Effects of LDLR variants rs5928, rs750518671 and rs879254797 on protein structure and functional activity in HepG2 cells transfected with CRISPR/Cas9 constructs

Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study.

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

Identification of pathogenic variants in the brazilian cohort with familial hypercholesterolemia using exon-targeted gene sequencing

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Genomics, epigenomics and pharmacogenomics of Familial Hypercholesterolemia (FHBGEP): A study protocol

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Association of variants in MYH7, MYBPC3 and TNNT2 with sudden cardiac death-related risk factors in Brazilian patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) and is one of the major causes of sudden cardiac death (SCD). An exon-targeted gene sequencing strategy was used to investigate the association of functional variants in sarcomeric genes (MYBPC3, MYH7 and TNNT2) with severe LVH and other SCD-related risk factors in Brazilian HCM patients. Clinical data of 55 HCM patients attending a Cardiology Hospital (Sao Paulo city, Brazil) were recorded. Severe LVH, aborted SCD, family history of SCD, syncope, non-sustained ventricular tachycardia and abnormal blood pressure in response to exercise were evaluated as SCD risk factors. Blood samples were obtained for genomic DNA extraction and the exons and untranslated regions of the MYH7, MYBPC3 and TNNT2 were sequenced using Nextera® and MiSEq® reagents. Variants were identified and annotated using in silico tools, and further classified as pathogenic or benign according to the American College of Medical Genetics and Genomics guidelines. Variants with functional effects were identified in MYBPC3 (n = 9), MYH7 (n = 6) and TNNT2 (n = 4). The benign variants MYBPC3 p.Val158Met and TNNT2 p.Lys263Arg were associated with severe LVH (p < 0.05), and the MYH7 p.Val320Met (pathogenic) was associated with family history of SCD (p = 0.037). Increased risk for severe LVH was found in carriers of MYBPC3 Met158 (c.472 A allele, OR = 13.5, 95% CI = 1.80-101.12, p = 0.011) or combined variants (MYBPC3, MYH7 and TNNT2: OR = 12.39, 95% CI = 2.14-60.39, p = 0.004). Carriers of TNNT2 p.Lys263Arg and combined variants had higher values of septum thickness than non-carriers (p < 0.05). Molecular modeling analysis showed that MYBPC3 158Met reduces the interaction of cardiac myosin-binding protein C (cMyBP-C) RASK domain (amino acids Arg215-Ala216-Ser217-Lys218) with tropomyosin. In conclusion, the variants MYBPC3 p.Val158Met, TNNT2 p.Lys263Arg and MYH7 p.Val320Met individually or combined contribute to the risk of sudden cardiac death and other outcomes of hypertrophic cardiomyopathy.

Effect of statins on lipid metabolism-related microRNA expression in HepG2 cells.

BACKGROUND: Statins are potent cholesterol-lowering drugs that prevent cardiovascular events. microRNAs (miRNAs) modulate the expression of genes involved in metabolic pathways and cardiovascular functions post-transcriptionally. This study explored the effects of statins on the expression of miRNAs and their target genes involved in lipid metabolism in HepG2 cells. METHODS: HepG2 cells were treated with atorvastatin or simvastatin (0.1-10 µM) for 24 h. The expression of 84 miRNAs and nine target genes, selected by in silico studies, was measured by qPCR Array and TaqMan-qPCR, respectively. RESULTS: Five miRNAs were upregulated (miR-129, miR-143, miR-205, miR-381 and miR-495) and two downregulated (miR-29b and miR-33a) in atorvastatin-treated HepG2 cells. Simvastatin also downregulated miR-33a expression. Both statins upregulated LDLR, HMGCR, LRP1, and ABCG1, and downregulated FDFT1 and ABCB1, whereas only atorvastatin increased SCAP mRNA levels. In silico analysis of miRNA-mRNA interactions revealed a single network with six miRNAs modulating genes involved in lipogenesis and lipid metabolism. The statin-dysregulated miRNAs were predicted to target genes involved in cellular development and differentiation, regulation of metabolic process and expression of genes involved in inflammation, and lipid metabolism disorders contributing to metabolic and liver diseases. CONCLUSIONS: Atorvastatin-mediated miR-129, miR-143, miR-205, miR-381, and miR-495 upregulation, and miR-29b, and miR-33a downregulation, modulate the expression of target genes involved in lipogenesis and lipid metabolism. Thus, statins may prevent hepatic lipid accumulation and ameliorate dyslipidemia.

