Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

Localization of types I, II, and X collagen in the transplanted tumor derived from human osteogenic sarcoma.

We have succeeded in transplanting human osteogenic sarcoma of the mandible into nude mice. As the transplanted tumor shows features of calcified chondrosarcoma, this tumor is thought to be an excellent model for study of the process of dystrophic endochondral calcification. Using this model, we studied relations between the expression of types I, II, and X collagen and chondrogenic differentiation of the transplanted tumor. Collagen distribution during the development and growth of the transplanted tumor was investigated by immunofluorescence with specific antibodies against type I, II or X collagen. Type X collagen was intensely stained in the mineralized region. Almost all tumor cells in this region were hypertrophic. Type II collagen was chiefly distributed in the unmineralized region where tumor cells showed chondrocytic or hypertrophic feature. These results indicate that the type of collagen changes from type II to type X in the hypertrophic region and the type X collagen may be synthesized by hypertrophic tumor cells. Type I collagen was localized in the marginal region of the tumor, though it disappeared in the mineralized region.

Clinical characteristics of double heterozygotes with familial hypercholesterolemia and cholesteryl ester transfer protein deficiency.

Coronary heart disease (CHD) in familial hypercholesterolemia (FH) may be modified by genetic and/or environmental factors. We described the effect of the cholesteryl ester transfer protein (CETP) gene on CHD in heterozygous FH caused by low density lipoprotein receptor (LDL-R) gene mutation. In 288 unrelated Japanese subjects with heterozygous FH, the allele frequency of an intron 14 G(+1)-to-A mutation (Int14 A) and a missense mutation in exon 15 (Asp442 to Gly, D442G) was 0.3 and 3.0%, respectively. HDL-C levels (1.55 +/- 0.08 mmol/l) in FH patients with heterozygous CETP deficiency were higher than those (1.19 +/- 0.08 mmol/l) in FH without CETP deficiency (P < 0.03), while LDL-C levels in FH with CETP deficiency were moderately reduced. However, two FH patients with CETP deficiency suffered myocardial infarction, and six patients had effort angina pectoris and/or coronary atherosclerosis. No difference in the score of coronary stenosis index (CSI) was found in FH with/without CETP deficiency, although CSI was inversely correlated with HDL-C levels (P < 0.05). Thus, the effect of increased HDL-C levels caused by partial deficiency of CETP is insufficient to prevent CHD in FH.

Reduction of lipoprotein(a) by LDL-apheresis using a dextran sulfate cellulose column in patients with familial hypercholesterolemia.

Lipoprotein(a) (Lp(a)) was eliminated by LDL-apheresis using a dextran sulfate cellulose column in 3 homozygous and 10 heterozygous familial hypercholesterolemic patients. Immediately after LDL-apheresis by the LA-15 system (continuous LDL apheresis), there were significant reductions in Lp(a) concentrations (28.6 +/- 11.8 mg/dl (mean +/- S.E.) to 9.6 +/- 5.6 mg/dl (P < 0.01)), and in LDL-cholesterol concentrations (156 +/- 32 mg/dl to 48 +/- 18 mg/dl (P < 0.01)). Immediately following LDL-apheresis, Lp(a) and LDL-cholesterol were reduced by 67.4% +/- 11.6% and 68.3% +/- 11.8%, respectively. The removal of Lp(a) paralleled that of LDL-cholesterol. The reduced levels of Lp(a) nearly returned to baseline within 7 days. In 6 of the heterozygous FH patients the rates of recovery of LDL cholesterol and Lp(a) were calculated, according to Apstein's equation after discontinuing lipid altering drug treatment for 4 weeks. Mean constant k values of LDL cholesterol and Lp(a) were 0.354 (range: 0.136-0.752) and 0.427 (range 0.112-0.933), respectively. The average concentration during the 7 days following LDL-apheresis was calculated. Average reductions were 28% in LDL cholesterol and 18% in Lp(a). Pravastatin treatment, which continued for 4 weeks, significantly decreased LDL cholesterol (P < 0.01); however, before LDL-apheresis pravastatin treatment significantly increased Lp(a) levels (P < 0.05) in a small number (n = 6) of the FH patients, who had been regularly treated with LDL-apheresis. These results suggest that LDL-apheresis using the dextran sulfate cellulose column is an effective treatment to reduce levels of serum Lp(a) and LDL proportionally. This therapy may be of value in the prevention and regression of coronary artery disease in FH patients.

Clinical efficacy of fluvastatin in the long-term treatment of familial hypercholesterolemia.

