[show abstract][hide abstract] ABSTRACT: Reverse cholesterol transport (RCT) has been inversely related to atherosclerosis and cardiovascular risk. The influence of menopause in the RCT process is poorly understood and the effects of cholesterol-lowering interventions, including statins and hormone therapy (HT), on genes controlling the RCT in postmenopausal women are also unknown.
The effects on serum lipids and expression profile of genes involved in RCT -APOA1, ABCA1, ABCG1, SCARB1 and LXRA- were evaluated by TaqMan(®) quantitative PCRin peripheral blood mononuclear cells (PBMC) from 87 postmenopausal hypercholesterolemic women treated with atorvastatin (AT, n=17), estrogen or estrogen plus progestagen (HT, n=34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT+AT, n=36).
Atorvastatin and HT treatments reduced the mRNA levels of APOA1 and SCARB1, respectively, whereas ABCA1 expression was reduced after all treatments. Although the expression of LXRA, an important transcription factor controlling the expression of genes involved in RCT, was not modified after any treatment, it was correlated with ABCA1, APOA1 and SCARB1 RNAm values before and after treatments, however no correlation with ABCG1 was observed. In a linear regression analysis, HT was related to an increase in apoAI levels after treatment when compared to atorvastatin and, moreover, higher SCARB1 and ABCA1 basal expression were also associated with decreased apoAI levels after treatments.
ABCA1 mRNA levels are decreased by atorvastatin and HT, however these treatments have a differential effect on APOA1 and SCARB1 expression in PBMC from postmenopausal women. Basal ABCA1 and SCARB1 expression profile could be helpful markers in predicting the effect of atorvastatin and HT on RCT, according to the changes in apoAI levels in this sample population.
The Journal of steroid biochemistry and molecular biology 09/2013; · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the relationship of short tandem repeats (STR) near genes involved in the leptin-melanocortin pathway with body mass index (BMI) and leptinemia.
Anthropometric variables and leptinemia were measured in 100 obese and 110 nonobese individuals. D1S200, D2S1788, DS11912, and D18S858 loci were analyzed by PCR and high-resolution electrophoresis.
Overall STR allele frequencies were similar between the obese and non-obese group (p > 0.05). Individual alleles D1S200 (17), D11S912 (43), D18S858 (11/12) were associated with obesity (p < 0.05). Individuals carrying these alleles showed higher BMI than non-carriers (p < 0.05). Moreover, a relationship between D18S858 11/12 alleles and increased waist circumference was found (p = 0.040). On the other hand, leptinemia was not influenced by the studied STRs (p > 0.05).
D1S200, D11S912, and D18S858 loci are associated with increased BMI and risk for obesity in this sample.
Arquivos brasileiros de endocrinologia e metabologia 02/2012; 56(1):47-53. · 0.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women. Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n=17), estrogen or estrogen plus progestagen (HT, n=34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT+AT, n=36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan(®) PCR. APOE ɛ2/ɛ3/ɛ4 genotyping was performed using PCR-RFLP. Total cholesterol (TC), LDL-c and apoB were reduced after each treatment (p<0.001). Triglycerides, VLDL-c and apoAI were reduced only after atorvastatin (p<0.05), whereas triglycerides and VLDL-c were increased after HT (p=0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT+AT groups (p<0.05). APOE mRNA expression was reduced after atorvastatin treatment (p=0.03). Although LXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before and after treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however the reduction on APOE expression was more pronounced in ɛ3ɛ3 than in ɛ3ɛ4 carriers. Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMC from postmenopausal women.
The Journal of steroid biochemistry and molecular biology 11/2011; 128(3-5):139-44. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apolipoprotein A-I gene (APOA1) polymorphisms have been associated with variations in serum low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol. We have investigated whether APOA1 common variants are also associated with variations in basal triglyceride serum concentrations and response to atorvastatin in individuals with hypercholesterolemia.
APOA1 G-75A and C83T polymorphisms and variations in serum lipids were evaluated in 150 hypercholesterolemic (HC) and 93 normolipidemic (NL) unrelated European-derived Brazilians treated with atorvastatin (10 mg/day for 4 weeks). Genomic DNA was extracted from blood leukocytes using a salting-out method and APOA1 polymorphisms were analyzed by polymerase chain reaction and restriction fragment length polymorphism.
