Abstract: Serum uric acid concentrations are correlated with gout and clinical entities such as cardiovascular disease and diabetes. In the genome-wide association study KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K (n = 1,644), the most significant SNPs associated with uric acid concentrations mapped within introns 4 and 6 of SLC2A9, a gene encoding a putative hexose transporter (effects: -0.23 to -0.36 mg/dl per copy of the minor allele). We replicated these findings in three independent samples from Germany (KORA S4 and SHIP (Study of Health in Pomerania)) and Austria (SAPHIR; Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk), with P values ranging from 1.2 x 10(-8) to 1.0 x 10(-32). Analysis of whole blood RNA expression profiles from a KORA F3 500K subgroup (n = 117) showed a significant association between the SLC2A9 isoform 2 and urate concentrations. The SLC2A9 genotypes also showed significant association with self-reported gout. The proportion of the variance of serum uric acid concentrations explained by genotypes was about 1.2% in men and 6% in women, and the percentage accounted for by expression levels was 3.5% in men and 15% in women.

Abstract: PURPOSE: Vascular endothelial growth factor (VEGF) is a key regulator of tumor-induced angiogenesis and is required for growth of tumors. We tested the hypothesis that VEGF gene polymorphisms may be associated with breast cancer. EXPERIMENTAL DESIGN: We performed a case-control study including 804 female incident breast cancer patients and 804 female age-matched healthy control subjects. We selected seven VEGF candidate polymorphisms and determined genotypes by 5'-nuclease (TaqMan) assays. Furthermore, VEGF plasma levels and genotypes were analyzed in a group of 81 healthy volunteers (64 men and 17 women). RESULTS: Haplotype analysis showed two separate blocks of high-linkage disequilibrium, formed by five polymorphisms upstream of the coding sequence (promoter and 5' untranslated region) and two polymorphisms downstream of the coding sequence. None of the single polymorphisms or haplotypes was significantly associated with the presence of breast cancer. After Bonferroni correction for multiple testing, only one statistical signifcant association between VEGF genotypes and haplotypes and tumor characteristics was observed (-634C allele and small tumor size; p < 0.001). In a multivariate regression analysis including sex, age, VEGF genotypes, and haplotypes as covariates and VEGF plasma level as dependent variable, none of the VEGF polymorphism or haplotypes was a significant predictor of VEGF plasma levels. CONCLUSIONS: Our findings do not support the hypothesis that VEGF polymorphisms are associated with breast cancer risk. The association of the VEGF -634C allele with small tumor size is in clear contrast to a previous publication and should be interpreted with caution until replicated by additional studies.

Abstract: OBJECTIVE: A previous epidemiological study showed an association of the insulin-induced gene 2 (INSIG2) gene with BMI. Additionally, experimental investigations in animals and cell culture provided evidence that this gene might be involved in lipoprotein and free fatty acid (FFA) metabolism. Therefore, the aim of this study was to examine the association between the rs7566605 variant near the INSIG2 gene and BMI and to extend it to other quantitative measures of obesity, as well as parameters of lipoprotein and FFA metabolism. METHODS AND PROCEDURES: We genotyped rs7566605 in a group of severely obese white patients (n = 1,026) with an average BMI of 46.0 kg/m(2) and a control group (n = 818) from Utah, as well as in the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study from Austria, which is based on a healthy working population (n = 1,696). RESULTS: We observed no difference in the genotype frequency of rs7566605 of INSIG2 between obese subjects and population-based controls from Utah. Furthermore, we did not find evidence of an association with measures of body composition (BMI, waist, waist-to-hip ratio, percentage body fat, amount of visceral and subcutaneous abdominal adipose fat) or lipoprotein metabolism (total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, triglycerides, and FFAs) in the Utah study population or in the independent SAPHIR study. DISCUSSION: Our results do not support an association of the INSIG2 gene with the regulation of body weight or parameters related to lipoprotein metabolism.

Abstract: BACKGROUND: Among other matrix metalloproteinases (MMPs), gelatinase B (MMP-9) is discussed to be associated with the pathogenesis of vascular diseases. Two single nucleotide polymorphisms (SNPs) of the MMP-9 gene, C-1562T in the promoter region and a G/A transition in exon 6 (R + 279Q), have been addressed in previous association studies which, however, produced conflicting results. MATERIAL AND METHODS: A novel multiplex RealTime PCR protocol for the fast and simultaneous detection of both polymorphisms is presented, which was used for genotyping 1737 participants of a prospective study investigating genetic factors influencing the progression of atherosclerosis. RESULTS: Haplotype analysis revealed -1562C/+279Q as the major haplotype in this population. Allelic distribution of the C-1562T polymorphism was consistent with data published for similar cohorts; however, we found that R + 279Q allelic distribution appears to vary significantly among Caucasian populations. Considering clinical data available from 1487 participants, we found significant associations between the presence of atherosclerotic plaque and the CA-haplotype in men (P = 0.028, phi = 0.08), and between the AG variant of exon 6 and common carotid artery intima-media thickness (CIMT) in women (P = 0.004, Eta(2) = 0.019). CONCLUSIONS: In summary, our results demonstrate associations of MMP-9 genotypes with different stages of carotid atherosclerosis.

Abstract: BACKGROUND AND PURPOSE: The independent contribution of insulin resistance to atherosclerosis is still under debate. We compared associations of 2 different indices of insulin resistance, the Homeostasis Model Assessment (HOMA) index and kITT from a short insulin tolerance test with carotid atherosclerosis. METHODS: A total of 1771 middle-aged white patients were investigated. Intima media thickness (IMT) and extent of carotid atherosclerosis were quantified by ultrasound. HOMA was calculated and an insulin tolerance test was performed. RESULTS: HOMA and kITT were significant predictors for average carotid IMT (P<0.001). After adjustment for age and the components of the metabolic syndrome, HOMA still remained an independent predictor for IMT(avg) (P=0.02), whereas kITT failed to do so. HOMA and kITT were also predictive (P=0.004 and P=0.024) for carotid plaques and extent of carotid atherosclerosis (P<0.001). After adjustment for age and the components of the metabolic syndrome, neither HOMA nor kITT were independently predictive any more. CONCLUSIONS: Our results provide evidence that HOMA rather than kITT is associated with carotid atherosclerosis and that the association is largely explained by the clustered expression of the components of the metabolic syndrome.

Abstract: The metabolic syndrome is associated with low high-density lipoprotein-cholesterol (HDL-C) and decreased low-density lipoprotein (LDL) particle size. The Taq1B-polymorphism in the cholesteryl ester-transfer protein (CETP)-gene influences HDL-C, CETP concentration, and LDL-size. We investigated the effect of the Taq1B-polymorphism on the risk of the metabolic syndrome in 1,503 participants (973 men, 530 women) of the Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk study. CETP concentration was determined in a subgroup (n = 486) by an enzyme-linked immunosorbent assay. Prevalence of the metabolic syndrome was 16.7% (18.5% in men, 13.5% in women). The Taq1B-polymorphism influenced significantly CETP concentrations, HDL-C levels, and LDL-size (P < 0.001 for all). The relative risk of the metabolic syndrome was reduced by 32% (odds ratio (OR) 0.68 (95% CI: 0.51-0.89), P = 0.005) in carriers of the B2 variant. This risk reduction persisted after adjustment for age and sex (OR 0.69 (0.53-0.92), P = 0.01) and after further adjustment for body mass index, waist-to-hip ratio, blood pressure, insulin resistance (IR), HDL-C, and triglycerides (TGs) (OR 0.43 (0.26-0.72), P = 0.001). Furthermore, the risk reduction was more pronounced in men than in women. We conclude that CETP plays an important role in the metabolic syndrome, possibly involving novel functions of CETP.

Abstract: CONTEXT: The sterol regulatory element-binding protein-1c (SREBP-1c) is a transcription factor involved in the regulation of lipid and glucose metabolism and has been implicated in the pathophysiology of type 2 diabetes mellitus (T2DM). OBJECTIVE: We aimed to confirm associations of the SREBF-1 gene with T2DM in an Austrian population and to study possible associations with diabetes-related quantitative traits. DESIGN, SETTINGS AND PARTICIPANTS: We genotyped a diabetic cohort (n=446) along with a control group (n=1524) for a common C/G variation that is located in exon 18c (rs2297508) and has been associated with obesity and T2DM in French populations. MAIN OUTCOME MEASURES: Body mass index (BMI), indices of insulin sensitivity and beta-cell function, plasma adiponectin, T2DM and single-nucleotide polymorphism rs2297508. RESULTS: Genotype distributions associated with rs2297508 differed by T2DM status (P=0.0045), but not by BMI. The variant G allele was associated with a modest, but significant, increase in the prevalence of T2DM after adjustment for age, sex and BMI (G/G: odds ratios (OR) (95% confidence intervals)=1.45 (0.99-2.11) and G/C: OR=1.37 (1.04-1.81)). In a cross-sectional population of non-diabetic subjects, associations of rs2297508 genotypes with plasma adiponectin levels adjusted for age, sex and BMI (P=0.0017) were observed in that the risk G/G genotype displayed the lowest adiponectin levels. CONCLUSIONS: We observed associations of rs2297508 with T2DM prevalence and plasma adiponectin. SREBP-1c has been implicated in the regulation of adiponectin gene expression. Our results therefore raise the possibility that sequence variations at the SREBF-1 gene locus might contribute to T2DM risk, at least in part, by altering circulating adiponectin levels.

Abstract: BACKGROUND: Increased plasma homocysteine (HCY) has been suggested as a novel risk factor for chronic heart failure (CHF). This study investigated the effect of a HCY lowering therapy by folic acid (FA) supplementation on N-terminal pro-brain natriuretic peptide (NT-proBNP) in healthy subjects. METHODS: We treated 61 healthy individuals (median age [25th-75th percentile]: 57 [53-69] years with placebo, 0.4, 1.0 or 5.0 mg FA daily for 2 months. Fasting blood samples were taken after 0, 4 and 8 weeks. Serum HCY, folate, vitamin B12 and NT-proBNP were studied. RESULTS: Baseline HCY, folate and NT-proBNP levels were 13.6 (10.0-16.4) micromol/L, 5.0 (3.8-6.4) microg/L and 40.4 (21.8-67.3) ng/L, respectively. Serum folate increased during supplementation (4 weeks of placebo, 0.4, 1.0, 5.0 mg of FA: -9%, +131%, +150%, +314%; 8 weeks: -72%, +152%, +185%, +62%) and HCY decreased (4 weeks: +2%, -12%, -20%, -15%; 8 weeks: -2%, -9%, -17%, -15%) in the treatment groups. NT-proBNP did not change within groups. Pooling FA treated subjects, individuals with a baseline NT-proBNP above the median of 40 ng/L exhibited a 20% decrease of NT-proBNP (significant on a 10% level) while HCY decreased by 15%. CONCLUSION: FA supplementation with doses between 0.4 and 5 mg does not affect NT-proBNP in healthy subjects with an NT-proBNP concentration <40 ng/L, but possibly lowers NT-proBNP in individuals with NT-proBNP levels >40 ng/L.

Abstract: Regular endurance exercise has favorable effects on cardiovascular risk factors. However, the impact of an exercise-induced change in aerobic fitness on blood lipids is often inconsistent. The purpose of this study was to investigate the effect of nine consecutive months of training on aerobic fitness and blood lipids in untrained adults. Thirty subjects 35-55 years of age (wt: 73.1 +/- 13.6 kg, height 171.1 +/- 9.0 cm, %body fat 24.6 +/- 6.3%, 14 males and 16 females) were randomly assigned to an exercise (EG) (N = 20) and control (CG) (N = 10) group. All subjects completed an incremental treadmill test, anthropometric measurements, and venous blood sample collection before and after the 9 months of exercise. Participants in the exercise group were supervised and adjusted for improvements in running performance, whereas no change was administered for the control group. One-way and multivariate ANOVA was conducted to determine significant differences in means for time and group in selected variables [body mass, % body fat, BMI; VO(2peak), km/h at 2.0 (v-LA2) and 4.0 (v-LA4) mmol l(-1) blood lactate (LA) concentration, km/h of the last load (v-max); TC, LDL-C, HDL-C, TG, Apo B, Apo A-1, and Lp (a)]. Correlation coefficients and multivariate regression analysis was used to determine the association between aerobic fitness and blood lipids. The exercise group improved significantly (P < 0.0001) in VO(2peak), v-LA2, v-LA4, v-max and exhibited a significant decrease in Apo B (P < 0.04) compared to the control group (NS). In 9 months, E achieved 24% increase in VO(2peak) and 18% reduction in Apo B, denoting the impact of cardiovascular fitness on cardiovascular risk.