Association of variants in MYH7, MYBPC3 and TNNT2 with sudden cardiac death-related risk factors in Brazilian patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) and is one of the major causes of sudden cardiac death (SCD). An exon-targeted gene sequencing strategy was used to investigate the association of functional variants in sarcomeric genes (MYBPC3, MYH7 and TNNT2) with severe LVH and other SCD-related risk factors in Brazilian HCM patients. Clinical data of 55 HCM patients attending a Cardiology Hospital (Sao Paulo city, Brazil) were recorded. Severe LVH, aborted SCD, family history of SCD, syncope, non-sustained ventricular tachycardia and abnormal blood pressure in response to exercise were evaluated as SCD risk factors. Blood samples were obtained for genomic DNA extraction and the exons and untranslated regions of the MYH7, MYBPC3 and TNNT2 were sequenced using Nextera® and MiSEq® reagents. Variants were identified and annotated using in silico tools, and further classified as pathogenic or benign according to the American College of Medical Genetics and Genomics guidelines. Variants with functional effects were identified in MYBPC3 (n = 9), MYH7 (n = 6) and TNNT2 (n = 4). The benign variants MYBPC3 p.Val158Met and TNNT2 p.Lys263Arg were associated with severe LVH (p < 0.05), and the MYH7 p.Val320Met (pathogenic) was associated with family history of SCD (p = 0.037). Increased risk for severe LVH was found in carriers of MYBPC3 Met158 (c.472 A allele, OR = 13.5, 95% CI = 1.80-101.12, p = 0.011) or combined variants (MYBPC3, MYH7 and TNNT2: OR = 12.39, 95% CI = 2.14-60.39, p = 0.004). Carriers of TNNT2 p.Lys263Arg and combined variants had higher values of septum thickness than non-carriers (p < 0.05). Molecular modeling analysis showed that MYBPC3 158Met reduces the interaction of cardiac myosin-binding protein C (cMyBP-C) RASK domain (amino acids Arg215-Ala216-Ser217-Lys218) with tropomyosin. In conclusion, the variants MYBPC3 p.Val158Met, TNNT2 p.Lys263Arg and MYH7 p.Val320Met individually or combined contribute to the risk of sudden cardiac death and other outcomes of hypertrophic cardiomyopathy.

Peripheral Blood miRome Identified miR-155 as Potential Biomarker of MetS and Cardiometabolic Risk in Obese Patients.

This study explored circulating miRNAs and target genes associated with metabolic syndrome (MetS) and cardiometabolic risk in obese patients. Small-RNA sequencing was used to assess the peripheral blood miRNome of 12 obese subjects (6 MetS and 6 non-MetS). Differentially expressed miRNAs and target genes were further analyzed by qPCR in a larger sample of obese patients (48 MetS and 32 non-MetS). miRNA:mRNA interactions were studied using in silico tools. miRNome analysis identified 10 downregulated miRNAs in MetS compared to non-Met patients (p < 0.05). In silico studies revealed three miRNAs (miR-155, miR-181a, and let-7a) and their predictive targets (CCAAT/enhancer-binding protein beta-CEBPB, KRAS proto-oncogene, GTPase-KRAS and suppressor of cytokine signaling 1-SOCS1) with a potential role in the insulin receptor signaling pathway. miR-155 expression was reduced and CEBPB mRNA levels were increased in MetS patients (p < 0.05), and these effects were correlated with the number of MetS diagnostic criteria (p < 0.05). Increased HOMA-IR (>7.6) was associated with low miR-155 levels, high CEBPB expression, and serum hsCRP (p < 0.05). miR-155 was negatively correlated with CEBPB, HOMA-IR, and plasma fibrinogen, and positively correlated with serum adiponectin (p < 0.05). Downregulation of circulating miR-155 is associated with insulin resistance, poor glycemic control, and increased MetS-related cardiometabolic risk, and these effects are potentially mediated by interaction with CEBPB.

