Mutsuko Takata

ATP-binding cassette transporter G8 M429V polymorphism as a novel genetic marker of higher cholesterol absorption in hypercholesterolaemic Japanese subjects.

The ratio of serum plant sterols to cholesterol is positively correlated with the fractional cholesterol absorption, whereas serum precursors of cholesterol synthesis are positively correlated with cholesterol synthesis. Recently, two ABC (ATP-binding cassette) transporters, ABCG5 and ABCG8, have been described as playing an important role in the absorption and excretion of sterols. In the present study, we tested the hypothesis that genetic variation in ABCG5/ABCG8 influences the levels of serum plant sterol (sitosterol) and cholesterol precursor (lathosterol) in Japanese primary hypercholesterolaemic patients (n = 100). We identified a novel mutation [859T/C (C287R)] and a novel polymorphism [1285A/G (M429V)] at the ABCG5/ABCG8 loci, as well as four polymorphisms reported previously [1810C/G (Q604E), 161G/A (C54Y), 1199C/A (T400K) and 1895C/T (A632V)]. In carriers of the novel M429V variant, the serum level of sitosterol and the sitosterol/cholesterol ratio were significantly higher than those in non-carriers (3.64 compared with 2.56 microg/ml, and 1.45 microg/mg compared with 1.00 microg/mg respectively; P < 0.01 for both), and serum lathosterol tended to be lower (1.95 microg/ml compared with 3.03 microg/ml; P = 0.08), whereas no significant difference was observed in other lipid profiles. These four polymorphisms (1810C/G, 161G/A, 1199C/A and 1285A/G) generated six haplotypes, and the C/G/C/G haplotype was significantly associated with a higher sitosterol level and sitosterol/cholesterol ratio compared with the other five haplotypes (P < 0.05 for both). We conclude that, in 8% of patients with hypercholesterolaemia, the novel ABCG8 M429V variant was associated with higher cholesterol absorption efficiency. Future studies should investigate whether these findings have implications for the optimal cholesterol-lowering drug treatment in hypercholesterolaemic patients.

Altered metabolism of low-density lipoprotein and very-low-density lipoprotein remnant in autosomal recessive hypercholesterolemia: results from stable isotope kinetic study in vivo.

Background— Autosomal recessive hypercholesterolemia (ARH) exhibits different responsiveness to statins compared with that in homozygous familial hypercholesterolemia (FH). However, few data exist regarding lipoprotein metabolism of ARH. Therefore, we aimed to clarify lipoprotein metabolism, especially the remnant lipoprotein fractions of ARH before and after statin therapy. Methods and Results— We performed a lipoprotein kinetic study in an ARH patient and 7 normal control subjects, using stable isotope methodology (10 mg/kg of [2H3]-leucine). These studies were performed at baseline and after the 20 mg daily dose of atorvastatin. Tracer/tracee ratio of apolipoprotein B (apoB) was determined by gas chromatography/mass spectrometry and fractional catabolic rates (FCR) were determined by multicompartmental modeling, including remnant lipoprotein fractions. FCR of low-density lipoprotein (LDL) apoB of ARH was significantly lower than those of control subjects (0.109 versus 0.450±0.122 1/day). In contrast, the direct removal of very-low-density lipoprotein remnant was significantly greater in ARH than those in control subjects (47.5 versus 2±2%). Interestingly, FCR of LDL apoB in ARH dramatically increased to 0.464 1/day, accompanying reduction of LDL cholesterol levels from 8.63 to 4.22 mmol/L after treatment with atorvastatin of 20 mg/d for 3 months. Conclusions— These results demonstrate that ARH exhibits decreased LDL clearance associated with decreased FCR of LDL apoB and increased clearance for very-low-density lipoprotein remnant. We suggest that increased clearance of remnant lipoprotein fractions could contribute to the great responsiveness to statins, providing new insights into the lipoprotein metabolism of ARH and the novel pharmacological target for LDLRAP1.

Infantile Cases of Sitosterolaemia with Novel Mutations in the ABCG5 Gene: Extreme Hypercholesterolaemia is Exacerbated by Breastfeeding.

Few data exists regarding the clinical impact of breastfeeding in infantile sitosterolaemic cases. We report four Japanese infantile cases of sitosterolaemia, an extremely rare inherited disease characterised by increased serum levels of plant sitosterol, presenting with severe hypercholesterolaemia and systemic xanthomas exacerbated by breastfeeding. In these four cases, genetic analyses were performed for low-density lipoprotein (LDL) receptor, proprotein convertase subtilisin/kexin type 9 (PCSK9), LDL receptor adaptor protein 1 and ATP-binding cassette (ABC) subfamily G member 5 and 8 (ABCG5 and ABCG8) genes. We assessed their clinical manifestations, including responsiveness to a variety of treatments, especially to weaning from breastfeeding and use of ezetimibe. Two pairs of mutations in the ABCG5 gene in each case, including two novel mutations (c.130C>T or p.Ser44Ala and c.1813_1817delCTTTT or p.Pro558GlufsX14) and two known mutations (c.1306G>A or p.Arg389His and c.1336C>T or p.Arg446X), were identified. Significant reductions in cholesterol levels were obtained by means of weaning from breastfeeding alone. Substantial reductions in sitosterol levels, without any apparent side effects, were observed with ezetimibe. In conclusion, we have identified infantile Japanese sitosterolaemic subjects with extreme hypercholesterolaemia exacerbated by breastfeeding. Their unique response to weaning from breastfeeding, as well as to use of ezetimibe, could provide insights into the metabolic basis of sterols in humans.