Abstract: BACKGROUND: Analysis of QT duration limited to a single heart rate correction formula might be problematic in cross-sectional studies. The aim of this study was to investigate five frequently used equations with respect to over- and under-correction of QT intervals in a middle-aged population without manifest atherosclerotic disease. MATERIAL/METHODS: A total of 1199 individuals (851 males, 348 females, aged 39 to 66 years) of the SAPHIR study (Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk) were investigated. Heart rate, RR, and QT intervals were measured automatically from resting ECGs with the 12SL ECG Analysis Program (GE Medical Systems). Five methods (Bazett, Fridericia, Framingham, Hodges, and Rautaharju) were used to correct the QT interval for heart rate. RESULTS: Females had shorter RR intervals than males (963+/-141 vs. 1022+/-158 ms, p<0.0005), with longer QT (410+/-28 vs. 404+/-28 ms, p=0.003) and QTc intervals using all five formulae (p<0.0005). No significant differences were observed between the gender groups for the QTc/RR slopes regardless of the formula. The Bazett formula performed the worst and the Fridericia the best in terms of rate adjustment success (slope B=-0.001). The optimal alpha of the best-fit equation (QTc=QT/RRalpha) was calculated as 0.326 for males and 0.328 for females. CONCLUSIONS: These results demonstrate that automatic QT measurements can be used reliably in cardiovascular prevention programs, encouraging further investigation of their clinical value in risk stratification. From an epidemiological perspective, however, the selected QT correction formula applied to a specific population has to be evaluated for appropriateness.

Abstract: BACKGROUND: The International Diabetes Federation (IDF) published a new definition of the metabolic syndrome (MetS). For this definition we compared frequency, concordance, clinical and laboratory stigmata and carotid atherosclerosis with those of the established definitions by the National Cholesterol Education Program (NCEP) and World Health Organization (WHO). MATERIALS AND METHODS: A total of 1518 subjects (943 men, 575 women) from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR), free of clinical atherosclerosis, were included in this study. To estimate insulin sensitivity two methods, i.e. homeostasis model assessment of insulin resistance (HOMA-IR) and the short insulin tolerance test, were employed. Carotid intima media thickness (IMT) and plaque extent were quantified for all subjects using high-resolution ultrasound. RESULTS: Prevalence of the MetS was 18.7% for men and 16.2% for women for the WHO definition, 18.9% and 17.0%, respectively, for the NCEP definition, and 25.8% and 19.5%, respectively, for the IDF definition. Concordance was lower between the definitions of WHO and IDF (< 50%) than between NCEP and IDF (> 67%). Compared to subjects identified by NCEP definition, subjects identified in excess by IDF (3.1-11.7%) showed less insulin resistance and lower IMT and plaque extent indistinguishable from MetS-free subjects. CONCLUSIONS: Our data suggest that the IDF definition includes subjects as MetS sufferers above these detected by NCEP or WHO, who exhibit considerably less insulin resistance and carotid atherosclerosis blurring the distinction between health and disease.

Abstract: Apolipoprotein A5 (APOA5) gene variants were reported to be associated with two components of metabolic syndrome (MetS): higher TG levels and lower HDL levels. Moreover, a recent Japanese case-control study found variant -1131T>C associated with MetS itself. Thus, our study systematically analyzed the APOA5 gene for association with lipid parameters, any other features of MetS, including waist circumference, glucose-related parameters, blood pressure, uric acid, and MetS itself in Caucasians. Ten polymorphisms were analyzed in a large fasting sample of the population-based Cooperative Health Research in the Region of Augsburg (KORA) survey S4 (n = 1,354; southern Germany) and in a second fasting sample, the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1,770; Austria). Minor alleles of variants -1131T>C, -3A>G, c.56C>G, 476G>A, and 1259T>C were significantly associated with higher TG levels in single polymorphism (P < 0.001) and haplotype (P <or= 6.6 x 10(-6)) analysis. Besides associations with lower HDL levels in SAPHIR (P <or= 0.001), there were no significant findings with any other features of MetS. Variant c.56C>G was associated with higher risk for MetS [odds ratio (95% confidence interval) = 1.43 (1.04, 1.99), P = 0.03 for KORA and 1.48 (1.10, 1.99), P = 0.009 for SAPHIR). Our study confirms the association of the APOA5 locus with TG and HDL levels in humans. Furthermore, the data suggest a different mechanism of APOA5 impact on MetS in Caucasians, as variant c.56C>G (not analyzed in the Japanese study) and not -1131T>C, as in the Japanese subjects, was associated with MetS.

Abstract: INTRODUCTION: The metabolic syndrome is a matter of immense public concern for atherosclerosis prevention. Key features are visceral obesity, dyslipidemia, hyperglycemia in the non-diabetic range, and arterial hypertension. Subclinical atherosclerosis is the clinical consequence of metabolic syndrome, which may influence the QT interval. The aim was to investigate the rate corrected QT interval in subjects with metabolic syndrome in comparison to those without cardiometabolic risk factor clusters, and to explore gender differences in cardiac repolarization between the two groups. PATIENTS, MATERIALS AND METHODS: Heart rate and QT interval were automatically measured from surface ECG in 1086 participants (767 men, 319 women) from the Salzburg-Atherosclerosis-Prevention-program-in-subjects-at-High-Individual-Risk (SAPHIR). To omit the QT adjustment bias inherent in Bazett's formula we used a QT adjustment method with linear scaling as described by Rautaharju. RESULTS: The prevalence of metabolic syndrome was 13.8% among males and 10% among females. Mean rate adjusted QT (QTa) intervals were longer in women than in men. Presence of metabolic syndrome, however, was associated with significantly prolonged QTa only in men but not in women. Adjustment for relevant confounders reduced the difference of mean QTa in men from 9.24 to 5.83 ms (95% CI 0.9-10.8), but this difference was still statistically significant (p = 0.021). The effect of metabolic syndrome on QTa was only partly mediated by hypertension and insulin resistance. In females, however, no relevant differences were detected for QTa interval between subjects categorized by presence or absence of metabolic syndrome. CONCLUSIONS: The findings indicate a significant association between metabolic syndrome and rate-invariant QT in middle-aged men after adjustment for other risk factors. QT measurement may provide additive diagnostic and prognostic information in populations undergoing cardiovascular risk screening. However, the effect of metabolic and hormonal factors on ventricular repolarization seems to differ between the sexes.

Abstract: BACKGROUND: Recently, increased plasma homocysteine (HCY) has been suggested as a novel independent risk factor for osteoporotic fractures. This study aimed to analyze the effect of a HCY lowering therapy by folic acid (FA) supplementation on biochemical bone markers in healthy subjects. MATERIAL AND METHODS: We treated 61 healthy individuals (mean age: 58+/-8 years) with placebo, 0.4, 1 or 5 mg FA daily for 2 months. Fasting blood samples were taken after 0, 4 and 8 weeks. Serum HCY, folate, vitamin B12, osteocalcin (OC), procollagen type I N-terminal propeptide (PINP) and C-terminal telopeptides of human collagen type I (CTX) were studied. RESULTS: Overall baseline HCY and folate levels were 13.4 +/- 3.6 micromol/L and 5.7 +/- 3.0 microg/L, respectively. Participants exhibited normal baseline OC, PINP and CTX levels. Serum folate increased during supplementation (4 weeks of placebo, 0.4, 1 and 5 mg of FA: -7, +160, +162 and +436 %; 8 weeks: -6, +305, +340 and +216 %) and HCY decreased (4 weeks of placebo, 0.4, 1 and 5 mg of FA: +2, -14, -21 and -17 %; 8 weeks: +2, -8, -20 and -17 %) in the treatment groups, but not in the placebo group. OC (placebo: 22.8 vs. 23.0 vs. 23.6; 0.4 mg FA: 21.6 vs. 22.1 vs. 24.1; 1 mg FA: 23.7 vs. 22.6 vs. 23.4; 5 mg FA: 24.1 vs. 20.5 vs. 20.9 microg/L), PINP (placebo: 43.5 vs. 51.3 vs. 46.5; 0.4 mg FA: 34.0 vs. 34.1 vs. 39.5; 1 mg FA: 43.6 vs. 39.7 vs. 43.2; 5 mg FA: 41.1 vs. 38.7 vs. 37.4 microg/L) and CTX (placebo: 258 vs. 360 vs. 321; 0.4 mg FA: 229 vs. 290 vs. 315; 1 mg FA: 319 vs. 325 vs. 301; 5 mg FA: 293 vs. 321 vs. 304 ng/L) did not change throughout the study. CONCLUSION: Short-term FA supplementation does not affect biochemical bone markers in non-osteoporotic subjects with a low folate status.

Abstract: Cyclooxygenase-2 (COX-2) is involved in carcinogenesis, immune response suppression, apoptosis inhibition, angiogenesis, and tumor cell invasion and metastasis. The gene for COX-2, designated as PTGS2, carries a common polymorphism at position 8473 in the 3'-untranslated region (PTGS2 8473T>C), which has been associated with susceptibility to malignant disease. To investigate the role of this polymorphism for breast cancer, we determined the prevalence of PTGS2 genotypes in 500 women with breast cancer and 500 sex- and age-matched healthy control subjects. Homozygous carriers of the 8473-CC genotype were more frequent among patients (12.4%) than among controls (6.6%; P = 0.002). The odds ratio for carriers of this genotype for breast cancer was 2.1 (95% confidence interval, 1.3-3.3). Among patients, estrogen receptor positivity was less frequent among carriers of a CC genotype (63.9%) than among carriers of a TT or TC genotype (76.9%; P = 0.028). Tumor size, histologic grade, presence of primary lymph node metastases, progesterone receptor positivity, or age at diagnosis were not associated with PTGS2 genotypes. We conclude that the homozygous PTGS2 8473-CC genotype may be associated with breast cancer risk.

Abstract: Low concentrations of adiponectin, the protein product of the APM1 gene, have been reported to be associated with obesity and insulin resistance. However, contrasting results have been described on the genetic variability in APM1 and characteristics of the metabolic syndrome and adiponectin serum concentrations. In the present study, we investigated the association of the two most well-known SNPs of APM1 (+45T>G and +276G>T) and their haplotypes, with serum adiponectin concentrations, metabolic parameters and intima-media thickness of the carotid arteries in 1,745 well-phenotyped asymptomatic unrelated Caucasian subjects of the SAPHIR cohort. The common T-allele (88.5%) of SNP +45T>G and the common G-allele (70.5%) of SNP +276G>T were associated with significantly lower serum adiponectin levels (P = 0.0008 and P = 0.00005, respectively). The most frequent haplotype TG (59.0%) defined by both loci showed a strong association with lower serum adiponectin concentrations (P = 0.000000002). A clear effect per copy of the respective haplotype was observed. This association was most pronounced in lean and insulin-sensitive subjects. The two less common haplotypes TT (29.5%) and GG (11.5%) were associated with higher serum adiponectin levels in a dose-dependent association. Interestingly, no significant association between the adiponectin 45-276 haplotypes and the majority of parameters of the metabolic syndrome or intima-media thickness of the carotid arteries was found in our study. In summary, we replicated a strong association of the adiponectin 45-276 genotypes and haplotypes with adiponectin levels in healthy Caucasians. However, we could not confirm an association of this gene locus with metabolic parameters of the insulin resistance syndrome.

