Mutation screening of the APOA5 gene in subjects with coronary artery disease.

OBJECTIVE: Hyperlipidemia is a risk factor for coronary artery disease (CAD). Apolipoprotein A5 (APOA5) is a member of the apolipoprotein APOA1/C3/A4/A5 gene cluster and a major determinant of plasma triglyceride levels in the population. Various studies have identified a number of common (APOA5 c.56C>G; p.S19W; rs 3135506 ) and rare variants in the APOA5 gene in individuals with hypertriglyceridemia. However, little is known on the impact of rare APOA5 mutations for the risk of coronary artery disease; therefore, we screened the APOA5 gene in subjects with CAD. METHODS: The coding region of the APOA5 gene was screened in 501 subjects (334 with CAD and 167 CAD-free) undergoing diagnostic coronary angiography by denaturing gradient gel electrophoresis. RESULTS: APOA5 p.S19W variant c.56 C>G was found in a total of 61 subjects, five of them homozygous. Beside this well-known mutation, the denaturing gradient gel electrophoresis screening identified only one subject with a synonymous APOA5 mutation, c.70C>A; p.R24R. APOA5 p.S19W was more frequent in patients with CAD (CAD, 14.4%; no CAD, 7.8%; P = 0.021); and in addition, all homozygous subjects (n = 5) for APOA5 p.S19W had CAD. Furthermore, carriers of the p.19W allele had significantly higher triglyceride levels (240 ± 149 vs 185 ± 118 mg/dL; P < 0.01). CONCLUSIONS: From these data, we conclude that (1) APOA5 p.S19W is a common variant, with very few additional APOA5 gene mutations; (2) APOA5 p.S19W plays a major role in triglyceride metabolism; and (3) APOA5 p.S19W is a CAD risk factor.

Genetic variation at chromosome 1p13.3 affects sortilin mRNA expression, cellular LDL-uptake and serum LDL levels which translates to the risk of coronary artery disease.

BACKGROUND: A single nucleotide polymorphism (SNP) rs599839 located at chromosome 1p13.3 has previously been associated with risk of coronary artery disease (CAD) and with serum levels of low-density lipoprotein cholesterol (LDL-C). A functional link explaining the association of SNP rs599839 with LDL-C levels and CAD risk has not yet been elucidated. METHODS: We analyzed the association of rs599839 with LDL-C in 6605 individuals across a wide age spectrum and with CAD in four case-control studies comprising 4287 cases and 7572 controls. Genome-wide expression array data was used to assess the association of SNP rs599839 with gene expression at chromosome 1p13. Finally, we overexpressed sortilin in transfected cells to study LDL-uptake in vitro. RESULTS: Each copy of the G-allele of rs599839 associated with a decrease of serum LDL-C by 0.14 mmol/L (90% confidence interval (CI) 0.09-0.17 mmol/L, p=2.6 x 10(-11)). Moreover, each copy of the G-allele associated with a 9% decrease of CAD risk (90% CI 4-14%) in the presently studied four case-control samples and with a 13% decrease (90% CI 10-17%, p=2.18 x 10(-9)) in a pooled meta-analysis including recent genome-wide association studies on CAD. The same allele was associated with higher mRNA-expression levels of the multiligand receptor sortilin (log transformed mRNA AA vs. GG=8.31 vs. 8.55; p=0.01). Overexpression of SORT1 cDNA resulted in a significant increase in LDL-particle uptake (+23%, p=0.01). CONCLUSIONS: Rs599839 associates with decreased LDL-C and a lower risk of CAD. Effects appear to be mediated by increased sortilin expression and subsequently enhanced LDL-uptake into cells.

Genetics and kinetics of familial hypercholesterolemia, with the special focus on FH-(Marburg) p.W556R.