G-75A polymorphism was associated with differences in serum concentrations of triglyceride and very low-density lipoprotein (VLDL)-cholesterol (p=0.026) in HC men. After atorvastatin treatment, women carrying the GG/CC haplotype had lower serum triglyceride and VLDL-cholesterol (p=0.020) than non-carriers. In men, the reduction in serum triglyceride in response to atorvastatin was found to be slightly lower in GG/CC haplotype carriers (p=0.051).
Our data suggest that APOA1 polymorphisms are associated with variations of baseline serum concentrations of triglyceride and VLDL-cholesterol and in response to atorvastatin in a gender-specific manner.
Clinical Chemistry and Laboratory Medicine 02/2005; 43(12):1339-45. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: An endothelial nitric oxide synthase gene (NOS3) polymorphism in exon 7 (G894T), resulting in Glu298Asp substitution at protein level, has been associated with myocardial infarction, hypertension and coronary atherosclerosis in some populations. This polymorphism is usually identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, the procedures described to date do not eliminate the possibility of misclassification and either require confirmation by DNA sequencing or are time-consuming. In this study, a PCR-RFLP procedure to detect the G894T polymorphism at the NOS3 was optimized by the introduction of a constitutive cleavage site in the amplification product. This cleavage site provides an internal control for enzymatic activity to avoid mistyping. The method was validated by the study of 35 white unrelated individuals with familial hypercholesterolemia and 70 controls. The frequency of the variant allele (T) was similar between both groups (27% vs. 22%, NS), and comparable to the frequency found in other white populations. However, future studies are necessary to confirm these data. In summary, the optimized procedure for detection of the G894T NOS3 polymorphism is rapid, simple, and does not require confirmatory tests. Using this method, we found no association between this polymorphism and familial hypercholesterolemia.
Clinical Chemistry and Laboratory Medicine 06/2002; 40(5):436-40. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: Low-density lipoprotein receptor (LDLR) gene mutations cause familial hypercholesterol-emia (FH), one of the most common single gene disorders. The spectrum of LDLR mutations in Brazil is not known. The aim of this study was the characterization of LDLR mutations in 35 unrelated Brazilian patients with heterozygous FH. The promoter region, the 18 exons and the flanking intron sequences of the LDLR gene were screened by PCR-SSCP analysis and by DNA sequencing. In addition, we have screened the apolipoprotein B gene (APOB) for known mutations (R3500Q and R3531C) that cause Familial defective apo B-100 (FDB) by PCR-RFLP procedure. We found two nonsense (E92X and C371X) and six missense LDLR mutations (R236W, G322S, G352D, A370T, C675W and C677Y), that were previously described in FH patients from other populations. We also found five novel missense [G(-20)R, T476P, V503G, D580H and S652R] and two novel frame shift LDLR mutations (FsR757 and FsS828). Four patients were found to carry two different mutations in the LDLR gene: G352D and A370T (one patient), S652R and C675W (one patient) and T476P and V503G (two patients). APOB mutations were not found. These findings demonstrate that there is a broad spectrum of mutations in the LDLR gene in FH individuals from Brazil.
Human Mutation 05/2002; 19(4):462-3. · 5.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have optimized a nonenzymatic, nontoxic procedure for efficient DNA extraction from fresh and cryopreserved clotted blood. Blood samples were obtained from 24 unrelated individuals who had given informed consent. We compared 10 paired samples of EDTA-anticoagulated blood and fresh blood clot and studied 14 samples of cryopreserved clot that had been frozen for 2 years at −20 °C. Blood clots were homogenized with 9 g/L NaCl, using a Potter-MARCONI MA 099 system, for 30 s. The homogenizing Teflon probe was cleaned three times with 700 mL/L ethanol and 9 g/L NaCl between samples to avoid cross-contamination. One milliliter of each homogenized sample was centrifuged at 1200g for 5 min, and the supernatant was removed. Blood cells were lysed with 1 mL of Tris buffer 1 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, and 25 mL/L Triton X-100). After centrifugation, the pellet was washed twice with Tris buffer 1 and lysed with 220 μL of Tris buffer 2 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, 0.4 mol/L NaCl, and 10 g/L sodium dodecyl sulfate) and incubated for 15 min at 56 °C. Cellular proteins were removed by precipitation, after addition of 100 μL of 5 mol/L NaCl. DNA was isolated by ethanol precipitation and solubilized in Tris-EDTA buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA). In conclusion, the protocol described here enables molecular biologists to obtain DNA from blood clots by use of stable and nonhazardous reagents. The method is simple, fast, and reliable for obtaining high quantities of DNA suited for clinical molecular testing.