CETP (cholesteryl ester transfer protein) promoter −1337 C>T polymorphism protects against coronary atherosclerosis in Japanese patients with heterozygous familial hypercholesterolaemia

CETP (cholesteryl ester transfer protein) and HL (hepatic lipase) play a role in the metabolism of plasma lipoproteins, but the effects of CETP and LIPC (gene encoding HL) genotypes on coronary atherosclerosis may be dependent on LDL (low-density lipoprotein)-receptor activity. Recently, the -1337 C>T polymorphism in the CETP gene has been reported in REGRESS (Regression Growth Evaluation Statin Study) to be a major determinant of promoter activity and plasma CETP concentration. In the present study, we have investigated the effects of the CETP promoter -1337 C>T and LIPC promoter -514 C>T polymorphisms on serum lipid profiles and risk of coronary atherosclerosis in 206 patients (154 males) with heterozygous FH (familial hypercholesterolaemia). To evaluate coronary atherosclerosis, we used CSI (coronary stenosis index) calculated from coronary angiograms. The CETP -1337 T allele was less frequent in subjects with a CSI > or =14 (mean value) in the group with coronary artery disease (P=0.04, as determined by chi(2) test). ANOVA revealed that HDL-C (high-density lipoprotein-cholesterol) and triacylglycerol (triglyceride) levels were not significantly higher in the presence of the CETP promoter -1337 T allele. Combined with LIPC promoter polymorphisms, HDL-C levels were highest and CSI were lowest with CETP -1337 CT+TT and LIPC -514 CC genotypes, but a significant interaction was not shown. A multiple logistic regression analysis revealed that, in patients with coronary atherosclerosis, the CETP- 1337 CC genotype was a significant genetic risk factor in FH (odds ratio=2.022; P=0.0256). These results indicate that the CETP promoter -1337C>T polymorphism is associated with the progression of coronary atherosclerosis in Japanese patients with FH, independent of HDL-C and triacylglycerol levels.

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

BACKGROUND The objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method. METHODS Our method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay. RESULTS Based on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n=20) and P664L, (n=5), and their LDLR activities was measured by this method, which was found to be 55.3+/-8.9% and 63.9+/-13.8%, respectively, of that of the control group (n=15). In comparison, the LDLR activity was 86.1+/-11.6% (K790X) and 73.3+/-6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p<0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4+/-13.2% (K790X) and 76.9+/-17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1+/-9.7% (K790X) and 70.3+/-26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used. CONCLUSION These results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.

High Frequency of a Retinoid X Receptor γ Gene Variant in Familial Combined Hyperlipidemia That Associates With Atherogenic Dyslipidemia

Objective—The genetic background of familial combined hyperlipidemia (FCHL) has not been fully clarified. Because several nuclear receptors play pivotal roles in lipid metabolism, we tested the hypothesis that genetic variants of nuclear receptors contribute to FCHL. Methods and Results—We screened all the coding regions of the PPAR&agr;, PPAR&ggr;2, PPAR&dgr;, FXR, LXR&agr;, and RXR&ggr; genes in 180 hyperlipidemic patients including 60 FCHL probands. Clinical characteristics of the identified variants were evaluated in other 175 patients suspected of coronary disease. We identified PPAR&agr; Asp140Asn and Gly395Glu, PPAR&ggr;2 Pro12Ala, RXR&ggr; Gly14Ser, and FXR −1g−>t variants. Only RXR&ggr; Ser14 was more frequent in FCHL (15%, P<0.05) than in other primary hyperlipidemia (4%) and in controls (5%). Among patients suspected of coronary disease, we identified 9 RXR&ggr; Ser14 carriers, who showed increased triglycerides (1.62±0.82 versus 1.91±0.42 [mean±SD] mmol/L, P<0.05), decreased HDL-cholesterol (1.32±0.41 versus 1.04±0.26, P<0.05), and decreased post-heparin plasma lipoprotein lipase protein levels (222±85 versus 149±38 ng/mL, P<0.01). In vitro, RXR&ggr; Ser14 showed significantly stronger repression of the lipoprotein lipase promoter than RXR&ggr; Gly14. Conclusion—These findings suggest that RXR&ggr; contributes to the genetic background of FCHL.