Abstract: Integrins are cell surface receptors, which mediate cell-to-cell and cell-to-extracellular matrix adhesion. Some of them, e.g. alpha(V)beta(3), alpha(IIb)beta(3) and alpha(2)beta(1), have been suggested as key players for cancer development and tumor metastasis. Two polymorphisms in the gene for the alpha(2) component, ITGA2 807C>T and 1648G>A, have been associated with the cell-surface density of integrin alpha(2)beta(1). The 176T>C polymorphism in the ITGB3 gene, encoding the beta(3) subunit of integrins alpha(IIb)beta(3) and alpha(V)beta(3), modifies a variety of traits of beta(3) expressing cells. To analyze the role of ITGA2 and ITGB3 polymorphisms for breast cancer risk and prognosis, we performed a case-control study including 500 female breast cancer patients and 500 healthy female age-matched control subjects. All study participants were of Caucasian origin (Austria, Middle-Europe). The ITGA2 1648_AA genotype was significantly associated with breast cancer (odds ratio 3.12; 95% confidence interval 1.11-8.77). Carriers of the most common ITGA2 haplotype (807C_1648G, 'wildtype') were at decreased risk for breast cancer (odds ratio 0.72; 95% confidence interval 0.53-0.98). A histological grade of 3 or 4 was found more often in ITGA2 807TT subjects (p=0.039 compared to CC+CT genotypes) and carriers of an ITGA2 1648A allele (p=0.017 compared to GG genotype). Carriers of the ITGA2 807C_1648G haplotype were less likely to have a histological grade 3 or 4 compared to non-carriers (p=0.003). The ITGB3 176T>C polymorphisms was not associated with breast cancer susceptibility. In a Cox-regression analysis, carriers of the homozygous ITGB3 176-CC genotype had a higher risk for metastasis (relative risk 2.3; 95% CI 1.3-4.2; p=0.005). We conclude that functional polymorphisms in integrin genes ITGA2 and ITGB3 influence the development and progression of breast cancer, respectively. The precise mechanism remains to be determined, but likely involves dysregulated signaling pathways.

Abstract: BACKGROUND: Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. METHODS: We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. RESULTS: Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. CONCLUSION: This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Abstract: The associations of the adiponectin (APM1) gene with parameters of the metabolic syndrome are inconsistent. We performed a systematic investigation based on fine-mapped single nucleotide polymorphisms (SNPs) highlighting the genetic architecture and their role in modulating adiponectin plasma concentrations in a particularly healthy population of 1,727 Caucasians avoiding secondary effects from disease processes. Genotyping 53 SNPs (average spacing of 0.7 kb) in the APM1 gene region in 81 Caucasians revealed a two-block linkage disequilibrium (LD) structure and enabled comprehensive tag SNP selection. We found particularly strong associations with adiponectin concentrations for 11 of the 15 tag SNPs in the 1,727 subjects (five P values <0.0001). Haplotype analysis provided a thorough differentiation of adiponectin concentrations with 9 of 17 haplotypes showing significant associations (three P values <0.0001). No significant association was found for any SNP with the parameters of the metabolic syndrome. We observed a two-block LD structure of APM1 pointing toward at least two independent association signals, one including the promoter SNPs and a second spanning the relevant exons. Our data on a large number of healthy subjects suggest a clear modulation of adiponectin concentrations by variants of APM1, which are not merely a concomitant effect in the course of type 2 diabetes or coronary artery disease.

Abstract: The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n = 1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility.

Abstract:

Abstract: The purpose of this study was to measure changes in plasma adiponectin (ApN) over 24 months of exercise intervention in middle age adults with a predisposition to metabolic syndrome and to determine if changes in ApN were more affected by physical activity or physical fitness. Thirty-six subjects completed a 24 months home-based exercise program (cycling>or=three times per week, >or=45 min/session at 50-65% of VO2peak). Body composition, blood samples, and physical fitness were studied at baseline and after 12 and 24 months of participation in the study. The prescribed physical activity was monitored via self-reported exercise diary to determine MET levels, hours, and exercise compliance. Two-tailed repeated measures ANOVA and Spearman Rank Correlation Coefficients were used to detect significant differences and associations between the variables. ApN increased significantly (P<0.05) after 12 months in males (n=17; 5.3+/-1.9-7.0+/-3.0 microg ml-1) but not in females (n=9; 8.6+/-3.8-11.5+/-4.0 microg ml-1). The net change in ApN over 24 months was significantly correlated to the net change in VO2peak (physical fitness) (r=0.66; P<0.001), whereas exercise intensity was negatively correlated to DeltaApN over 12 months (r=-0.4; P<or=0.04) and 24 months (r=-0.45; P<or=0.02). Based on our results, an improvement in cardiorespiratory fitness of 15% increased plasma ApN concentration. Our findings suggest that moderate physical activity performed over many months induces positive changes in the plasma ApN concentration in adults with a predisposition to metabolic syndrome.

Abstract: BACKGROUND AND PURPOSE: Peroxisome proliferator activated receptor gamma coactivator 1alpha (PGC-1alpha, PPARGC1A) integrates the transcriptional program of mitochondrial biogenesis. Mitochondria are the main source of cellular reactive oxygen species implicated in atherogenesis. We therefore ascertained associations of PPARGC1A polymorphisms with asymptomatic carotid atherosclerosis. METHODS: Eight single nucleotide polymorphisms tagging two haplotype blocks within PPARGC1A were studied in 1379 participants of the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk. Early atherosclerosis was assessed by intima-media thickness and extent of plaques (B-score) of the carotid arteries. RESULTS: No associations of carotid artery intima-media thickness measurements with block 1 or 2 haplotype distributions or individual haplotypes were observed. However, the block 1 haplotype carrying the variant C nucleotide at -3974 relative to the transcription start site was associated with disease status defined by the presence of more than one minimal lesion and the -3974 C allele was associated with decreased risk (odds ratio=0.60, P=0.007) after adjustment for linkage disequilibrium between single nucleotide polymorphisms. CONCLUSIONS: These result are consistent with the concept that risk factors for distinct carotid phenotypes may vary and suggest, but do not prove, that PGC-1alpha may contribute to the regulation of atherogenic pathways.

Abstract: OBJECTIVE: Low high-density lipoprotein cholesterol (HDL-C), hypertriglyceridemia, and small dense-low density lipoprotein (LDL) are key components of metabolic syndrome (MS). Cholesteryl ester transfer protein (CETP) mediates the transfer of triglycerides (TGs) from TG-rich lipoproteins to HDL and LDL particles in exchange for cholesteryl esters, leading to low HDL-C and small dense-LDL. The aim of this study was to investigate the role of CETP in subjects with MS. RESEARCH METHODS AND PROCEDURES: In a cross-sectional cohort of 234 middle-aged men and 252 women randomly selected from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study, MS was diagnosed according to the National Cholesterol Education Program guidelines. CETP mass was determined by enzyme-linked immunosorbent assay and LDL size-by-gradient polyacrylamide gel electrophoresis. RESULTS: Men and women with MS had lower HDL-C (45 +/- 7 vs. 58 +/- 13 and 48 +/- 10 vs. 71 +/- 14 mg/dL for men and women, respectively; p < 0.001 for all) and higher TG levels (222 +/- 71 vs. 98 +/- 54 and 167 +/- 67 vs. 90 +/- 35 mg/dL for men and women, respectively; p < 0.001 for all) than healthy subjects. LDL size was lower in subjects with MS (256 +/- 11 A vs. 267 +/- 11 A and 262 +/- 10 A vs. 273 +/- 8 A for men and women, respectively; p < 0.001 for all). CETP mass was higher in men with MS (1.87 +/- 0.78 vs. 1.40 +/- 0.65 mug/mL; p < 0.001) but not in women (1.74 +/- 0.79 vs. 1.62 +/- 0.62 mug/mL). CETP mass correlated inversely with LDL size in both men and women (r = -0.19, p < 0.01 and r = -0.13, p < 0.05 in men and women, respectively). DISCUSSION: MS is associated with increased CETP mass in men. Increased CETP mass may be responsible for reduced HDL-C and reduced LDL particle diameter in MS.

Abstract: An association between susceptibility to rheumatoid arthritis (RA) and a common -168A>G polymorphism in the MHC2TA gene with differential major histocompatibility complex (MHC) II molecule expression was recently reported in a Swedish population. The objective of the present study was to replicate this finding by examining the -168A>G polymorphism in an Austrian case-control study. Three hundred and sixty-two unrelated RA cases and 351 sex-matched and age-matched controls as well as 1,709 Austrian healthy individuals were genotyped. All participants were from the same ethnic background. Genotyping was performed using 5' allelic discrimination assays. The association between susceptibility to RA and the -168A>G single nucleotide polymorphism was examined by chi-square test. Comparison was made assuming a dominant effect (AG + GG genotypes versus AA genotype). In contrast to the primary report, the frequency of MHC2TA -168G allele carriers was not significantly different between patients and controls in the Austrian cohort. The homozygous MHC2TA -168 GG genotype was more frequent in matched controls than in Austrian RA patients. There was no association between the presence of RA-specific autoantibodies and the MHC2TA -168 GG genotype. In this cohort of Austrian patients, no association between the MHC2TA polymorphism and RA was found.

Abstract: OBJECTIVES: To investigate the relationship between cardiac repolarization (QT interval duration) and intima media thickness (IMT) of the carotid arteries as surrogate measures of subclinical atherosclerosis. DESIGN: Prospective study with consecutive subjects enrolled in the SAPHIR program (Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk). SETTING: The analysis of the material was performed at the departments of medicine and neurology of a university hospital. SUBJECTS: The study cohort comprises a population-based sample of 1199 clinically healthy subjects (851 men and 348 women; age 39-66 years). Exclusion criteria were cardiovascular disease, diabetes, atrial fibrillation, bundle branch block and use of medication affecting QT interval duration. MAIN OUTCOME MEASURES: IMT of common (CCA) and internal carotid arteries (ICA) was measured by B-mode ultrasound. QT interval duration was determined in the resting 12-lead electrocardiogram by an automatic analysis program. The QT intervals were corrected for heart rate with five standard equations (QTc-Bazett, -Fridericia, -Framingham, -Hodges and -Rautaharju) and tested for their relationship with carotid IMT after adjustment for clinical and metabolic variables. Results. Females had higher heart rates than males (64 +/- 10 b min(-1) vs. 60 +/- 9 b min(-1), P <0.0005), with longer mean QT (410 +/- 28 ms vs. 404 +/- 28 ms, P=0.003) and QTc intervals in all correction formulae (P <0.0005). Significant correlations between QT/QTc and ICA IMT (r=0.14-0.16) were found in males. In the general linear model the association between QTc (except for Bazett) and ICA IMT remained significant after adjusting for age, BMI and further cardiovascular risk factors. In females the crude correlations between QT/QTc and ICA IMT were lower than those with CCA IMT. Only the correlation between uncorrected QT and CCA IMT (r=0.15, P=0.006) remained significant after adjustment for covariates. CONCLUSIONS: The results of the present study demonstrate that QT and QTc prolongation are in part associated with IMT of carotid arteries, which is an established risk marker of subclinical atherosclerosis. In men the data support the hypothesis of an association between QTc and ICA IMT. In women a statistically significant relationship was found between the uncorrected QT interval and CCA IMT. These findings suggest that differences in carotid IMT and ventricular repolarization between genders might be related to hormonal and nonhormonal effects.

Abstract: OBJECTIVE: Reactive oxygen species (ROS) contribute to atherogenesis. Uncoupling protein 2 (UCP2) reduces mitochondrial ROS generation and protects against the disease in animal models. A common -866G/A promoter polymorphism that has been associated with obesity and beta-cell function may also affect UCP2 gene expression in cells of the arterial wall. METHODS AND RESULTS: Genotype distributions of the -866G/A and of a 45nt-del/ins polymorphism in the 3'-untranslated region of the UCP2 gene were determined in 1334 participants of the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR). We observed a modest association of the -866G/A promoter polymorphism and 2-loci haplotypes with asymptomatic carotid atherosclerosis in female study participants. Functional studies revealed increased expression of the -866G wild-type allele in human umbilical vein endothelial cells and differentiated THP-1 cells. Electrophoretic mobility shift assay studies and antibody-interference assays performed with nuclear extracts of various cell lines showed binding of cell-type specific protein complexes to the region encompassing the -866 site and suggested involvement of hypoxia inducible factor 1alpha in the regulation of UCP2 gene expression in endothelial cells and macrophages. CONCLUSIONS: Our results suggest a role of UCP2 in atherogenesis as originally proposed from studies in animal and cell culture models.