The long-term clinical efficacy of fluvastatin was assessed in 24 patients with familial hypercholesterolemia over a total treatment period of 104 weeks. Patients received an initial fluvastatin dose of 20 mg/day for 8 weeks, which was increased to 30 mg/day for a further 16 weeks. From week 24, if serum total cholesterol remained > or = 230 mg/dL, the fluvastatin dose could be increased to 40 or 60 mg/day, as necessary. By the end of treatment, 4 patients were receiving 30 mg/day fluvastatin, 1 patient was receiving 40 mg/day, and 19 patients were receiving 60 mg/day. Serum total cholesterol and low density lipoprotein cholesterol (LDL-C) levels showed a significant decrease from baseline at week 104 (total cholesterol, -26.8 +/- 2.4%; LDL-C, -33.1 +/- 3.3%; p < 0.001). The reductions in total cholesterol and LDL-C were dose-related. Statistically significant (p < 0.05) increases in serum high density lipoprotein cholesterol (HDL-C) were observed at week 24 (12.1 +/- 5.0%) and at week 76 (11.0 +/- 3.3%), although the effect was variable. Nevertherless, at the end of treatment the LDL-C: HDL-C ratio showed a 35% reduction from baseline. Changes in triglyceride levels failed to achieve statistical significance, with a reduction from baseline of -13.9 +/- 7.3% at week 104. Changes in apolipoprotein A-I were variable, with statistically significant (p < 0.01) increases observed at week 24 (7.6 +/- 2.3%) and week 76 (8.4 +/- 2.7%). By contrast, a significant reduction from baseline in apolipoprotein B was achieved by week 12 (-15.0 +/- 2.3%; p < 0.001) and was maintained throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma homocysteine level and development of coronary artery disease.

BACKGROUND: The plasma level of homocysteine is an independent risk factor for atherosclerotic vascular disease. The relationship between plasma homocysteine level and the onset of coronary artery disease (CAD) has not been established. OBJECTIVE: To investigate the relationship between plasma homocysteine level and the age at which CAD was diagnosed. METHODS: Fifty-seven male patients aged < or = 65 years (mean age 53 years) with angiographically proven symptomatic CAD seen consecutively and 138 age-matched male control subjects (mean age 52 years) free from atherosclerotic vascular disease were studied. They were divided into two subgroups, a group of younger subjects (aged < or = 55 years) and a group of older subjects (aged 56-65 years). RESULTS: Plasma homocysteine levels in CAD patients significantly exceeded those of control subjects (means 13.4 versus 10.6 nmol/ml, P = 0.0002). Plasma homocysteine level of subjects in younger CAD group was significantly higher than that of subjects in older CAD group (15.0 versus 11.3 nmol/ml, P = 0.03), and age and logarithmically transformed plasma homocysteine level exhibited a significant negative correlation (r = -0.28, P = 0.03) for subjects in CAD group. Among control subjects, members of our two age subgroups had similar plasma homocysteine levels. Younger CAD patients had significantly higher plasma homocysteine levels than did younger controls (15.0 versus 10.4 nmol/ml, P < 0.0001). However, for older groups there was no significant difference between plasma homocysteine levels in CAD patients and controls (11.3 versus 10.9 nmol/ml). Multiple regression analysis showed that only logarithmically transformed plasma homocysteine level was a significant predictor for age of onset of CAD. CONCLUSION: An elevated level of plasma homocysteine is more important in the development of premature CAD than it is in that of late-onset CAD among men.

Hyperhomocysteinemia, diabetes mellitus, and carotid atherosclerosis independently increase atherosclerotic vascular disease outcome in Japanese patients with end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) have high mortality from atherosclerotic/atherothrombotic vascular disease (AVD). However, the role of an elevated plasma total homocysteine (tHcy) level as a risk factor is uncertain in ESRD. METHODS: We enrolled 55 ESRD patients in a prospective follow-up study in order to evaluate the prognostic significance of their tHcy levels, common methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, and other atherosclerotic risk factors, in combination with the results of B mode ultrasound for carotid arteries. RESULTS: Mean intima-media thickness of the common carotid artery (CCA-IMT) in ESRD patients was thicker than that in 102 age- and sex-matched healthy controls. Carotid plaque was more frequently present in patients compared with controls, as was calcified plaque more common in patients (p < 0.001). Plasma tHcy levels (mean +/- SD) in patients (39.1 +/- 27.2 nmol/ml) were higher than that (8.8 +/- 2.7 nmol/ml) in controls (p < 0.001). Folic acid was the major determinant of elevated tHcy levels in ESRD patients. During the follow-up period of 31 +/- 3 months, 14 patients had one or more AVD complications, and 10 consequently died from AVD causes. Proportional hazards modeling showed that 5-year intervals of age (relative risk of 2.95, 95% CI 1.62 - 5.37), 10 nmol/ml intervals of tHcy levels (relative risk of 2.31, 95% CI 1.31 - 4.08), and presence of diabetes mellitus (relative risk of 6.62, 95% CI 1.07 +/- 40.8) were independent predictors of future AVD events, and tHcy levels (relative risk of 2.67, 95% CI 1.29 - 5.52) and age (relative risk of 2.10, 95% CI 1.15 - 3.83) were those of AVD mortality. We also found a significant association between carotid plaque prevalence and AVD events (X(2) = 11.6, p = 0.001). CONCLUSION: Hyperhomocysteinemia, diabetes mellitus, and carotid atherosclerosis appeared to contribute independently to increase the risk of AVD outcome in Japanese patients with ESRD.