OBJECTIVE: Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder, caused by mutations in the low density lipoprotein receptor (LDLR) gene. FH is characterized by elevated plasma LDL cholesterol, premature atherosclerosis and high risk of premature myocardial infarction. Extended work has been done to understand both, the primary genetic defect as well as the in vivo kinetic consequences of this disease. Both approaches, genetics and kinetics, are challenging but also fruitful approaches for a better understanding of this devastating disease. For this we reviewed the recent literature and used our in vitro and in vivo data on one of the most frequently occurring types of FH, the FH(Marburg) p.W556R. METHODS: To identify the primary genetic defect of the FH(Marburg) we used denaturing gradient gel electrophoresis (DGGE) mutation analysis. In vivo kinetic studies were performed in a heterozygote FH(Marburg) subject and in 5 healthy control subjects utilizing a stable isotope tracer kinetic approach with 3D-leucine. RESULTS: DGGE screening of the LDLR gene identified a tryptophan (W) to arginine (R) substitution at residue 556 (p.W556R) in the fifth conserved YWTD repeat of the LDLR-beta-propeller in FH(Marburg). In vivo kinetic studies in a heterozygote FH subject for FH(Marburg) and in 5 healthy control subjects demonstrated a severe decrease in LDL FCR and a mild increase of LDL PR in FH compared to healthy controls. CONCLUSIONS: The LDLR mutation p.W556R is a frequent and severe defect for FH. This defect has a major influence on the in vivo lipoprotein kinetics and lipid levels. In a heterozygote FH patient we found a dual defect for the increase in LDL cholesterol, namely a decrease in the fractional catabolic rate (FCR) of LDL but also an increase in LDL production rate (PR). By this a well defined, single genetic defect may have a series of different in vivo metabolic consequences which could be used for potential therapeutic approaches to this disease.

Periodontal microbiota in patients with coronary artery disease measured by real-time polymerase chain reaction: a case-control study.

BACKGROUND: Recent data have shown that periodontal disease may increase the risk of occurrence of coronary heart disease in which inflammation initiated by bacteria and their compounds might be a common causal factor. This case-control study aimed at studying the relationship between periodontal disease and coronary artery disease (CAD) based on clinical and periodontal microbiologic parameters. METHODS: A total of 90 male subjects, 48 to 80 years of age, were included in this study. Forty-five men had CAD (CAD+), which was confirmed by coronary angiography. Forty-five age-matched controls showed no history or symptoms of CAD (CAD-). All subjects underwent a clinical periodontal examination including assessment of tooth loss, probing depth, clinical attachment level, and bleeding on probing. In the CAD+ group, this examination took place 1 day before coronary angiography. Subgingival microbial samples were taken and evaluated by means of real-time polymerase chain reaction (RT-PCR) for the total amount of bacteria and the following periodontopathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra (formerly Micromonas micros), Dialister pneumosintes, and Campylobacter rectus. RESULTS: Compared to control subjects, CAD+ subjects had significantly deeper pockets (2.28 mm versus 2.96 mm; P <0.001) and greater attachment loss (2.85 mm versus 3.65 mm; P <0.001), and this difference remained statistically significant after adjusting for smoking. No significant differences were observed between cases and controls with regard to the number of teeth present. P. intermedia was the only periodontal pathogen that showed significantly higher mean counts in CAD+ subjects compared to CAD- subjects. Higher counts of total bacteria, P. micra, D. pneumosintes, and C. rectus were found in the CAD- group. CONCLUSION: The results suggest that a relationship between periodontal disease and coronary heart disease exists, although P. intermedia was the only periodontopathogen related to CAD.

Molecular basis of obesity and the risk for cardiovascular disease.

Atherosclerosis and cardiovascular disease (CVD) are the main causes of death in the Western world, for both men and women. The onset and development of diseases of the cardiovascular and cerebrovascular system are strongly dependent on multiple risk factors that promote pathologic conditions like atherosclerosis, hypertension and thrombosis. Besides genetic factors also environmental influences such as diet composition are known to be closely related to CVD. In this context obesity has been postulated as an independent cardiovascular risk factor. Data from the Framingham Heart Study have consistently shown that increasing degrees of obesity are accompanied by greater rates of CVD. At present, obesity affects 10-35% of the European and US population and increases steadily. As obesity is a serious health problem which promotes metabolic abnormalities (insulin resistance, hyperinsulinemia and dyslipidemia) and dramatically increases the risk for CVD, this review will focus on the epidemiologic and genetic background of obesity. Furthermore, the molecular mechanisms involved in obesity development and their contribution to CVD will be discussed.

The 103I variant of the melanocortin 4 receptor is associated with low serum triglyceride levels.