Abstract: Interleukin-10 (IL-10) is an immunosuppressive cytokine which may facilitate development of cancer by supporting tumor escape from the immune response. A [TCATA] haplotype formed by polymorphisms at positions -3575, -2763, -1082, -819 and -592 in the promoter of the IL-10 gene is a strong determinant for IL-10 expression. The presence of this haplotype can be determined by analysis of the -592C > A polymorphism. Aim of the present study was to analyze the role of the IL-10 [TCATA] haplotype for breast cancer. We performed a case-control study including 500 female patients with histologically confirmed breast cancer and 500 female, age-matched, healthy control subjects from population-based screening studies. The -592C > A polymorphism was determined by a 5'-nuclease assay (TaqMan). Frequency of the homozygous -592 AA genotype, indicating homozygosity for the [TCATA] haplotype, was 4.2% among patients and 7.3% among controls (p=0.038; odds ratio 0.56; 95% confidence interval 0.32-0.97). IL-10 genotypes were not associated with tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. Therefore we conclude that the IL-10 -592C > A promoter polymorphism may be associated with a reduced breast cancer risk.

Abstract:

Abstract: BACKGROUND AND PURPOSE: The metabolic syndrome (MetS) is associated with an increased risk for subsequent development of type 2 diabetes mellitus, cardiovascular disease, and stroke. Type 2 diabetes increases the risk of stroke and coronary heart disease in women to a greater extent than in men, and thus the question arises whether there are sex differences in the association of early atherosclerosis and MetS. METHODS: 1588 middle-aged Austrian subjects (1001 males, 587 females) were included in the present study. MetS was defined by the criteria of the National Cholesterol Education Program Adult Treatment Panel III. Early atherosclerosis was assessed by intima-media thickness (IMT) and extent of plaques (B-score) of the carotid arteries. RESULTS: B-score and carotid artery IMT parameters were significantly higher in subjects with the MetS. After adjustment for established risk factors, the difference in B-score remained significant only in women. Computed common carotid artery IMT values using general linear model equations with age, body mass index, and low-density lipoprotein cholesterol as covariates displayed the highest values for men with MetS (811.8+/-9.5 microm). Women with MetS (797.6+/-15 microm) and men without the syndrome (788.8+/-5 microm) showed similar IMTs, whereas women without the MetS presented significantly lower values (735.6+/-7 microm). Among the subcomponents of the MetS, high-density lipoprotein cholesterol showed the strongest impact on IMT in men, whereas blood glucose ranked first in women. CONCLUSIONS: The effect of MetS on early atherosclerosis is more pronounced in females. The impact of the components of MetS on carotid IMT differs between men and women.

Abstract: BACKGROUND AND PURPOSE: Adipose tissue produces and secretes a number of bioactive molecules, conceptualized as adipocytokines. Adiponectin has been identified as one of the adipocytokines, and hypoadiponectinemia was demonstrated in patients with obesity, diabetes mellitus, and coronary artery disease. Whether decreased adiponectin levels are cause or consequence is an important issue in the discussion on the association between adiponectin and atherosclerosis. In the present study, we investigated the association of plasma adiponectin levels with sonographic phenotypes of subclinical atherosclerosis, which may represent different stages of disease as well as common and distinct determinants. METHODS: A total of 1515 middle-aged healthy white subjects (940 males and 575 females) were included. Common carotid artery intima-media thickness (CIMT) and presence of atherosclerotic plaques were assessed by B-mode ultrasound. RESULTS: After adjustment for established risk factors, per 1 microg/mL decrease in adiponectin CIMT increased on the average by 3.48 microm in males (95% CI, 1.23 to 5.73 microm) and by 2.39 microm in females (95% CI, 0.50 to 4.27 microm). After dichotomizing adiponectin levels at the median and adjustment for established risk factors, the mean difference of CIMT between subjects with low and high adiponectin levels was 20.42 microm in men (95% CI, 6.80 to 34.04; P=0.003) and 20.75 microm in women (95% CI, 1.08 to 40.42; P=0.039). No significant relationship was found between adiponectin levels and presence of atherosclerotic plaques. CONCLUSIONS: Our results demonstrate an independent negative association of adiponectin levels and CIMT, whereas no relationship with presence of atherosclerotic plaques was found, thus suggesting hypoadiponectinemia as a risk factor in the development of early atherosclerosis.

Abstract: Peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator implicated in transcriptional programs of hepatic gluconeogenesis, oxidative phosphorylation, and insulin release by beta-cells. To study associations of the PGC-1alpha gene locus with carbohydrate metabolism and type 2 diabetes in humans, we identified several polymorphisms in the promoter region that were located in a haplotype block distinct from a second haplotype block containing part of intron 2 and extending beyond exon 13. Each block contained five common haplotypes. Oral glucose tolerance testing revealed associations of promoter haplotype combinations with 30- and 60-min postload plasma glucose levels, whereas haplotypes in both blocks were associated with indexes of beta-cell function. The associations of promoter haplotypes are supported by functional studies showing that some polymorphisms are located in transcription factor binding sites and affect transactivation in an allele-specific manner. By comparing patients with type 2 diabetes and control subjects, we observed borderline significant differences of four-loci haplotype distributions in the downstream haplotype block. Moreover, the haplotype that was associated with the strongest insulin response to glucose conferred the lowest risk of type 2 diabetes (P < 0.01). Thus, the PGC-1alpha gene locus influences carbohydrate metabolism and contributes to type 2 diabetes in the population studied.

Abstract: The 825C>T polymorphism in the gene for the G-protein beta3 subunit (GNB3) has been linked to the occurrence of a splice variant of GNB3 and distinct cellular and metabolic features and may be associated with malignant disease. 500 patients with histologically confirmed breast cancer and 500 female age-matched healthy control subjects were genotyped for the GNB3 polymorphism to analyze its role for breast cancer. Prevalences of GNB3 CC, CT and TT genotypes were similar among patients (49.7, 39.8, 10.5%) and controls (50.1, 42.4, 7.5%, P = 0.25). The GNB3 genotype was furthermore not linked to tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. In an exploratory analysis, carriage of a 825-T allele was associated with a longer metastasis-free period in patients with primary low-grade breast cancer, but not in those with primary high-grade breast cancer (Cox regression, P = 0.025). We conclude that the GNB3 825C>T polymorphism does not appear to be associated with breast cancer risk, but may influence development of metastasis in low-grade tumors.

Abstract:

Abstract: PURPOSE: The matrix metalloproteinase 3 (MMP3), also known as stromelysin-I, is a key-player for carcinogenesis and tumor growth. A 5A/6A promoter polymorphism is associated with differences in MMP3 activity and has been linked to cancer susceptibility in some studies. In the present study we evaluated the role of this polymorphism for breast cancer risk. EXPERIMENTAL DESIGN: A case-control study was performed including 500 patients with histologically confirmed breast cancer and 500 female, age-matched, healthy control subjects from population-based screening studies. The MMP3 5A/6A polymorphism was determined by a 5'-nuclease (TaqMan) assay. RESULTS: Prevalences of 5A/5A, 5A/6A, and 6A/6A genotypes were similar among patients (20.6, 51.8, and 27.6%, respectively) and controls (23.3, 47.3, and 29.4%, P = 0.34). The odds ratio of carriers of a MMP3 5A allele for breast cancer was 1.09 (95% confidence interval, 0.83-1.44). Patients with the 5A/5A genotype had a higher proportion of lymph-node metastases than those with a 5A/6A or 6A/6A genotype (P = 0.010). CONCLUSIONS: The MMP3 5A/6A promoter polymorphism does not appear to influence breast cancer susceptibility but may be linked to a higher risk for metastasizing among breast cancer patients.

Abstract: The cholesteryl ester transfer protein (CETP) is responsible for the exchange of triglycerides and cholesteryl esters between lipoprotein particles leading to an increased hepatic clearance of HDL-cholesteryl esters. A high CETP activity reduces serum HDL levels, whereas persons without CETP activity have high HDL levels. We investigated the association of the TaqIB CETP polymorphism and various parameters of the insulin resistance syndrome in a cross sectional population based study. We included 1029 persons without known cardiovascular disease or diabetes mellitus consecutively enrolled in our SAPHIR program (Salzburg Atherosclerosis Prevention program in persons with a High Infarction Risk). Numerous clinical and laboratory data were accomplished. Insulin sensitivity was measured by a short insulin tolerance test. The TaqIB CETP polymorphism was determined by PCR, TaqI restriction and electrophoresis. 35.2% were homozygous for the prevalence (B1B1), 46.7% were heterozygous (B1B2), and 18.1% homozygous for the absence (B2B2) of the restriction site. HDL cholesterol and apolipoprotein A1 were lower and small dense low-density lipoproteins (sdLDL) higher in B1B1 compared to B2B1 and B2B2 persons. In women, we found a significant interaction effect between CETP genotype and adiposity for HDL cholesterol. B1B1 women with a BMI and a waist circumference above the median had 9.7 mg/dl lower HDL than B1B2 and 9.1 mg/dl lower HDL than B2B2 women (P < 0.001). In men, no interaction effect but a marked genotype to HDL correlation was found. There was a high CETP effect on sdLDL detected in men (P = 0.001). B1B1 men had sdLDL in 36%, B1B2 in 24.6%, and B2B2 in only 14.5%. Men with adiposity and insulin resistance had twice as many sdLDL as insulin sensitive men. We found a significant sex specific effect of the TaqIB CETP polymorphism on the insulin resistance parameters HDL-cholesterol and sdLDL in an Austrian population based study.

Abstract: Sulfotransferase 1A1 (SULT1A1), also designated as phenol-preferring sulfotransferase, is involved in the bioactivation and detoxification of a variety of potential carcinogens, including iodothyronines, hydroxylated aromatic amines, and phenolic xenobiotics. A common arginine (R) to histidine (H) polymorphism at amino acid position 213 influences SULT1A1 activity and has been suggested as risk factor for a different types of cancers. To investigate the role of this polymorphism for breast cancer risk, SULT1A1 genotype was determined in 500 women with clinically verified breast cancer and 500 female age-matched healthy control subjects. Frequencies of heterozygous (controls: 42.5% patients: 50.2%) or homozygous (controls: 12.6%; patients: 9.4%) carriers of the 213H variant were not significantly different between groups. The SULT1A1 genotype was furthermore not associated with tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. The SULT1A1 213H variant was associated with the presence of lymph node metastases (p = 0.002). We conclude that the SULT1A1 R213H polymorphism is not a general risk factor for breast cancer, but may be involved in lymph node metastazing in breast cancer patients.

Abstract:

Abstract:

Abstract: Peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARGC1/PGC-1) is a transcriptional coactivator of nuclear hormone receptors implicated in blood pressure regulation. We therefore ascertained whether the PPARGC1 gene locus is associated with hypertension. We studied associations of 3 polymorphisms in PPARGC1 transcripts with hypertension in 683 middle-aged men and 530 middle-aged women of a cross-sectional Austrian population. Hypertension was defined by average values of systolic or diastolic ambulatory blood pressure readings (taken between 7 AM and 10 PM) above 140 and/or 90 and/or use of antihypertensive medication. Among the 3 polymorphic sites, genotype distributions associated with Gly482Ser differed by hypertension status in men (P=0.0038), but not in women. The less common Ser482 allele was associated with a modest, but significant, reduction in the prevalence of hypertension in men. The distribution of 3 loci haplotypes also differed in men with and without hypertension (P=0.015). Despite its moderate effect, but because of its high frequency (approximately 64%), the more common risk allele contributed to hypertension in 35% (95% CI 16% to 54%) of our male population. These results suggest, but do not prove, that PPARGC1 participates in blood pressure control, and sequence substitutions at its gene locus confer an increased risk of hypertension to a substantial proportion of men.