Impact of severe coronary disease associated or not associated with diabetes mellitus on outcome of interventional treatment using stents: results from HERZ (Heart Research Group of Kanazawa) analyses.

Percutaneous coronary intervention (PCI) using a drug-eluting stent (DES) leads to less re-stenosis than PCI using a bare metal stent (BMS), however there is still controversy whether use of a DES for severe coronary disease leads to an acceptable outcome in patients with diabetes mellitus (DM). In this study 8159 lesions were treated in 6739 patients (mean age 68.9 years) with coronary artery disease. Use of a DES significantly decreased the re-stenosis rate compared with BMS in both DM (9.6% versus 21.3%) and non-DM (9.5% versus 17.1%) patients. The re-stenosis rate was significantly higher in DM than in non-DM patients in the BMS group but not in the DES group. There was no statistically significant difference in event-free survival after stenting of patients with left main coronary artery (LMCA) disease between the BMS and DES groups. It was concluded that, compared with BMS, DES reduced re-stenosis in patients with DM, however, we advise careful treatment after using DES for severe coronary disease, including LMCA lesions, in patients with DM.

Expression of collagen species in a cartilaginous tumor derived from a human osteogenic sarcoma.

We have succeeded in transplanting human osteogenic sarcoma into nude mice. Morphologically, the transplanted tumor is chondrosarcoma and manifests calcification, but not ossification. This tumor is thought to be an excellent model for studying the process of morbid endochondral calcification. In this study, we have used in situ hybridization to examine expression of collagen type I, II, and III mRNAs in this tumor. In situ hybridization was carried out using biotinylated DNA probes. Hybridized probes were detected using a streptavidin-biotin-alkaline phosphatase reagent. The results showed that collagen type I and II mRNAs were produced by cells of the transplanted tumor. Collagen type I mRNA was chiefly localized in the marginal region of the tumor. Collagen type II mRNA, which was predominantly found in the premineralized region of the transplanted tumor, gradually decreased toward the mineralized region. Collagen type III mRNA was not expressed in the transplanted tumor. These results suggest that the character of progenitor chondrogenic cells might be transferred to the transplanted tumor, and that the tumor cells may change the expression of collagen genes with the differentiation or maturation.

Long-term efficacy of immunoadsorbent plasmapheresis in a patient with Budd-Chiari syndrome due to antiphospholipid syndrome: case report with nine-year follow-up.

We describe the safety and efficacy of long-term immunoadsorbent plasmapheresis (IAPP) with dextran sulfate-cellulose bead columns in antiphospholipid syndrome (APS). IAPP was administered to a 38-year old male Japanese patient with APS with Budd-Chiari syndrome (BCS), who had presented with refractory lower leg skin ulcers and arterial and venous thromboses including BCS. After hepatic vein transluminal angioplasty was performed, the combination of corticosteroid, aspirin and IAPP was administered because of an underlying bleeding tendency related to liver dysfunction. From February 1994 to February 2003, a total of 228 procedures were performed. No further thrombosis-related symptoms or bleeding have occurred for more than nine years, suggesting that IAPP with dextran sulfate cellulose columns is safe and effective for APS in preventing additional thrombotic events. This IAPP supplements anticoagulation, antiplatelet, corticosteroid and immunosuppressant therapies.

Rapid detection and prevalence of cholesteryl ester transfer protein deficiency caused by an intron 14 splicing defect in hyperalphalipoproteinemia.

