Atorvastatin and hormone therapy influence expression of ABCA1, APOA1 and SCARB1 in mononuclear cells from hypercholesterolemic postmenopausal women.

BACKGROUND: 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. METHODS: The effects on serum lipids and expression profile of genes involved in RCT - APOA1, ABCA1, ABCG1, SCARB1 and LXRA - were evaluated by TaqMan(®) quantitative PCR in peripheral blood mononuclear cells (PBMC) from 87 postmenopausal hypercholesterolemic women treated with atorvastatin (AT, n=17), estrogen or estrogen plus progestin (HT, n=34) and estrogen or estrogen plus progestin associated with atorvastatin (HT+AT, n=36). RESULTS: 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. CONCLUSION: 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.

Relationship of short tandem repeats flanking leptin-melanocortin pathway genes with anthropometric profile and leptinemia in Brazilian individuals.

OBJECTIVE: To investigate the relationship of short tandem repeats (STR) near genes involved in the leptin-melanocortin pathway with body mass index (BMI) and leptinemia. SUBJECTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: D1S200, D11S912, and D18S858 loci are associated with increased BMI and risk for obesity in this sample.

Relationship of short tandem repeats flanking leptin-melanocortin pathway genes with anthropometric profile and leptinemia in Brazilian individuals / Relação de short tandem repeats (STR) em genes envolvidos na via da leptina-melanocortina com o perfil antropométrico e a leptinemia em brasileiros

OBJECTIVE: To investigate the relationship of short tandem repeats (STR) near genes involved in the leptin-melanocortin pathway with body mass index (BMI) and leptinemia. SUBJECTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: D1S200, D11S912, and D18S858 loci are associated with increased BMI and risk for obesity in this sample.

OBJETIVO: Investigar a relação de short tandem repeats (STR) em genes envolvidos na via da leptina-melanocortina com índice de massa corporal (IMC) e leptinemia. SUJEITOS E MÉTODOS: Variáveis antropométricas e leptinemia foram medidas em 100 indivíduos obesos e 110 não obesos. Os loci D1S200, D2S1788, DS11912 e D18S858 foram analisados por PCR e eletroforese de alta resolução. RESULTADOS: As frequências globais dos alelos da STR foram similares entre os grupos obeso e não obeso (p > 0,05). Alelos individuais de D1S200 (17), D11S912 (43), D18S858 (11/12) foram associados com obesidade (p < 0,05). Indivíduos portadores desses alelos apresentaram valores de IMC maiores que os dos não portadores (p < 0,05). Além disso, a presença dos alelos D18S858 11/12 foi relacionada com circunferência abdominal elevada (p = 0,040). Por outro lado, a leptinemia não foi influenciada pelos STRs estudados (p > 0,05). CONCLUSÕES: Os loci D1S200, D11S912 e D18S858 são associados com IMC aumentado e risco de obesidade nesta amostra populacional.

Atorvastatin and hormone therapy effects on APOE mRNA expression in hypercholesterolemic postmenopausal women.

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.

Atorvastatin and hormone therapy effects on APOE mRNA expression in hypercholesterolemic postmenopausal women

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis andcardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profilein postmenopausal women.Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expressionwere evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatmentwith atorvastatin (AT, n = 17), estrogen or estrogen plus progestagen (HT, n = 34) and estrogen or estrogenplus progestagen associated with atorvastatin (HT + AT, n = 36). RNA was extracted from peripheralblood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan® PCR. APOE 2/ 3/ 4genotyping 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 wereincreased after HT (p = 0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT + ATgroups (p < 0.05). APOE mRNA expression was reduced after atorvastatin treatment (p = 0.03). AlthoughLXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before andafter treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however thereduction on APOE expression was more pronounced in 3 3 than in 3 4 carriers.Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMCfrom postmenopausal women.

APOA1 polymorphisms are associated with variations in serum triglyceride concentrations in hypercholesterolemic individuals.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

A method to detect the G894T polymorphism of the NOS3 gene. Clinical validation in familial hypercholesterolemia.

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.

Molecular basis of familial hypercholesterolemia in Brazil: Identification of seven novel LDLR gene mutations.

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.

Molecular Basis of Familial Hypercholesterolemia in Brazil: Identification of Seven Novel LDLR Gene Mutations

Low-density lipoprotein receptor (LDLR) gene mutations cause familial hypercholesterolemia (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 missenseLDLR mutations (R236W, G322S, G352D, A370T, C675W and C677Y), that were previously described in FH patients from other populations. We also found five novelmissense [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 mutationsin 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 findingsdemonstrate that there is a broad spectrum of mutations in the LDLR gene in FH individuals from Brazil. © 2002 Wiley-Liss, Inc.