Limited effects of long-term enzyme replacement therapy on the cardiac conduction system in Fabry disease

The long-term effects of enzyme replacement therapy (ERT) on cardiac function and the conduction system in Fabry disease are not clearly understood. We report a case of a 48-year-old man with non-classical Fabry disease treated with ERT for 11 years. He was diagnosed with Fabry disease at age 27 years based on the presence of decreased alpha-galactosidase A activity in the peripheral leukocytes and of the causal alpha-galactosidase A mutation (Val339Gln). Subsequently, peritoneal dialysis was initiated for renal failure at age 35 years. ERT was initiated at age 39 years to halt the progression of cardiac dysfunction. Electrical conduction disturbances progressed gradually to complete atrioventricular block with atrial standstill during 9 years of ERT despite the lack of progression of ventricular hypertrophy. Although he underwent permanent pacemaker implantation to prevent sudden cardiac death, the atrioventricular junctional rhythm remained, thereby lowering the ventricular pacing rate. Based on this case, we recognize that the effects of ERT are limited for inhibiting the progression of Fabry disease and especially for inhibiting arrhythmia and conduction disturbances. Early diagnosis of Fabry disease and early initiation of ERT might be the key to further improvements in this disease and its associated conditions. <Learning objective: We encountered a patient with Fabry disease treated with long-term enzyme replacement therapy (ERT) in whom conduction disturbances progressed without progression of left ventricular hypertrophy. This case suggests that ERT is limited for inhibiting the progression of Fabry disease and especially of arrhythmia and conduction disturbances. Early diagnosis of Fabry disease and initiation of ERT may be important for providing further improvements of this condition.>.

Acute necrotizing eosinophilic myocarditis complicated by complete atrioventricular block promptly responded to glucocorticoid therapy

Acute myocarditis is frequently accompanied with conduction disturbances. Complete atrioventricular (AV) block may occur in acute myocarditis, but rarely in eosinophilic myocarditis. Acute necrotizing eosinophilic myocarditis, the most severe form of eosinophilic myocarditis, is generally fatal, and rarely complicated by complete AV block. We report a case of a 66-year-old woman with acute necrotizing eosinophilic myocarditis who presented with general malaise and nausea. She suddenly fell into cardiogenic shock because of complete AV block and worsened heart failure. Ultrasound cardiography revealed pericardial effusion, edematous myocardium, and reduced contractility of the left ventricle. The biopsied specimens showed marked interstitial infiltration with predominant eosinophils accompanied with myocardial necrosis. Oral administration of glucocorticoid in moderate dose promptly resolved the complete AV block, her clinical symptoms, and cardiac function. We recognized that acute necrotizing eosinophilic myocarditis can be complicated by complete AV block. Steroid therapy could be effective in the treatment of conduction disturbance as well as myocardial inflammation. <Learning objective: We experienced a case of acute necrotizing eosinophilic myocarditis complicated by complete atrioventricular block. This case report documents the rare complication of acute necrotizing eosinophilic myocarditis and the great benefit of early steroid therapy for the condition.>

Abrupt progression of ventricular septal perforation after primary angioplasty for acute myocardial infarction

A 61-year-old-man was transferred to our hospital because of progressive heart failure after reperfusion for acute myocardial infarction (MI). When he visited the local hospital with severe chest pain associated with inferior MI, transthoracic echocardiography revealed small ventricular septal perforation (VSP). The patient had emergent coronary angiography, which revealed total occlusion of the mid-portion of the right coronary artery. Primary angioplasty was successful for reperfusion. However, because of hemodynamic instability the patient was transferred to our hospital. Under these conditions, transthoracic echocardiography which was undertaken 3 hours after primary angioplasty, demonstrated progressive enlargement of the VSP probably due to reperfusion injury. The rupture site, which was further enlarged at the time of operation, was repaired using the patch exclusion technique. The patient could discharge without complications. We suggest that primary angioplasty may potentially induce late reperfusion injury in patients with VSP complicating MI.

P-214 Clinical Impact of Heterozygous Carrier of Autosomal Recessive Hypercholesterolemia on Asymptomatic Hyperlipidemic Patients

Velocity (baPWV) and conventional coronary risk factors. The relationship between this new parameter and coronary artery sclerosis was evaluated in comparison with other indexes. Results: CAD patients were characteristic of more hypertensive and diabetic with higher CAVI and baPWV than nonCAD subjects. Multivariate logistic analysis after adjustment for hypertension, diabetes mellitus, Dyslipidemia, current smoke, sex and age, still verified that CAD risk was significantly related to CAVI (OR; 3.60, for the difference between the first and the fourth quartile [95% Confidence Interval; 1.60 8.10, p = 0.002]), whereas the same relationship with baPWV statistically diminished (OR; 1.99, 95% Confidence Interval; 0.88 4.49, p = 0.097). CAVI also showed a more remarkable ability to predict CAD than baPWV and age among patients without hypertension and diabetes mellitus under age of 70 (CAVI; OR = 3.98, p = 0.020, baPWV; OR = 0.90, p = 0.631, age; OR = 1.15, p = 0.043). Conclusions: CAVI is more useful in predicting coronary artery stenosis than baPWV in all CAD-suspected subjects and even in their relative low-risk groups, which may lead to a possibility of CAVI for helping detecting CAD among those who are not easily screened out by coronary risk factors and baPWV.