A deficiency of plasma cholesteryl ester transfer protein (CETP) is one of the genetic causes of increased serum high density lipoprotein (HDL)-cholesterol levels (hyperalphalipoproteinemia). A splicing defect (G-->A mutation) at the +1 position of intron 14 of the human CETP gene is a common mutation in the Japanese CETP deficiency. A rapid screening method for the splicing defect by means of primer-specified restriction map modification was described. The frequency of the mutation in hyperalphalipoproteinemia was determined, and its frequency in the general population was estimated. During polymerase chain reaction (PCR) with a modified primer, a novel NdeI restriction endonuclease site was created from the mutated allele in the PCR products, which could be visualized after electrophoresis of the digested products. As a result, 21 of 121 unrelated hyperalphalipoproteinemic subjects with HDL-cholesterol > or = 60 mg/dl (1.55 mmol/l), were found to have the G-->A mutation. Of the 21 individuals, 8 were found to be homozygous for the mutation. Allele frequency of the mutation was 1.5% (1/68), 2.8% (2/72), 7.1% (4/56), and 47.8% (22/46) in the groups with HDL-cholesterol levels of 60-79 mg/dl, 80-99 mg/dl, 100-119 mg/dl, and > or = 120 mg/dl, respectively. Based on the percentage of the area under the computed normal distribution curve of serum HDL-cholesterol, the frequency of the mutated allele in the general population was estimated to be 0.81% from the present results. This rapid detection method facilitates large-scale screening of CETP deficiency caused by the splicing defect. The mutation was frequent in Japanese subjects with hyperalphalipoproteinemia, especially in the group with HDL-cholesterol > or = 120 mg/dl.

Residual cardiomyocytes and scintigraphic findings in advanced coronary artery disease: correlation with technetium-99m-tetrofosmin and thallium-201 single photon emission computed tomography.

A 68-year-old man suffering from chronic heart failure due to coronary artery disease (CAD) underwent rest technetium-99m (99mTc)-tetrofosmin and thallium-201 (201Tl) with reinjection studies, but died thereafter. The heart was removed and sectioned into short-axis slices and examined by gross and microscopic pathologic methods. A close correlation between the amount of residual cardiomyocytes and the level of regional tracer activity in the left ventricular wall was obtained for redistribution 201Tl, reinjection 201Tl and rest 99mTc tetrofosmin images. The correlation coefficients were r=0.901 for the 201Tl redistribution images, r=0.913 for the 201Tl reinjection images and r=0.917 for the rest 99mTc-tetrofosmin images. This case report provides further evidence of the validity of SPECT tetrofosmin imaging for the determination of myocardial viability in CAD.

Carotid artery intima-media thickness and brachial artery flow-mediated vasodilation in asymptomatic Japanese male subjects amongst apolipoprotein E phenotypes.

BACKGROUND: Epidemiological studies suggest that apolipoprotein E (apoE) polymorphism influences plasma lipoprotein levels and the development of cardiovascular disease. OBJECTIVE: To clarify the role of apoE polymorphism as a risk factor for early atherosclerosis. DESIGN: Using a high-resolution ultrasound method, we investigated the association between apoE phenotypes, carotid intima-media thickness (CCA-IMT), and flow-mediated dilation in the brachial artery (brachial-FMD) in 96 healthy asymptomatic Japanese men (mean +/- SD age, 50 +/- 8 years). RESULTS: Serum cholesterol and LDL-cholesterol levels in subjects with E3E4 were highest and those with E2E3 were lowest (P < 0.05 and P < 0.05, respectively). The CCA-IMT in E3E4 subjects (0.76 +/- 0.17 mm) was greater than that in E2E3 and E3E3 (0.61 +/- 0.15 and 0.64 +/- 0.14 mm, respectively; P < 0.01). In contrast, there was no difference between brachial-FMD and apoE phenotypes (P=0.15). By univariate analysis, CCA-IMT was positively correlated with age (r=0.51, P < 0.01), LDL-chol/HDL-chol ratio (r=0.37, P < 0.01), triglycerides (r=0.23, P < 0.05), and negatively correlated with HDL-cholesterol (r=-0.31, P < 0.01). An association between CCA-IMT and the presence of E4 allele was also found (P < 0.05). Logistic regression analysis revealed that the presence of E4 allele was a higher risk for increased IMT (relative risk of 4.4, 95% CI 1.5-12.5), even after adjustment for age, LDL-cholesterol, blood pressure and other known risk factors. A negative correlation between brachial-FMD and CCA-IMT was also found in all subjects (r=-0.21, P < 0.05), being most apparent in the E3E4 subjects (r=-0.53, P < 0.02). CONCLUSION: ApoE4 phenotype was independently associated with an increased risk of carotid atherosclerosis and elevated LDL-cholesterol levels in asymptomatic middle-aged Japanese men.