity of CAD, suggesting that it does not play an important role in the development of this disease. (Circ J 2005; 69: 793 - 797)

The relationship between single nucleotide polymorphisms (SNPs) and clinical outcomes has been intensively studied. We intended to determine SNPs of CYP2C19 and 23S rRNA of Helicobacter pylori by using rapid urease test (RUT)-positive gastric mucosal samples.One hundred thirty-nine patients with H pylori -positive results based on RUT completed 1-week treatment with lansoprazole 30 mg twice a day, clarithromycin 200 mg 3 times daily, and amoxicillin 500 mg 3 times daily. SNPs from adenine to guanine at positions 2142 and 2143 of 23S rRNA of H pylori (A2142G and A2143G) and SNPs from guanine to adenine at positions 681 in exon 5 (* 2 ) and 636 in exon 4 (* 3 ) of CYP2C19 were determined by the serial invasive signal amplification reaction assay by using DNAs extracted from gastric tissue samples already used for RUT. Minimum inhibitory concentrations of clarithromycin for H pylori were determined by culture test. CYP2C19 genotypes were classified into the rapid metabolizer (* 1 /* 1 ), intermediate metabolizer (* 1 /* 2 or * 1 /* 3 ), and poor metabolizer (* 2 /* 2 , * 2 /* 3 , or * 3 /* 3 ) groups.H pylori strains with A2142G or A2143G mutation had higher minimum inhibitory concentrations for clarithromycin. Cure rates in rapid, intermediate, and poor metabolizer groups were 57.8% (95% confidence interval, 42.1%-72.4%), 88.2% (78.1%-94.8%), and 92.3% (74.9%-99.1%), respectively ( P.001). Cure rates in strains with and without A2142G or A2143G mutation were 48.3% (29.4%-67.5%) and 87.3% (79.5%-92.7%), respectively ( P.001).SNPs of CYP2C19 and 23S rRNA of H pylori using RUT-positive gastric mucosal samples could be predictable determinants for H pylori eradication by triple therapy.

Background: A lectin‐like oxidized low‐density lipoprotein (LDL) receptor‐1 (LOX‐1) is the major receptor of oxidized LDL in endothelial cells. The expression of LOX‐1 was shown to be upregulated in atherosclerotic lesions. Recently, LOX‐1 gene polymorphism (G501C) was reported to be associated with myocardial infarction (MI). Hypothesis: Our study was undertaken to elucidate the association between this polymorphism and coronary artery disease (CAD). Methods: We evaluated LOX‐1 gene polymorphism using Invader assay in 586 patients undergoing coronary angiography. Results: Study patients were categorized into three groups: normal/minimal stenosis (≤25%) (n = 128); mild stenosis (26‐50%) (n = 39); and significant stenosis (> 50%) (n = 419). Of the 419 patients with significant stenosis, 163 had single‐vessel, 165 had double‐vessel, and 91 had triple‐vessel disease. Myocardial infarction was present in 171 patients. The frequency of LOX‐1 gene variants (C/C or C/G) was lower in patients with significant than in those with normal/minimal stenosis (36 vs. 49%, p < 0.01). The frequency of LOX‐1 gene variants did not differ between patients with and without MI (34 vs. 37%). However, a stepwise decrease in the frequency of such variants was found depending on the severity of CAD: 49% in normal/minimal stenosis, 41% in mild stenosis, 39% in single‐vessel, 35% in double‐vessel, and 32% in triple‐vessel disease. Multivariate analysis demonstrated LOX‐1 gene variants to be inversely associated with the presence of significant stenosis (odds ratio = 0.61; 95% confidence interval = 0.41‐0.92). Conclusions: The LOX‐1 gene variants at 501 were found to be inversely associated with the severity of CAD. This polymorphism may be modifying the severity of CAD.

Background: In individuals heterozygous for ABCA1 transporter mutations, defective reverse cholesterol transport (RCT) causes low HDL-cholesterol and premature coronary heart disease (CHD). However, the extent to which impaired RCT underlies premature CHD in others with low HDL-cholesterol is not known. The primary acceptors of cell cholesterol are a minor subclass of lipid-poor pre-β-HDLs. These are generated during remodeling of α-HDLs, which account for almost all HDL-cholesterol. We studied the strength of the association of CHD with pre-β-HDL concentrations in Japanese men.Methods: Blood was collected from 42 men with clinical CHD and 44 healthy controls 40–70 years of age. Pre-β-HDL was assayed by crossed immunoelectrophoresis.Results: Cases had lower HDL-cholesterol (−23%), total apolipoprotein A-I (−26%), and pre-β-HDL (−55%; all P Conclusions: Our results suggest that inefficient RCT, secondary to a low pre-β-HDL concentration and production rate in plasma, contributes to premature CHD in Japanese men with low HDL-cholesterol.

Despite progress in molecular characterization, specific diagnoses of disorders belonging to a group of in-herited hypoalphalipoproteinemias, i.e., apolipoprotein AI deficiency, lecithin-cholesterol acyltransferase deficiency, Tangier disease (TD), and familial high-density lipoprotein (HDL) deficiency, remain difficult on a purely clinical basis. Several TD patients were recently found to be homozygous for mutations in the ABCA1 gene. We have documented here a clinical variant of TD in a Japanese patient who manifested corneal lipidosis and premature coronary artery disease as well as an almost complete absence of HDL-cholesterol, by identifying a novel homozygous ABCA1 mutation (R1680W). We propose that patients with apparently isolated HDL deficiency who are found to carry ABCA1 mutations may in fact belong to a category of TD patients whose phenotypic features are only partially expressed, and that a number of hidden clinical variants of TD might exist among other HDL deficiency patients who have escaped correct clinical diagnosis.

Cholesteryl ester transfer protein (CETP) defi- ciency is one of the most important and common causes of hyperalphalipoproteinemia (HALP) in the Japanese. CETP deficiency is thought to be a state of impaired reverse cho- lesterol transport, which may possibly lead to the develop- ment of atherosclerotic cardiovascular disease despite high HDL-cholesterol (HDL-C) levels. Thus, it is important to in- vestigate whether HALP is caused by CETP deficiency. In the present study, we identified two novel missense muta- tions in the CETP gene among 196 subjects with a marked HALP (HDL-C � 2.59 mmol/l � 100 mg/dl). The two mis- sense mutations, L151P (CTC → CCC in exon 5) and R282C (CGC → TGC in exon 9), were found in compound het- erozygous subjects with D442G mutation, whose plasma CETP levels were significantly lower when compared with those in D442G heterozygous subjects. In COS-7 cells ex- pressing the wild type and mutant CETP, these two mutant CETP showed a marked reduction in the secretion of CETP protein into media (0% and 39% of wild type for L151P and R282C, respectively). These results suggested that two novel missense mutations cause the decreased secretion of CETP protein into circulation leading to HALP. By using the Invader ® assay for seven mutations, including two novel mutations of the CETP gene, we investigated their fre- quency among 466 unrelated subjects with HALP (HDL-C � 2.07 mmol/l � 80 mg/dl). Two novel mutations were rare, but L151P mutation was found in unrelated subjects with a marked HALP. Furthermore, we demonstrated that CETP deficiency contributes to 61.7% and 31.4% of marked HALP and moderate HALP in the Japanese, respec

Abstract —Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B–containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the −69 nucleotide of the promoter region (−69 G→A), corresponding to the second nucleotide of the PEA3/ETS binding site (C G GAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol ≥2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the −69 G→A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the −69 G→A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel −69 G→A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 TC), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population. Hum Mutat 14:87, 1999. © 1999 Wiley-Liss, Inc.