Abstract: BACKGROUND: The Gly972Arg mutation in the IRS-1 gene has been found to be associated with insulin resistance and type II diabetes. A recently published study described an association between the Arg allele and an increased risk for coronary artery disease. In the present study we asked whether the presence of the codon 972 mutation in the IRS-1 gene is associated with higher IMT values of the carotid arteries. MATERIALS AND METHODS: To address this question, genotypes of the codon 972 polymorphism were determined in 1018 healthy unrelated individuals aged 40-65 years. Three homozygous carriers of the mutation were excluded for statistical analysis. In all subjects, intima media thickness (IMT) and B-scores of carotid arteries as well as a large number of metabolic parameters were determined. RESULTS: Heterozygous carriers of the Arg972 allele exhibited significantly lower IMT and B-score values than noncarriers. Total cholesterol, LDL-cholesterol and serum levels of apolipoprotein B were significantly lower in the carriers. Furthermore, a significant interaction between Gly972Arg-carrier status and mean daytime 24-h systolic blood pressure with regard to IMT could be observed; carriers with a systolic blood pressure above the median had lower IMT values than carriers with a systolic blood pressure equal or below the median. All these effects were more pronounced in females and remained significant after adjustment for sex, age, BMI, systolic blood pressure and serum apolipoprotein B levels. No significant differences between the carriers and the noncarriers could be found for BMI, insulin sensitivity or frequency of type II diabetes. CONCLUSIONS: The results of our study demonstrate that the presence of the Arg972 allele is associated with lower IMT values of the carotid arteries. This finding is partly explained by lower serum levels of apolipoprotein B in carriers. The protective effect of the Gly972 Arg mutation seems to be stronger in the presence of a higher systolic blood pressure. Our data contradict previous findings suggesting an increased risk for insulin resistance, type II diabetes and atherosclerotic vascular disease in carriers of the mutation.

Abstract: BACKGROUND AND PURPOSE: The C825T dimorphism of the gene encoding the human G protein beta3 subunit (GNB3) is associated with hypertension and obesity. Although these findings suggest an association with insulin resistance and atherosclerosis, this hypothesis has yet been tested only partially. METHODS: To investigate this hypothesis, the C825T dimorphism was determined in a population of 932 middle-aged white subjects of middle European (Austrian) origin. Insulin sensitivity was measured with the short insulin tolerance test; intima-media thickness of the carotid artery and morphological plaque burden were measured by ultrasound. RESULTS: Insulin sensitivity was found to be significantly lower in carriers of the T allele (3.55+/-1.27 versus 3.92+/-1.30%/min, P=0.012) in the group of male subjects with abdominal body fat distribution (waist-to-hip ratio >0.9). No effect was observed in women or men with a waist-to-hip ratio <0.9. Advanced carotid artery plaques were more frequent (odds ratio, 1.606; 95% confidence interval, 1.002 to 2.575; P=0.04) in carriers of the T allele regardless of sex. No effect was observed with regard to carotid artery intima-media thickness. CONCLUSIONS: In summary, our results demonstrate that the GNB3 825T allele is associated with reduced insulin sensitivity in men with abdominal fat distribution and with more advanced carotid atherosclerosis in middle-aged white men and women.

Abstract: OBJECTIVE: CX(3)CR1 is a novel chemokine receptor located on monocytes. Recently, two polymorphisms were linked to coronary artery disease (CAD), V249I and T280M. Carriers of at least one I-allele or one M-allele were found less frequently among patients with CAD compared to controls. The aim of the present study was to investigate the influence of these polymorphisms on the development of peripheral arterial disease (PAD). METHODS: 522 human subjects with documented PAD and 522 age and sex matched controls were genotyped by polymerase chain reaction followed by restriction digestion. RESULTS: Adjusted odds ratio (OR) of carriers of the I-allele for PAD was 1.34 (95% confidential interval (CI) from 0.86 to 2.09; P=0.19). The OR associated with the M-allele for PAD was 0.65 (95% CI from 0.41 to 1.04; P=0.07), when tested in the same regression analysis with the V249I genotypes. The genotypes were not linked to age at onset or severity of the disease. A subgroup of 137 CAD patients of whom 131 could be genotyped and who did not differ in baseline parameters from the remaining PAD patients, showed VV-genotype in 52.0%, VI in 42.7% and II in 5.3% CAD (OR associated with the I-allele for CAD: 1.29; 95% CI: 0.66-2.51; P=0.46). The distribution of the T280M genotypes was 67.1, 29.8, 3.1% (TT, TM, MM) also showing no association with CAD (OR=0.77; 95% CI 0.36-1.46; P=0.37). CONCLUSION: In this study we could not detect a difference in genotype frequencies of the V249I and T280M polymorphisms in CX(3)CR1 between PAD patients and controls. CAD concomitant with PAD was also not affected by the I- or the M-allele.

Abstract: Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Abstract: OBJECTIVES: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are key enzymes in lipoprotein metabolism by mediating the transfer and exchange of phospholipids (PL) and neutral lipids between lipoproteins. Lipoprotein lipase (LPL) deficiency is associated with low HDL-cholesterol (HDL-C) levels in both, the homozygous and heterozygous state. In the present study we set out to investigate the role of lipid transfer proteins, which are known to strongly determine HDL-C levels, in LPL deficiency. DESIGN/SUBJECTS: Phospholipid acceptor and donor properties of lipoproteins, PLTP activity, CETP mass, activity and cholesteryl ester (CE) transfer were determined in two homozygous and six heterozygous LPL-deficient subjects and in 10 healthy, normolipidaemic controls, respectively. RESULTS: The HDL isolated from LPL-deficient subjects showed strongly increased PL-acceptance when compared with controls (homozygotes versus heterozygotes versus control: 26.46 +/- 15.26 vs. 3.41 +/- 1.61 vs. 1.89 +/- 0.33 micromol mL-1 h-1/micromol mL-1 PL; all P < 0.05). Phospholipid transfer from apolipoprotein B containing lipoproteins was increased in heterozygotes when compared with controls (46.66 +/- 23.3 vs. 28.91 +/- 18.05 micromol mL-1 h-1/micromol mL-1 PL, P = 0.05). PLTP activity, however, was similar in LPL-deficient subjects and controls. CETP mass was highest in homozygotes, whilst enzyme activity was similar in LPL-deficient subjects and controls. CE transfer was highest in homozygotes (72.5 +/- 8.8%) and lowest in controls (28.7 +/- 5.2%, P < 0.01). CONCLUSIONS: In conclusion, PL and CE transfer are increased in LPL deficiency and thus, partly explain low HDL-levels in LPL-deficient subjects. Enhanced transfer seems rather to be the result of altered lipoprotein composition and concentration than altered enzyme activity. Our findings on mechanisms leading to low HDL-C levels might show another aspect in atherogenesis in LPL deficiency.

Abstract: A common 936 C/T polymorphism in the gene for the vascular endothelial growth factor (VEGF) has been associated with VEGF plasma levels. In our case-control study, we investigated the role of this polymorphism for breast cancer risk. VEGF genotype was determined in 500 women with breast cancer and 500 sex- and age-matched healthy control subjects. Carriers of a 936T-allele were more frequent among controls (29.4%) than among patients (17.6%; p = 0.000014). The odds ratio for carriers of a 936T-allele for breast cancer was 0.51 (95% confidence interval 0.38-0.70). Additionally, VEGF plasma levels were determined in 21 nonsmoking post-menopausal controls; carriers of a 936T allele had significantly lower levels (median 23 pg/ml; range 6-50 pg/ml) than noncarriers (37; 21-387; p = 0.034). We conclude that carriers of a VEGF 936T-allele are at decreased risk for breast cancer, this, however, requiring further confirmation in a larger study.

Abstract: A common 870G > A polymorphism in the gene for cyclin D1, CCND1, has been linked to alternative splicing and cancer susceptibility. To analyze its role for breast cancer, we determined the CCND1 genotype in 500 breast cancer patients and 500 controls. CCND1 genotype frequencies were similar among patients and controls. The CCND1 genotype was furthermore not associated with tumor characteristics. We conclude that the CCND1 870G > A polymorphism is not associated with breast cancer.

Abstract: Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).

Abstract: Transforming growth factor-beta 1 (TGF-beta1) is a potent inhibitor of proliferation of epithelial, endothelial and hematopoietic cells and acts as a tumor suppressor. The gene for TGF-beta1, TGFB1, carries a common T/C variation of nucleotide 29, resulting in a leucine (L) to proline (P) polymorphism at codon 10 (TGFB1 L10P). The less common 10P allele has repeatedly been linked to higher TGF-beta1 levels and in at least one study to a lower incidence of breast cancer. To further analyze the role of this polymorphism for breast cancer risk, 500 patients with histologically confirmed breast cancer and 500 sex-and age-matched healthy control subjects were genotyped for the TGFB1 L10P polymorphism by an allele-specific polymerase chain reaction assay. TGFB1 LL, LP and PP genotype frequencies were not significantly different for patients (39.6, 44.2, 16.2%) and controls (36.5, 45.9, 17.6%). We conclude that the TGFB1 L10P polymorphism is not associated with breast cancer risk.

Abstract: Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and plays a role in DNA biosynthesis, methylation, and repair in actively dividing cells. A common 677C>T polymorphism in the gene for MTHFR, leading to a thermolabile enzyme with decreased activity, has been associated with reduced plasma folate levels and elevated homocysteine levels and could be a risk factor for breast cancer. In the present case-control study, MTHFR genotype was determined in 500 women with clinically verified breast cancer and 500 female age-matched healthy control subjects. The homozygous TT genotype was found in 13.0% patients and 13.1% controls (P = n.s.). The odds ratio of TT homozygotes for breast cancer was 0.99 (95% confidence interval 0.68-1.43). The MTHFR genotype was furthermore not associated with tumor size, histological grading, estrogen or progesterone receptor status and age at diagnosis. In a subgroup of 116 premenopausal patients, no increased frequency of the homozygous 677T genotype was found (13.8%). Therefore, we conclude that the MTHFR 677C>T polymorphism is not associated with individual susceptibility to breast cancer.

Abstract: OBJECTIVE: Conflicting data exists about the possible contribution of the homozygous Asp/Asp genotype of the Glu298Asp polymorphism of endothelial nitric oxide synthase to human atherosclerotic vascular disease. We investigated the polymorphism in two independent study populations: a case-control study including patients with angiographically verified coronary artery disease (CAD) on the one hand and a cross-sectional epidemiological study on the other hand. METHODS: The Glu298Asp polymorphism was determined by PCR-RFLP as established. In the case-control study (240 patients and 248 controls) a possible association between the polymorphism and CAD, and age of onset of CAD and myocardial infarction was investigated. In the cross-sectional epidemiological study (932 subjects) intima-media thickness (IMT) of the carotid artery as well as morphological plaque burden and forearm vascular reactivity (peak postischemic reactive hyperaemia, determined by venous occlusion plethysmography) were measured. RESULTS: In the case-control study genotype distribution (Glu/Glu; Glu/Asp; Asp/Asp) was not different between the CAD patients (43/46/11%) and the controls (49/41/10%, P = NS). No association of the polymorphism with age of onset of CAD or myocardial infarction was found. In the epidemiological study no influence of the genetic variant on IMT was observed after correction for classical determinants of IMT (average IMT: Asp/ Asp: 0.077 +/- 0.011 mm; Glu/Glu and Glu/Asp: 0.080 +/- 0.012 mm, P = NS). Forearm vascular reactivity was also not different between homozygous Asp/Asp subjects and Glu/Glu and Glu/Asp subjects (peak-reactive hyperaemia 20.1 +/- 7.3 mL min-1 100 mL-1 vs. 20.0 +/- 6.5 mL min-1 100 mL-1, P = NS). CONCLUSIONS: Our results suggest that there is no association of the Glu298Asp polymorphism with coronary or carotid atherosclerosis or forearm vascular reactivity in these populations recruited in a country with a rather high risk for atherosclerosis. We suggest additional investigations to be performed in populations at different risk for coronary events to further elucidate the possible contribution of this polymorphism to vascular disease.