Micrométodo para extraçäo de DNA genômico útil no diagnóstico molecular da Hipercolesterolemia Familial / A micromethod for DNA extraction useful in the molecular diagnostic of Familial Hypercholesterolemia

Os métodos para para extraçäo de DNA empregados hoje em dia na rotina laboratorial, utilizam grandes volumes de sangue, o que constitui um problema quando se usam amostras pediátricas. Outros métodos envolvem várias etapas, näo sendo adequadas para rotina laboratorial. No presente trabalho, foi desenvolvido um mátodo rápido de extraçäo de DNA de sangue total para pequenos volumes de amostra. Brevemente, volumes iguais de sangue total e NaI a 6M foram misturados. A seguir, as proteínas e debris celulares foram removidas por adiçäo de uma mistura de clorofórmio/álcool isoamílico (24:1). O DNA foi isolado da fase aquosa e purificado com isopropanol. Amostras de DNA foram também extraídas pelo método de precipitaçäo salina, previamente desenvolvido em nosso laboratório. A quantificaçäo e a pureza do DNA foram realizadas por espectrofotometria em 260 e 280 nm, sendo a sua integridade verificada por eletroforese em gel de agarose a 1 porcento. A quantidade de DNA de sangue total obtida pelo método proposto foi 1,5 vezes superior à obtida pela técnica de precipitaçäo salina (28,6 ñ 7,8 vs. 19,1 ñ 3,5µg/ml de sangue, respectivamente; p=0,0003). A pureza do DNA foi também superior (A subscrito 260/280=1,73ñ0,18 vs. 1,61ñ0,06, respectivamente; p=0,0164). Utilizando o DNA extraído pelo micrométodo, obtivemos sucesso na triagem de alteraçöes genéticas no receptor da LDL e na apolipoproteína B em pacientes com Hipercolesterolemia Familial (HF). Em resumo, o procedimento de extraçäo de DNA é simples, rápido e aplicável para obtençäo de DNA genômico de alta qualidade em 30 minutos, sendo portanto, útil no diagnóstico molecular da HF

Avaliaçäo do desempenho analítico da determinaçäo de uréia sérica por método enzimático / Evaluation of the analytical performance of the urea assay in serum by enzymatic method

A uréia é um produto do metabolismo dos aminoácidos no organismo humano e sua análise é relevante na avaliação das funções renal e hepática. A determinação da uréia é usualmente realizada por métodos colorimétricos e enzimáticos, que devem ser avaliados para implantação na rotina laboratorial. Com a finalidade de avaliar o desempenho analítico de método enzimático no UV para a determinação da concentração de uréia sérica, por procedimentos manual e automatizado, foram analisados materiais de referência, de controle e 30 amostras de soro provenientes do Hospital Universitário da USP. Embora os dados de desempenho analítico tenham mostrado que o procedimento automatizado apresenta maior grau de precisão e exatidão (C.V.-1,9 por cento, C.D.-3,3 por cento) que o manual (C.V.-5,0 por cento, C.D.-5,0 por cento) não se observou diferença significativa entre os resultados obtidos pelos dois procedimentos, utilizando materiais de referência certificado ou materiais de controle...

Extração de DNA de coágulo sanguíneo humano como método alternativo para estudos de genotipagem / DNA extraction from clotted human blood as a alternative method for genotyping studies

Foi otimizado um método de extração de DNA de coágulo sangüíneo. Amostras de sangue total colhidas em EDTA e de coágulo foram obtidas de 10 indivíduos não aparentados. Os coágulos foram homogeneizados (sistema Potter-Elvelyn NSE-11R). O DNA foi extraído pelo método de precipitação salina modificado (Clin.Chem.42-S298,1996). A quantificação e a pureza do DNA foram realizadas por espectrofotometria em 260 e 280 nm, sendo a sua integridade verificada por eletroforese em gel de agarose. A qualidade do DNA foi avaliada por amplificação da região polimórfica Ava II do gene do receptor da LDL, por PCR. A quantidade de DNA de coágulo foi semelhante a de sangue total (61,5 ± 11,9 e 60,6 ± 12,6 mg/ml de sangue, respectivamente). A pureza do DNA de coágulo foi semelhante a de sangue total (A260/280=1,87 ± 0,19 e 1,96 ± 0,17, respectivamente). As amostras de DNA apresentaram-se intactas no gel de agarose e sem contaminação com RNA. O rendimento do DNA obtido por este método dói maior que o dos descritos em outros estudos e mostrou-se adequado para análise do polimorfismo genético do receptor da LDL. Portanto, o procedimento de extração de DNA, otimizado em nosso laboratório, é simples, rápido e aplicável para obtenção de DNA de alta qualidade de amostras de coágulo sangüíneo, sendo útil em estudos de genotipagem.