Genomics, epigenomics and pharmacogenomics of familial hypercholesterolemia (FHBGEP): A study protocol.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Associação de variantes dos genes MYH7, MYBPC3 e TNNT2 com fatores de risco para morte súbita cardíaca em pacientes com cardiomiopatia hipertrófica / Association of MYH7, MYBPC3, and TNNT2 gene variants with risk factors for sudden cardiac death in patients with hypertrophic cardiomyopathy

INTRODUÇÃO: A Cardiomiopatia Hipertrófica (CMH) caracteriza-se por hipetrofia do ventrículo esquerdo (HVE) na ausência de outras doenças cardíacas que justifiquem a hipertrofia. A CMH é um dos principais causadores de Morte Súbita Cardíaca, um desfecho trágico de muitas doenças cardíacas. Mutações nos genes que codificam proteínas sarcoméricas, principalmente nos genes MYH7, MYBPC3 e TNNT2 têm sido associados com HCM em diversas populações. OBJETIVO: Investigar variantes nos genes MYH7, MYBPC3 e TNNT2 e suas associações com HVE severa (espessura maior que 30mm) e demais fatores de risco para SCD em pacientes brasileiros com HCM. MÉTODOS: Dados clínicos de 65 pacientes foram coletados em um hospital de cardiologia do estado de São Paulo, bem como características como HVE severa, MSC recuperada, histórico familiar de MSC antes dos 40 anos (HFMSC), síncope, taquicardia ventricular não-sustentada e resposta anormal da pressão sanguíne no exercício foram avaliados como fatores de risco para MSC e para classificação em grupos. Amostras de sangue foram coletadas para extração de DNA genômico e sequenciamento exômico dos genes MYH7, MYBPC3 e TNNT2, utilizando protocolo Nextera e plataforma MiSeq. RESULTADOS: Das 19 variantes identificadas com potencial de serem associadas com os fatores de risco, a variante MYBPC3 rs3729986 foi associada com HVE severa, cujos pacientes apresentaram 14 vezes mais chance de apresentarem tal características, e também com ausência de episódios de síncope. A variante MYH7 rs376897125 foi associada com o grupo HFMSC, grupo que também apresentou alta frequência de mulheres. A elevada frequência de indivíduos mulheres também foi encontrada no grupo síncope. CONCLUSÕES: Os resultados sugerem que a variante MYBPC3 rs3729986 poderia representar uma das principais variantes de risco para o desenvolvimento de HVE severa, e que a variante MYH rs376897125 poderia estar relacionada com o elevado risco de FHMSC, principalmente em indivíduos do sexo feminino. Portanto, portadores dessas variantes poderiam apresentar elevado grau de risco para MSC, considerando que ambas as características são consideradas fatores de risco para MS|C. (AU)

Polymorphisms in Genes Involved in the Leptin-Melanocortin Pathway are Associated with Obesity-Related Cardiometabolic Alterations in a Southern Chilean Population.

BACKGROUND: Polymorphisms in genes encoding proteins of the leptin-melanocortin pathway have been associated with obesity. The involvement of these polymorphisms with changes in body mass index (BMI) and anthropometric measures could also imply a contribution to the risk of metabolic syndrome (MetS) and metabolic alterations. We evaluated the relationship of leptin-melanocortin system polymorphisms with obesity, MetS, and other metabolic alterations in Southern Chilean individuals. METHODS: Two-hundred individuals were grouped as normoweight (BMI 18.0-24.9 kg/m2), overweight (BMI 25.0-29.9 kg/m2), and obese (BMI ≥ 30 kg/m2) or according to MetS status. Anthropometric measures (BMI, abdominal circumference, waist-to-hip ratio [WHR]) and biochemical parameters (glycemia and lipid profile) were evaluated. Polymorphisms LEP rs7799039, LEPR rs1137101, MC3R rs3746619 and rs3827103, and MC4R rs17782313 were evaluated by real-time PCR using allelic discrimination assays. RESULTS: LEPR rs1137101 GG genotype was related to reduced risk of obesity (odds ratio [OR] 0.26, 95% confidence interval [CI] 0.08-0.79; p = 0.018) and MetS (OR 0.36, 95% CI 0.15-0.88; p = 0.024), but it was not significant after Bonferroni correction for multiple tests as compared to the AA genotype (p > 0.01). Moreover, LEPR rs1137101 allele G (AG + GG) was related to lower BMI and WHR (p < 0.01). Further multiple linear regression analysis demonstrated that this genotype was also responsible for reduced BMI in 2.44 kg/m2 and WHR in 0.033 units. MC4R rs17782313 allele C (TC + CC) was slightly associated with diminished risk of MetS (OR 0.48, 95% CI 0.23-0.98; p = 0.040) and reduced BMI values in 1.95 kg/m2 (p < 0.05). Regarding lipid profile, LEPR rs1137101 allele G carriers had lower triglycerides and very-low-density lipoprotein (VLDL) cholesterol, whereas individuals carrying the MC4R rs17782313 allele C had higher high-density lipoprotein (HDL) cholesterol (p < 0.01). LEP rs7799039 allele A (GA + AA) was slightly associated with reduced total and low-density lipoprotein (LDL) cholesterol (p < 0.05). CONCLUSIONS: These results suggest that polymorphisms at LEP, LEPR, and MC4R may be useful biomarkers of obesity-related cardiometabolic alterations in our population.