CONTEXT: The melanocortin 4 receptor (MC4R) is an essential regulator of energy intake and body weight. Recently, the V103I polymorphism of MC4R has been shown to be negatively associated with body mass index. This suggests that serum lipids and blood pressure in individuals carrying the 103I allele might be influenced as well. OBJECTIVE: The objective of this study was to determine whether the most common polymorphism of the MC4R, V103I, affects serum lipid levels and/or blood pressure. DESIGN, SETTING, AND PARTICIPANTS: The study participants were 1173 consecutive patients undergoing cardiac catheterization; they were genotyped for the rs2229616 G-->A substitution at codon 103 (V103I polymorphism) of the MC4R gene. Patients had strictly fasted for at least 12 h before blood samples were drawn. The average age of the patients was 60.9 yr; 72% were males. MAIN OUTCOME MEASURES: The main outcome measures were body mass index, serum lipids, aortic and systolic blood pressure, and MC4R polymorphism V103I. RESULTS: Heterozygous carriers of the 103I allele had significantly lower triglyceride levels than individuals homozygous for the wild-type allele (127 vs. 168 mg/dl mean total triglyceride; P = 0.001 or 0.009 after Bonferroni adjustment for seven tests). No homozygous carriers of the 103I allele were present in the study population. CONCLUSIONS: Our study suggests an influence of MC4R activity on triglyceride levels in cardiovascular patients.

Lipids and lipoproteins in women.

Atherosclerosis and coronary artery disease (CAD) are the main causes of death in the Western world, for both men and women. However, in premenopausal women CAD is less frequent than in men, but in elderly women (e.g., > 75 years) myocardial infarction (MI) occurs even more often than in men. In summary, women suffer from CAD and MI but at a later age than men. Therefore it is important to observe and compare the cardiovascular risk factors in women and men. The typical CAD risk factors such as hyperlipidemia, smoking, arterial hypertension, diabetes mellitus, obesity, physical inactivity, and unhealthy nutrition are increasingly important for both genders. Many of these factors are comparable between men and women, but due to hormonal influences especially the lipoprotein metabolism shows some striking differences between men and women, but interestingly enough also between pre- and postmenopausal women. Therefore this paper will focus especially on the gender-specific differences in lipid metabolism as a potential target which might explain both the gender-specific and also pre- and postmenopausal differences in the occurrence of CAD.

Risk of coronary artery disease associated with polymorphism of the cytochrome P450 epoxygenase CYP2J2.

BACKGROUND: Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. METHODS AND RESULTS: All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at -50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; P=0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1+/-2.4% decrease in CYP2J2 promoter activity (P<0.01). Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP. CONCLUSIONS: A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary artery disease.

Osteoprotegerin gene polymorphisms in men with coronary artery disease.

Osteoprotegerin (OPG) antagonizes receptor activator of nuclear factor-kappaB ligand (RANKL), the principal regulator of osteoclasts. Of note, OPG-deficient mice display osteoporosis and arterial calcification. Recently, OPG gene polymorphisms have been associated with osteoporosis and early predictors of cardiovascular disease. In this study, we examined OPG gene polymorphisms in 468 men who had absence of coronary artery disease (CAD) or single-, double-, or triple-vessel disease on coronary angiography. Denaturing gradient gel electrophoresis followed by DNA sequencing revealed nucleotide substitutions 149 T-->C, 163 A-->G, 209 G-->A, 245 T-->G, 950 T-->C (all promoter), 1181 G-->C (exon 1), and 6890 A-->C (intron 4), respectively. Although single polymorphisms were not associated with CAD, linkage of polymorphisms 950 and 1181 revealed that haplotypes were overrepresented in men with CAD (chi(2) = 17.05; P = 0.03) with an increased risk of CAD in carriers of genotypes 950 TC/1181 GC and 950 CC/1181 CC (odds ratio, 1.67; 95% confidence interval, 1.02-2.72; P = 0.04). Furthermore, serum OPG levels were correlated with the presence of a C allele at position 950 (P = 0.02). In summary, linkage of genetic variations of the OPG gene at positions 950 and 1181 may confer an increased risk of CAD in Caucasian men.

A new but frequent mutation of apoB-100-apoB His3543Tyr.

ApolipoproteinB 100 (apoB-100) is an important component of atherogenic lipoproteins such as LDL and serves as a ligand for the LDL-receptor. Familial defective apolipoproteinB 100 (FDB) is caused by a R3500Q mutation of the apoB gene and results in decreased binding of LDL to the LDL-receptor. So far FDB is the most frequent and best studied alteration of apoB-100. Apart from this, three other apoB mutations, R3500W, R3531C and R3480W, affecting binding to the LDL-receptor are known to date. We screened the apoB gene segment of codons 3448-3561 by denaturing gradient gel electrophoresis (DGGE) analysis in a total of 853 consecutively sampled German patients undergoing diagnostic coronary angiography for suspected CAD. By this, a new single base mutation was detected and confirmed by DNA sequencing. The mutation, CAC(3543)TAC results in a His3543Tyr substitution in apoB-100 (H3543Y). The prevalence of heterozygotes for H3543Y in the study population was 0.47% compared to 0.12% for the known Arg 3500 Gln (R3500Q) mutation. In conclusion, the new mutation is four times more frequent than "classical" FDB and thus appears to be the most common apoB mutation in Germany.