Abstract: OBJECTIVE: In obesity, plasma leptin is high and soluble leptin receptor (sOb-R) levels are low, resulting in a low fraction of bound leptin. The aim of this study was to investigate the influence of insulin resistance (IR) and the metabolic syndrome (MS) on sOb-R concentration and the bound-free ratio of leptin. RESEARCH METHODS AND PROCEDURES: sOb-R, leptin levels, and homeostasis model assessment (HOMA) index for IR were determined in 76 middle-aged obese or overweight men. RESULTS: Concentration of sOb-R and soluble receptor-bound fraction of leptin were lowest in the highest tertile of HOMA-IR. sOb-R and the bound-free ratio of leptin correlated with HOMA-IR, leptin concentration, and waist-to-hip ratio independently of age, BMI, and fat mass. Leptin and waist-to-hip ratio were the sole independent determinants of sOb-R concentration, and BMI, HOMA-IR, and visceral adipose tissue were independent determinants of the bound fractin of leptin. sOb-R concentration and the bound fraction of leptin decreased with increasing numbers of components of the MS, resulting in lower sOb-R concentration and a lower fraction of bound leptin in men with the MS. DISCUSSION: IR and abdominal obesity are associated with low sOb-R concentration and low bound-free ratio of leptin independent of fat mass. Low sOb-R concentration and low bound-free ratio of leptin segregate with components of the MS. We suggest that low sOb-R levels and a low fraction of specifically bound leptin are markers of leptin resistance, which is independently associated with IR and abdominal obesity and may constitute an additional component of the MS.

Abstract: Peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARGC1) is a transcriptional coactivator that has been implicated in the regulation of genes involved in energy metabolism. We studied associations of two polymorphisms identified in PPARGC1 transcripts with obesity indices in 591 middle-aged men and 467 middle-aged women of a cross-sectional Austrian population. Because neither polymorphic site was likely to be a functional site, we analyzed sex-specific associations of two loci haplotype combinations with obesity indices. Significant associations with BMI (P = 0.006), waist (P = 0.01) and hip circumference (P = 0.03), and total body fat (P = 0.005) and borderline significant associations with abdominal visceral and subcutaneous fat were observed in women but not men. In women, plasma triglycerides, HDL cholesterol, and glucose significantly differed by haplotype combinations, but these associations were not maintained after statistical consideration of BMI. The haplotype combination of the double-variant allele with the double-wild-type allele was associated with the lowest obesity indices, whereas homozygosity for the double-variant allele was not discriminatory among haplotype combinations. These studies suggest functional differences of PPARGC1 haplotypes in human energy metabolism and support a role of PPARGC1 in obesity.

Abstract: AIMS/HYPOTHESIS: Several studies have investigated the lipoprotein phenotype in heterozygous carriers of a defective lipoprotein lipase allele. We studied whether heterozygosity for lipoprotein lipase deficiency also affects glucose metabolism beyond its effect on plasma lipids. METHODS: To address this question 85 heterozygous carriers of either a missense mutation (Gly188Glu) or a splice site mutation (C-->A in position -3 at the acceptor splice site of intron 6) in the LPL gene which both result in a catalytically inactive product were compared with 108 unaffected subjects from the same families. RESULTS: Carriers for one of these mutations had higher fasting insulin levels but only a trend towards increased fasting blood glucose concentrations could be detected. HOMA index values were significantly higher in carriers than in non-carriers. Furthermore, in carriers, a significantly higher BMI and a trend towards higher systolic and diastolic blood pressure were observed. Carriers also had significantly higher fasting triglycerides, lower HDL cholesterol, and lipoprotein lipase particles of smaller size, confirming previous reports. Among carriers, subjects with one rare allele of the SstI polymorphism in the apo CIII gene had significantly higher plasma triglyceride levels than those with two common SstI alleles. This difference could not be observed in non-carriers of a mutant lipoprotein-lipase allele. The mean intima media thickness of the carotid arteries was slightly, but not significantly higher in carriers when compared with non-carriers. CONCLUSION/INTERPRETATION: This study shows that carrier status of one defective lipoprotein-lipase allele is associated with impaired insulin sensitivity, an atherogenic lipoprotein profile and other characteristics of the metabolic syndrome, which are risk factors for atherosclerotic vascular disease. A higher incidence of atherosclerotic vascular disease, however, could not be firmly established in carriers of this study population.

Abstract: BACKGROUND: An insertion/deletion (I/D) polymorphism of the gene for angiotensin-converting-enzyme (ACE) is associated with ACE plasma levels and activity. Conflicting results have been reported about the relevance of this polymorphism for atherosclerotic vascular disease. The aim of the present study was to analyze the role of this polymorphism for peripheral arterial disease (PAD). METHODS: The study was designed as a case-control study including 522 patients with documented PAD and 522 sex- and age-matched controls. ACE genotype was determined by size-analysis of polymerase chain reaction products. RESULTS: ACE genotype frequencies were similar between patients (II: 23.4%; ID: 44.8%; DD: 31.8%) and controls (II: 23.8%; ID: 48.3%; DD: 27.9%, P=0.37). The adjusted odds ratio of carriers of the DD genotype for PAD was 1.29 (95% confidence interval 0.95-1.75). The polymorphism was furthermore not associated with age at onset of PAD (P=0.56), Fontaine stage of the disease (P=0.68) or ankle/brachial index of patients (P=0.86). CONCLUSION: The ACE I/D polymorphism is not a significant risk factor for PAD.

Abstract: Obesity is the most common nutritional disorder in Western society. Uncoupling protein-2 (UCP2) is a recently identified member of the mitochondrial transporter superfamily that is expressed in many tissues, including adipose tissue. Like its close relatives UCP1 and UCP3, UCP2 uncouples proton entry in the mitochondrial matrix from ATP synthesis and is therefore a candidate gene for obesity. We show here that a common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo and results in increased transcription of a reporter gene in the human adipocyte cell line PAZ-6. In analyzing 340 obese and 256 never-obese middle-aged subjects, we found a modest but significant reduction in obesity prevalence associated with the less-common allele. We confirmed this association in a population-based sample of 791 middle-aged subjects from the same geographic area. Despite its modest effect, but because of its high frequency (approximately 63%), the more-common risk allele conferred a relatively large population-attributable risk accounting for 15% of the obesity in the population studied.

Abstract: AIMS/HYPOTHESIS: Phospholipid transfer protein plays a key role in lipoprotein metabolism by catalysing the transfer of phospholipids from triglyceride-rich lipoproteins to high-density lipoproteins and, also, within the high-density lipoprotein family, from particle to particle. This transfer results in a change of HDL particle size and the generation of pre-beta-high-density lipoproteins which function as initial lipid acceptors in the process of reverse cholesterol transport. Because adipose tissue is a source of phospholipid transfer protein we investigated the influence of obesity and insulin sensitivity on phospholipid transfer protein activity. METHODS: Using an exogenous substrate assay phospholipid transfer protein activity was measured in plasma specimens of 190 normolipidaemic, non-diabetic subjects with BMI ranging from 19 to 43 kg/m2. Insulin sensitivity was measured by the short insulin tolerance test. RESULTS: Phospholipid transfer protein activity was associated with BMI (r = 0.46, p < 0.01), body fat mass (r = 0.39, p < 0.01), subcutaneous fat area (r = 0.32, p < 0.01) and plasma leptin concentration (r = 0.24, p < 0.01) but not with insulin sensitivity expressed as the k(s) of the insulin tolerance test (kITT value) (r = -0.14, p = 0.40). Accordingly, phospholipid transfer protein activity was higher in obese than in nonobese subjects. As determined by linear regression analysis, BMI was the sole predictor of phospholipid transfer protein activity in plasma explaining 22.2% of the activity (p< 0.01). CONCLUSIONS/INTERPRETATIONS: This data suggests that increased phospholipid transfer protein activity in obese subjects is a consequence of obesity itself without the contribution of insulin resistance and can be explained by increased synthesis of phospholipid transfer protein from the enlarged mass of adipose tissue.

Abstract: Obesity is a prevalent disorder that increases the risk for premature cardiovascular disease. The adipose tissue itself plays an active role in the regulation of fuel metabolism and energy homeostasis by expressing a number of regulatory genes, such as leptin, peroxisome proliferator-activated receptor-gamma (PPARgamma), and CCAAT/enhancer binding protein-alpha (C/EBPalpha). To study the in vivo relationships among these genes and their associations with cardiovascular risk factors, plasma levels of leptin, lipids, apolipoproteins (apo), insulin, and glucose were measured in 216 obese, 165 nonobese, and 36 weight-losing postobese subjects. mRNA expression of leptin, PPARgamma, and C/EBPalpha in the extraperitoneal and intraperitoneal adipose tissue was quantified in subsets of subjects. In obese individuals, plasma leptin was associated with apoA-I (r=0.2346, P<0.001) and insulin (r=0.2125, P<0.002). Leptin and C/EBPalpha mRNA expression in extraperitoneal and intraperitoneal adipose tissue of obese patients was higher than in the respective tissues of nonobese or postobese subjects. No significant differences among the study groups were found for PPARgamma mRNA expression. Leptin, PPARgamma, and C/EBPalpha mRNA levels correlated with each other in the intraperitoneal and extraperitoneal fat of obese subjects, but multivariate analysis revealed that only C/EBPalpha was a predictor of leptin expression in extraperitoneal tissue (partial r=0.6096, P<0.001). Intraperitoneal PPARgamma expression was inversely related to fasting insulin (r=-0.2888, P<0.017) and a fasting insulin resistance index (r=-0.2814, P<0.021) in obese subjects. In postobese patients, intraperitoneal PPARgamma expression was associated with plasma HDL cholesterol (r=0.5695, P<0.018) and apoA-I (r=0.6216, P<0.008) but was inversely related to LDL cholesterol (r=-0.5101, P<0.03) and apoB (r=-0.6331, P<0.007). These findings suggest a relationship between plasma leptin and HDL metabolism as well as adipose-tissue site-dependent associations among leptin, C/EBP-alpha, and PPAR-gamma mRNA expression. Furthermore, our results suggest that C/EBP-alpha enhances leptin expression in vivo and that PPARgamma mRNA expression is inversely associated with cardiovascular risk factors.

Abstract: Two novel mutations in the lipoprotein lipase (LPL) gene are described in an Austrian family: a splice site mutation in intron 1 (3 bp deletion of nucleotides -2 to -4) which results in skipping of exon 2, and a missense mutation in exon 5 which causes an asparagine for histidine substitution in codon 183 and complete loss of enzyme activity. A 5-year-old boy who exhibited all the clinical features of primary hyperchylomicronemia was a compound heterozygote for these two mutations. Nine other family members were investigated: seven were heterozygotes for the splice site mutation, one was a heterozygote for the missense mutation, and one had two wild-type alleles of the LPL gene. LPL activity in the post-heparin plasma of the heterozygotes was reduced to 49;-79% of the mean observed in normal individuals. Two of the heterozygotes had extremely high plasma triglyceride levels; in three of the other heterozygotes the plasma triglycerides were also elevated. As plasma triglycerides in carriers of one defective LPL allele can be normal or elevated, the heterozygotes of this family have been studied for a possible additional cause of the expression of hypertriglyceridemia in these subjects. Body mass index, insulin resistance, mutations in other candidate genes (Asn291Ser and Asp9Asn in the LPL gene, apoE isoforms, polymorphisms in the apoA-II gene and in the apoAI-CIII-AIV gene cluster, and in the IRS-1 gene) could be ruled out as possible factors contributing to the expression of hypertriglyceridemia in this family. A linkage analysis using the allelic marker D1S104 on chromosome 1q21;-q23 suggested that a gene in this region could play a role in the expression of hypertriglyceridemia in the heterozygous carriers of this family, but the evidence was not sufficiently strong to prove this assumption. Nevertheless, this polymorphic marker seems to be a good candidate for further studies.