Polymorphisms in Genes Involved in the Leptin-Melanocortin Pathway are Associated with Obesity-Related Cardiometabolic Alterations in a Southern Chilean Population

BACKGROUND:Polymorphisms in genes encoding proteins of the leptin-melanocortin pathway have been associated with obesity. The involvement of these polymorphisms with changes in body mass index (BMI) and anthropometric measures could also imply a contribution to the risk of metabolic syndrome (MetS) and metabolic alterations. We evaluated the relationship of leptin-melanocortin system polymorphisms with obesity, MetS, and other metabolic alterations in Southern Chilean individuals.METHODS:Two-hundred individuals were grouped as normoweight (BMI 18.0-24.9 kg/m2), overweight (BMI 25.0-29.9 kg/m2), and obese (BMI ≥ 30 kg/m2) or according to MetS status. Anthropometric measures (BMI, abdominal circumference, waist-to-hip ratio [WHR]) and biochemical parameters (glycemia and lipid profile) were evaluated. Polymorphisms LEP rs7799039, LEPR rs1137101, MC3R rs3746619 and rs3827103, and MC4R rs17782313 were evaluated by real-time PCR using allelic discrimination assays.

Effects of short-term add-on ezetimibe to statin treatment on expression of adipokines and inflammatory markers in diabetic and dyslipidemic patients.

AIM: The influence of short-term add-on ezetimibe to simvastatin treatment on expression of adipokines and inflammatory markers was investigated in diabetic and nondiabetic patients with hypercholesterolemia. METHOD: Hypercholesterolemic nondiabetic (HC, n = 37) and diabetic (DM, n = 47) patients were treated with simvastatin (SV, 10 or 20 mg/d/8-wk) and then SV plus ezetimibe (SV + EZ, 10 mg each/d/4 wk). Serum lipids, glycemic profile, and inflammatory markers (hsCRP, adiponectin, resistin, VCAM-1, and ICAM-1) were evaluated before and after the add-on ezetimibe therapy. mRNA expression of ADIPOR1, ADIPOR2, RETN, VCAM1, and ICAM1 was measured by real-time PCR in peripheral blood mononuclear cells (PBMC). RESULTS: Serum concentrations of LDL and HDL cholesterol, and adiponectin were higher in HC than DM patients (P < .05). The add-on ezetimibe therapy reduced total and LDL cholesterol, apoB and adiponectin serum levels in HC and DM groups, and resistin in HC subjects (P < .05). DM patients showed higher expression of ADIPOR1, ADIPOR2, RETN, and VCAM1 in PBMC than subjects in HC group, before and after add-on ezetimibe therapy (P < .05). PBMC RETN mRNA expression was reduced by add-on ezetimibe therapy in HC individuals (P < .05), but not in DM subjects. CONCLUSION: Short-term add-on ezetimibe to simvastatin treatment suppressing effects on hypercholesterolemia and adiponectinemia is independent of the diabetes status. Resistin serum levels and leukocyte mRNA expression are influenced by add-on ezetimibe to statin treatment.

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.

INTRODUCTION: Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. AIMS: To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. METHODS: TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. RESULTS: Clopidogrel at 20 µmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form. CONCLUSIONS: Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process.