[Excessive hyperchylomicronemia--a rare cause of acute retrosternal and epigastric pain in pregnancy]. / Exzessive Hyperchylomikronämie--eine seltene Ursache für akuten Thorax- und Oberbauchschmerz in der Schwangerschaft.

CASE REPORT: A 24-year-old woman in her 13th gestational was admitted to our department with acute retrosternal and epigastric pain. She had been transferred from the gynecologic department where she was treated for vaginal bleeding because of abortus imminens. A cardiac cause was excluded by ECG and echo. Clinical chemistry and abdominal ultrasound confirmed the diagnosis of acute pancreatitis. The woman was known in our outpatient department for hyperchylomicronemia and had already had an earlier episode of acute pancreatitis under oral contraception years ago. At current admission, triglycerides were 11,500 mg/dl. To reduce plasma triglycerides, selective lipid apheresis was performed. Apheresis was well tolerated, and the patient became free of pain within the first 30 min of treatment. Triglycerides decreased to 6,600 mg/dl at this session. Keeping to a low-fat diet (< 30 g fat per day), the patient remained healthy and completed pregnancy with the delivery of a healthy girl in her 39th week of pregnancy. CONCLUSION: Selective lipid apheresis is a safe and effective option in the treatment of hyperlipidemic pancreatitis, even in pregnant patients.

Increased osteoprotegerin serum levels in men with coronary artery disease.

Osteoprotegerin (OPG) regulates osteoclast and immune functions and appears to represent a protective factor for the vascular system. However, the role of OPG in human atherosclerosis has not been evaluated. In this study, we assessed OPG serum levels in 522 age-matched men who, on the basis of coronary angiography, had either absence of coronary artery disease (CAD) or presence of single-vessel disease, double-vessel disease, or severe triple-vessel disease. OPG serum levels were positively correlated with age (r = 0.28; P < 0.001) and were higher in men with diabetes mellitus (P < 0.01). OPG serum levels in men without CAD were 5.4 +/- 2.0 pmol/liter, compared with 6.1 +/- 2.1 pmol/liter in single-vessel disease (P < 0.005), 5.9 +/- 2.4 in double-vessel disease (P < 0.05), and 6.3 +/- 2.3 pmol/liter in triple-vessel disease (P < 0.001). Moreover, OPG serum levels were positively correlated with the severity of CAD as determined by a CAD scoring system (r = 0.17; P < 0.01). In conclusion, our data underline that OPG serum levels are associated with the severity of CAD and are increased in elderly men and patients with diabetes mellitus. We conclude that increased OPG serum levels may reflect advanced cardiovascular disease in men.

Molecular mechanisms involved in atherosclerosis.

BACKGROUND: Research in atherosclerosis is a good example how helpful different disciplines such as clinicians, epidemiologists and basic science can collaborate. In recent years our knowledge on cellular and subcellular mechanisms involved in initiation and progress of atherosclerosis has expanded due to the shared knowledge of different disciplines and thanks to new technologies in molecular biology. PATHOPHYSIOLOGY OF LDL AND HDL METABOLISM: The understanding of the molecular basis of inborn errors of LDL metabolism - such as familial hypercholesterolemia due to a defect of the LDL receptor - provided us new insights in physiology and pathophysiology of LDL metabolism. Most recently we have learned much about the vasoprotective HDL cholesterol. HDL is the major player in reverse cholesterol transport and some of its receptors such as ABCA1 and SR-BI were identified. This knowledge gives us a deeper understanding of the complex system which performs reverse cholesterol transport from peripheral tissue and the vessel wall back to the liver. PLAQUE FORMATION: Furthermore the process of formation and progression of the atherosclerotic plaque has been the focus of recent research. The stability or instability of plaques is depending on the complex interaction of adhesion molecules, monocytes, macrophages, endothelial cells, cytokines, transmitters and proteinases. Since we are unable to prevent plaque formation completely, the stabilization of plaques is a major goal for the coming years. Despite some success (such as the use of statines and ACE inhibitors) there is still a long way to go.