Abstract:

Abstract: A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (-5262 to -899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints.

Abstract: Lowering of LDL-cholesterol by 25 to 30% with statins resulted in a highly significant reduction of coronary event rates in 2 large primary prevention trials. In the West of Scotland Primary Prevention Study (WOSCOPS) hypercholesterolemic asymptomatic men were treated with either 40 mg of pravastatin or placebo, in the Airforce/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS) 6605 men and women with average levels of LDL-cholesterol and low levels of HDL-cholesterol were treated with either 20 to 40 mg of lovastatin or placebo. Moreover, in the WOSCOP study a marked reduction of total mortality was observed which approached the level of statistical significance. Several groups of experts have recently developed guidelines for the use of statins in prevention of atherosclerotic vascular disease. There are major differences in the goals for lowering of LDL-cholesterol and in the levels at which initiation of lipid lowering by drugs is advocated. In most of these recommendations graded target levels for LDL-cholesterol are suggested which are guided by the level of global risk. According to the recommendations of the American National Cholesterol Education Program (NCEP) LDL-cholesterol should be lowered below 130 mg/dl in asymptomatic individuals at high absolute risk and below 160 in individuals with a moderate increase in risk. The Joint Task Force of European and other Societies on Coronary Prevention recently developed guidelines, which suggest that in primary prevention lipid lowering by drugs should be restricted to individuals whose 10 year CHD risk exceeds 20% or will exceed 20% if projected to age 60. In these individuals LDL-cholesterol levels should be lowered to less than 115 mg/dl. The International Task Force for Prevention of Coronary heart disease recently published recommendations which suggest, that LDL-cholesterol should be reduced below 100 mg/dl in asymptomatic individuals at very high coronary risk, while it should be lowered below 135 mg/dl in individuals at moderately increased risk and below 160 mg/dl in subjects with a small increase in risk. In conclusion, results of 2 landmark trials in primary prevention of coronary heart disease demonstrated that lowering of LDL-cholesterol by statins is one of the most effective strategies to reduce coronary risk. It should be applied most aggressively in subjects at the highest overall risk. Nevertheless, non-pharmacologic measures are still considered as the preferred strategy for the reduction of coronary risk in the setting of primary prevention.

Abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma), a fatty acid-activated nuclear receptor, is implicated in adipocyte differentiation and insulin sensitisation. In view of the association of dietary fat intake and bowel disease, the expression of PPARgamma in rodent and human intestine was studied. Expression of PPARgamma mRNA was examined by Northern blot hybridisation, RNase protection, and/or competitive RT-PCR assays, whereas PPARgamma protein levels were evaluated by immunoblotting and immunohistochemistry. PPARgamma mRNA and protein were abundantly expressed in colon relative to the small intestine both in rodents and in man. Interestingly, expression of PPARgamma was primarily localised in the more differentiated epithelial cells in the colon. The level of expression of PPARgamma in colon was similar to the levels seen in adipose tissue. Expression of PPARgamma increased from proximal to distal segments of the colon in man. In Caco-2 and HT-29 human adenocarcinoma cells, PPARgamma expression increased upon differentiation, consistent with PPARgamma being associated with a differentiated epithelial phenotype. High-level expression of PPARgamma was observed in the colon, but not in the small intestine, suggesting a potential role of this nuclear receptor in the colon.

Abstract: An insertion/deletion polymorphism in the gene coding for the angiotensin-converting enzyme (ACE) is strongly associated with ACE activity. This polymorphism may be a marker for an increased risk for cardiovascular events. Our study examined a possible relationship between the D/I polymorphism and myocardial release of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Ninety-six individuals with normal or impaired left ventricular function were included in the study. ANP and BNP plasma levels were measured at rest and after exposure to physical stress. At rest no association of ACE genotypes with ANP and BNP was found. After exercise homozygotes with the genotype DD had significantly higher ANP plasma levels than homozygotes with the genotype II. In contrast to ANP, BNP levels were not significantly different between genotype groups after exercise. Differences in site of production and mode of release between ANP and BNP might explain this difference. We hypothesize that our result might represent a variability gene effect of the ACE gene locus on endocrine processes in the heart during exposure to physical stress.

Abstract: Although the association between insulin resistance and hypertriglyceridemia has long been recognized, the question of the causal relationship of these two entities is still a matter of debate. To gain more insight into the relationship between hypertriglyceridemia and insulin resistance, we studied insulin sensitivity in two severely hypertriglyceridemic subjects in whom insulin resistance as a cause for hypertriglyceridemia could be positively ruled out. Rather, lipoprotein lipase deficiency due to a mutation in the lipoprotein lipase gene was identified as the cause. In the two study subjects, whole body glucose utilization was measured during a continuous infusion of somatostatin, glucose and insulin. Mean values of plasma glucose and insulin concentrations at 150, 160, 170 and 180 minutes were used to calculate steady state plasma glucose (SSPG) and steady state plasma insulin (SSPI) concentrations. SSPG of the two hypertriglyceridemic patients was in the range of those reported in the literature for healthy subjects without insulin resistance did not differ from those of two control subjects with normal plasma lipid levels. Therefore, the dyslipidemic state of the two patients, characterized by extreme elevation of triglyceride rich plasma lipoproteins and a severe reduction of HDL cholesterol, was clearly not associated with insulin resistance. From these findings we conclude that hypertriglyceridemia per se is not an obligatory cause for insulin resistance.

Abstract: Genetic hemochromatosis (GH) is the most common autosomal-recessive disorder (1 in 300 in populations of Celtic origin). Homozygosity for a C282Y mutation in the hemochromatosis (HFE) gene is the underlying defect in approximately 80% of patients with GH, and 3. 2-13% of Caucasians are heterozygous for this gene alteration. Because the high frequency of this mutation may result from a selection advantage, the hypothesis was tested that the C282Y mutation confers protection against iron deficiency in young women. To address this question the genotype of codon 282 was determined in a cohort of 468 unrelated female healthcare workers, ages 18-40 years. In all study participants, a complete blood count was obtained, and erythrocyte distribution width, serum iron, transferrin, transferrin saturation, and ferritin were measured. Two individuals were homozygous for the C282Y mutation, 44 were heterozygous, and 416 were homozygous for the wild-type allele. Heterozygous women had significantly higher values for hemoglobin (P = 0.006), serum iron (P = 0.013), and transferrin saturation (P = 0. 006) than women homozygous for the wild-type allele. Our data provide evidence for a protective role of the C282Y mutation in the HFE gene against iron deficiency in young women and suggest that a more efficient utilization of nutritional iron may have contributed to the high prevalence of the mutation in Caucasian populations.

Abstract: The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.

Abstract: BACKGROUND/AIMS: Genetic haemochromatosis is the most common autosomal recessive disorder in Northern European populations. A major histocompatibility complex class I-like gene, HLA-H, has been proposed to be responsible for genetic haemochromatosis. The prevalence of HLA-H gene mutations 282(TGC; Cys/TAC; Tyr) and 63(CAT; His/GAT; Asp) was determined in patients of Austrian origin. METHODS: DNA extracted from the blood of 40 Austrian patients and 271 controls was used to amplify HLA-H gene fragments by the polymerase chain reaction method. The base changes responsible for mutations Cys282Tyr and His63Asp alter recognition sites for restriction enzymes SnaB I and Bcl I, respectively. Digestion products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. RESULTS: Thirty-one (77.5%) genetic haemochromatosis patients were homozygous for mutation Cys282Tyr and three compound heterozygous for mutations Cys282Tyr and His63Asp. One patient was homozygous for mutation His63Asp but normal for mutation Cys282Tyr. Four patients were normal at both genetic loci and one patient was heterozygous for mutation His63Asp. One control subject homozygous for mutation Cys282Tyr was found on investigation to fulfill diagnostic criteria for haemochromatosis. Eight control subjects homozygous for mutation His63Asp showed no biochemical or clinical evidence of haemochromatosis indicating that this variant is not directly responsible for haemochromatosis. Absence of the Cys282Tyr mutation in six genetic haemochromatosis patients with distinct haplotypes indicates mutations within the HLA-H gene or at alternative genetic loci are the cause of genetic haemochromatosis in these patients. CONCLUSIONS: The HLA-H Cys282Tyr defect is likely to play a key role in the pathogenesis of haemochromatosis in most patients. Predominance of a single HLA-H gene mutation in haemochromatosis allows presymptomatic screening by genotypic analysis.

Abstract: Apolipoprotein B (apoB) is the major protein component of low density lipoproteins, and plays a central role in cholesterol transport and metabolism. The apoB gene is transcribed in the liver and in the intestine in humans. Although much is known about the DNA sequence elements and protein factors that are important for transcription of the human apolipoprotein B gene in the liver, less is known about the mechanisms that control transcription of this gene in the intestine. The sucrose isomaltase gene (SI), is expressed exclusively in the intestine. Two sequences from the promoter region of the SI gene, namely SIF-1 and SIF-3, are essential for promoter activity of the SI gene in intestinal cells. Sequences displaying a high degree of similarity to those of SIF-1 and SIF-3 are present in the third intron of the apoB gene. Rather than stimulating apoB promoter activity, the BSIF-1 and BSIF-3 sequences repressed transcription in CaCo-2 cells. Gel retardation studies demonstrated that BSIF-1, like SIF-1, binds to proteins related to the caudal family of proteins such as mCdx-4 and mCdx-2. These proteins appear to repress transcription from the apoB promoter by a mechanism that involves an interaction with members of the C/EBP family of proteins, that bind to a target sequence for the repressor in the segment from -139 to -111 of the apoB promoter. On the other hand, BSIF-3, like SIF-3, binds to HNF-1 and also represses transcription from the apoB promoter.

Abstract: A sample of 66 healthy, unrelated males with normal blood pressure were studied for a possible association between an insertion/deletion polymorphism in the gene coding for the angiotensin converting enzyme and blood pressure response to physical exercise. No association was found between the polymorphism and systolic blood pressure at rest and during stress. Statistically significant associations between the polymorphism and diastolic blood pressure were observed during exercise and post-stress. At maximal workload, among homozygotes for the deletion, the mean diastolic blood pressure was 93 (+/- 10) mmHg, among homozygotes for the insertion it was 82 (+/- 8) mmHg, among heterozygotes it was 85 (+/- 10) mmHg. The difference was still statistically significant 3 minutes post-stress. The angiotensin converting enzyme polymorphism may be a marker for genetically determined differences in the response of the cardiovascular system to physical stress.

Abstract: This study examined a possible relationship between genetic variation in the gene coding for the angiotensin converting enzyme (ACE) and increased risk for coronary heart disease (CHD) in an Austrian population. Polymerase chain reaction (PCR) was used to determine the genotypes for an insertion/deletion polymorphism in intron 16 of the ACE gene in 315 patients with CHD and in 149 normal controls. In the control group, the relative allele frequencies of the polymorphism were similar to those of previously published European studies. The genotype distribution among our patients was not significantly different from that among controls. We were not able to show a significant association of the DD genotype with coronary heart disease in subgroups containing patients considered at low coronary risk. There was no association of lipid parameters and ACE genotype. From these data we conclude that, in the Austrian population, the insertion/deletion polymorphism in the ACE gene cannot be used as a marker for coronary risk assessment.

Abstract: In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C-->A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188-->Glu substitution.

Abstract: In 16 members of two Austrian families affected by a missense mutation at codon 188 of the lipoprotein lipase (LPL) gene (8 heterozygous and 8 normal subjects), carrier status for the mutation as determined by DNA analysis was related to LPL activity in postheparin plasma, to the magnitude of postprandial lipemia, and to concentration, composition, and size of the major lipoprotein classes of postabsorptive plasma. Carriers exhibited clearly reduced LPL activity, normal fasting triglycerides, but pronounced postprandial lipemia. The carriers' impaired triglyceride tolerance, as evident in the postprandial state of challenge only, was associated with a fasting lipoprotein constellation characterized by (a) enrichment of HDL2 with triglycerides, (b) reduced HDL2-cholesterol, (c) enrichment of VLDL and intermediate density lipoprotein (IDL) with cholesteryl esters, (d) elevated IDL levels, and (e) small-sized LDL. Within any given individual, the degrees of expression of these characteristics were quantitatively and continuously related with each other as well as with the magnitude of lipemia and with LPL activity.

Abstract: Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

Abstract: The positions of several DNase I-hypersensitive (DH) sites have been mapped in the second and third introns of the human apolipoprotein B gene. Two such DH sites, I and V, are present both in human hepatoma (HepG2) and colon carcinoma (CaCo-2) cells that express the gene but absent from HeLa cells that do not express the gene. These DH sites map near sequence elements that have been highly conserved between the human and mouse genes. A PvuII-EcoRI fragment (+1064 to +2977) from the hypersensitive region exhibited enhancer activity, which was further localized by means of deletion experiments to a 155-base pair segment located entirely within the third intron and flanked by two DH sites. Three DNase I footprints were observed within this core enhancer, one of which contains putative binding sites for three liver specific nuclear proteins. Experiments are presented that suggest that this enhancer operates by a similar mechanism as that described previously for the strong second intron enhancer, involving an interaction with the basal transcriptional machinery. Digestions with low levels of micrococcal nuclease were performed to ascertain whether nucleosomes were present in the DNase I sensitive enhancer region. Nine different micrococcal nuclease-hypersensitive (MH) sites were detected in HepG2 cells but not in HeLa cells; one MH site was common to both cell types, and HeLa cells exhibited three unique MH sites. The first six MH sites (I-VI) are spaced approximately 200 base pairs apart, suggesting the presence of positioned nucleosomes in that region. MH sites VI-X are more closely spaced, suggesting either additional cutting sites within the core particle or the absence of one or two nucleosomes in this segment of the third intron enhancer.

Abstract: To determine the molecular basis for type I hyperlipoproteinemia in two Austrian families, the lipoprotein lipase (LPL) gene of two patients exhibiting LPL deficiency was analyzed by Southern blotting and by direct genomic sequencing of DNA amplified by polymerase chain reaction (PCR). All exons of the LPL gene except part of the noncoding region of exon 10, all splice donor and acceptor sites, as well as 430 basepairs of the 5'-region including the promotor were sequenced. A homozygous substitution of adenine for guanine in the fifth exon at cDNA position 818 of the LPL gene was found in both patients. Our sequencing strategy largely ruled out a linkage disequilibrium of the identified nucleotide change with another defect potentially causing the clinical phenotype. The base change described abolishes a normally present AvaII restriction site allowing the identification of carriers of the mutant allele by AvaII digestion of PCR fragments of exon 5; three members of the two families were homozygous for this mutation and ten members were heterozygous. The activity of LPL in postheparin plasma was almost completely absent in homozygotes and about half normal in heterozygotes. The loss of activity was related to LPL protein structure. This mutation alters the amino acid sequence at residue 188 from Gly to Glu. The conformational preferences of the protein chain around position 188 were calculated with the use of a knowledge-based computerized method. The most probable conformation is a beta-turn formed by residues 189-192. The mutation seems to destabilize the beta-turn and/or a yet larger domain critical for substrate alignment.

Abstract: In a group of 110 subjects with severe coronary artery disease, two were heterozygous for the apolipoprotein (apo) B arginine3,500----glutamine mutation that characterizes familial defective apo B-100. Both affected subjects were moderately hypercholesterolemic, and their low density lipoproteins (LDLs) were deficient in binding to the LDL receptor. Pedigree analysis of the two probands' families established a correlation between the apo B mutation, defective LDL, and a particular apo B haplotype that was characterized by 10 apo B gene markers. In addition to having one allele carrying the arginine3,500----glutamine mutation, one family member may harbor a second mutant apo B allele that causes its gene product to be present in plasma at a lower than normal level, despite the fact that the affinity of the protein for the LDL receptor appears to be normal. The metabolic basis for the underrepresentation of this second allotype remains to be elucidated.

Abstract: In the preceding paper (Paulweber, B., Onasch, M. A., Nagy, B. P., and Levy-Wilson, B. (1991) J. Biol. Chem. 266, 24149-24160) we demonstrated that the segment of the apolipoprotein B promoter extending from -260 to -85 is functionally different in hepatic (HepG2) and intestinal (CaCo-2) cells. These functional differences could be explained at least in part by differences in binding of three nuclear proteins to the region from -111 to -88. In this article we present further evidence suggesting that the mechanisms involved in transcriptional control of the apolipoprotein B gene in HepG2 and CaCo-2 cells are different, by demonstrating that an enhancer and a reducer can alter the activity of various apolipoprotein B promoter segments in widely differing ways in these two cell lines. Furthermore, we have localized a 329-base pair segment that is found within a negative regulatory region in the 5' distal portion of the gene. It displays a strong reducer effect upon promoter sequences that exhibit maximal transcriptional activity in CaCo-2 cells, but not in HepG2 cells.

Abstract: The arrangement of regulatory elements along the apolipoprotein B promoter region (positions -898 to +1) has been examined in transient transfection experiments performed in HepG2 and Hep3B (hepatic) and CaCo-2 (intestinal) cell lines, all of which express the apoB gene, and also in Chinese hamster ovary cells, which do not express the gene. The overall distribution of positive and negative regulatory segments was very similar in the two hepatoma cell lines (HepG2 and Hep3B) but different from that observed in the colon carcinoma cells (CaCo-2). Thus, whereas 260 base pairs of 5'-flanking sequence were sufficient for maximal expression of the promoter in HepG2 cells, only 139 nucleotides were required for maximal expression in CaCo-2 cells. Promoter activity in Chinese hamster ovary cells was exhibited by short constructs, with maximal activity for the -85 construct. DNase I footprinting of the apolipoprotein B promoter region using hepatic and intestinal extracts revealed multiple sites of interaction between the DNA and nuclear proteins. Gel retention experiments using the region from -262 to -88 (the region of greatest contrast between HepG2 and CaCo-2 cells) revealed interesting variations in the relative abundance of various nuclear proteins between the two cell types. A major functional difference between HepG2 and CaCo-2 cells was localized to the region between -111 and -88, which harbors the sequence TGTTTGCT, a motif present in the promoter region of several liver-specific genes. The molecular basis for the functional differences between these two cell types may be attributable to a difference in the relative abundance of three proteins that bind to sequences between -111 and -88.

Abstract: We have identified a negative regulatory region between positions -1802 and -3211 of the human apolipoprotein B gene that reduces expression of the gene. This "reducer" effect was detected in transient transfection experiments performed with hepatic (HepG2 and Hep3B) cells as well as intestinal (CaCo-2) cells. It appears to be specific for the apolipoprotein B promoter because it did not affect expression from the heterologous thymidine kinase promoter. This reducer segment operated in the presence or absence of the transcriptional enhancer from the second intron of the apolipoprotein B gene, suggesting that the protein factors involved in the enhancer and reducer effect can interact with the transcriptional machinery independently of each other. Deletion experiments further localized the positions of the negative regulatory elements in HepG2 cells: sequences between positions -2738 and -2470 together with those from -2118 to -1802 are required for the reducer activity. DNase I footprinting of these two reducer DNA segments identified several DNA sequences that are bound by nuclear proteins from HepG2 cells. Analysis of the nucleotide sequences within these footprints demonstrated a high degree of similarity with sequences within negative regulatory regions of other genes.

Abstract: The role of genetic variation at the 3' end of the apolipoprotein B gene locus in the development of coronary heart disease and the regulation of the serum levels of various lipoproteins was studied by using two common restriction fragment length polymorphisms detected with the enzymes Xba I and EcoR I. A group of 106 male patients with coronary heart disease and 118 matched controls of Austrian origin were investigated. The frequency of the R2 allele of the EcoR I polymorphism at cDNA position 12,669 defined by the absence of the polymorphic EcoR I cutting site was significantly higher among patients than among controls. The controls with the R2 allele had significantly higher levels of total triglycerides, very low density lipoprotein (VLDL) triglycerides, and VLDL cholesterol than did the controls without this allele. Among the patients, the R2 allele was associated with higher serum VLDL apolipoprotein B levels. The chemical composition of VLDL in individuals with different genotypes for the EcoR I polymorphism did not differ significantly. For the Xba I polymorphism at cDNA position 7673, no correlation with coronary risk could be demonstrated. Patients and controls homozygous for the X2 allele characterized by the presence of the polymorphic Xba I cutting site showed a higher total and low density lipoprotein cholesterol level than did subjects with the genotype X1X1 or X1X2. This difference, however, was not statistically significant. These findings indicate that the R2 allele of the EcoR I polymorphism is associated with the occurrence of coronary heart disease and that variation at the 3' end of the apo B gene is involved in the regulation of VLDL metabolism.

Abstract: Several recent reports have examined whether there is a correlation between the presence of some minor alleles of the highly polymorphic apolipoprotein B gene and atherosclerosis and premature heart disease. The present study extends this investigation. A high-resolution method was used to study the allele frequencies of a hypervariable minisatellite region close to the apolipoprotein B gene in 110 patients with severe coronary disease and in 117 normal controls. Alleles containing 38, 44, 46, or 48 hypervariable elements showed an association with coronary heart disease. These alleles were also associated with elevated serum levels of total cholesterol and apolipoprotein B among patients and with elevated serum levels of total triglycerides among controls. The hypervariable region showed strong linkage disequilibrium with a polymorphic EcoRI site in exon 29 and was in linkage equilibrium with a polymorphic MspI site in exon 26. Two patients carried a base change at codon 3500 that results in an arginine-to-glutamine substitution; the base change was linked in both instances to the allele with 48 hypervariable elements.

Abstract: An indirect immunofluorescence test (IIF) has been developed for detecting Campylobacter pylori in gastroduodenal biopsies. This test was compared with standard methods of C. pylori diagnosis, namely Gram staining and urease test, in a study population of 226 patients; 121 of the biopsy specimens were cultured for C. pylori as well. C. pylori colonization was detected in 154 of 226 patients (68%) by at least one of these methods (IIF, 96%; Gram staining, 78%; urease test, 60%; cultivation, 55%). Serum samples from 191 patients of the study population were screened for circulating antibodies to C. pylori by an indirect enzyme-linked immunosorbent assay with whole, untreated bacteria as antigen. Of these serum specimens, 140 (73%) revealed absorbance readings above the limit of positivity, which was determined as an optical density of greater than 0.35 at 405/620 nm. Of 132 serum specimens, 128 (97%) from patients with C. pylori detected in biopsies, but only 12 (20%) of 59 specimens from those without C. pylori detection showed elevated specific antibody levels. Our data revealed that IIF proved to be the superior rapid, sensitive, and specific diagnostic method. The correlation between microbiological findings and the immune response favors our enzyme-linked immunosorbent assay as an additional tool in C. pylori diagnosis.

Abstract:

Abstract: Six RFLPs in the apolipoprotein (apo) AI-CIII-AIV gene region detected with the restriction enzymes XmnI, MspI, PstI, SstI and PvuII were used to study the role of genetic variation at this locus in the development of coronary heart disease and in the regulation of serum levels of various lipid and lipoprotein parameters in the Austrian population. 106 male patients with coronary heart disease and 118 matched controls were investigated. None of the alleles defined by these RFLPs was associated with increased coronary risk. In the patients, but not in the control group individuals with the genotype P1P2 for the PstI polymorphism in the 3' flanking region of the apo AI gene had significantly lower serum levels of high density lipoprotein (HDL)-cholesterol and apo AI levels than those with the genotype P1P1. The S2 allele of the SstI polymorphism at the 3' end of the apo CIII gene was significantly associated with elevated serum levels of triglycerides in the patient, but not in the control group. Controls with the genotype V2V2 for the PvuII(A) polymorphism at the 5' end of the apo CIII gene had significantly higher serum levels of apo B than those with V1V1 or V1V2. This association did not exist among the patients. These findings suggest that variation associated with some of these RFLPs is contributing to the determination of lipid levels in patients and controls, but that the RFLPs themselves cannot be used as markers for increased coronary risk in the Austrian population.

Abstract: Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man.