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects

NTRODUCTION: Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. AIMS: To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. METHODS: TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. RESULTS: Clopidogrel at 20 μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form...

Effects of short-term add-on ezetimibe to statin treatment on expression of adipokines and inflammatory markers in diabetic and dyslipidemic patients

AIM: The influence of short-term add-on ezetimibe to simvastatin treatment on expression of adipokines and inflammatory markers was investigated in diabetic and nondiabetic patients with hypercholesterolemia. METHOD: Hypercholesterolemic nondiabetic (HC, n = 37) and diabetic (DM, n = 47) patients were treated with simvastatin (SV, 10 or 20 mg/d/8-wk) and then SV plus ezetimibe (SV + EZ, 10 mg each/d/4 wk). Serum lipids, glycemic profile, and inflammatory markers (hsCRP, adiponectin, resistin, VCAM-1, and ICAM-1) were evaluated before and after the add-on ezetimibe therapy. mRNA expression of ADIPOR1, ADIPOR2, RETN, VCAM1, and ICAM1 was measured by real-time PCR in peripheral blood mononuclear cells (PBMC). RESULTS: Serum concentrations of LDL and HDL cholesterol, and adiponectin were higher in HC than DM patients (P < .05). The add-on ezetimibe therapy reduced total and LDL cholesterol, apoB and adiponectin serum levels in HC and DM groups, and resistin in HC subjects (P < .05). DM patients showed higher expression of ADIPOR1, ADIPOR2, RETN, and VCAM1 in PBMC than subjects in HC group, before and after add-on ezetimibe therapy (P < .05). PBMC RETN mRNA expression was reduced by add-on ezetimibe therapy in HC individuals (P < .05), but not in DM subjects...

Modulation of adhesion molecules by cholesterol-lowering therapy in mononuclear cells from hypercholesterolemic patients.

INTRODUCTION: Cholesterol-lowering therapy has been related with several pleiotropic effects including anti-inflammatory action in vascular endothelium; however, their influence on monocyte adhesion molecules is poorly described. AIMS: To investigate the effect of inhibitors of synthesis (statins) and absorption (ezetimibe) of cholesterol on expression of adhesion molecules L-selectin, PSGL-1, VLA-4, LFA-1, and Mac-1 in mononuclear cells in vivo and in vitro using THP-1 cells. METHODS: The influence of simvastatin (10 mg/day), ezetimibe (10 mg/day), and their combination (10 mg each/day) on mRNA expression of adhesion molecules was analyzed in peripheral blood mononuclear cells (PBMC) from hypercholesterolemics. The effects of atorvastatin, simvastatin, and ezetimibe on mRNA and protein expression of adhesion molecules were also evaluated in THP-1 cells. RESULTS: Simvastatin/ezetimibe combination, but not the monotherapies, reduced the mRNA expression of the PSGL-1, LFA-1, and Mac-1 genes in PBMC from hypercholesterolemics. Total and LDL cholesterol in serum correlated with PSGL-1 mRNA expression, whereas HDL cholesterol negatively correlated with mRNA levels of L-selectin and VLA-4 genes (P < 0.05). Plasma hsCRP was also correlated with mRNA levels of VLA-4, LFA-1, and Mac-1 (P < 0.05). Atorvastatin and simvastatin at 10 µM reduced mRNA and protein expression of L-selectin, PSGL-1, and VLA-4 in THP-1 cells (P < 0.05). CONCLUSION: Cholesterol-lowering therapy modulates gene expression of adhesion molecules in PBMC from hypercholesterolemics and THP-1 cells. Simvastatin/ezetimibe combination gives more benefits by reducing to a larger extent the expression of adhesion molecules in mononuclear cells.

ADIPOQ and IL6 variants are associated with a pro-inflammatory status in obeses with cardiometabolic dysfunction.

BACKGROUND: Polymorphisms in genes encoding adiponectin (ADIPOQ) and interleukin-6 (IL6) have been associated with adiposity and obese-related phenotypes. This study investigated the relationship of ADIPOQ and IL6 gene polymorphisms with pro-inflammatory and cardiometabolic markers in obese patients. METHODS: Anthropometric and body composition parameters were measured in 249 Brazilian subjects (30 to 68 yr). Metabolic and inflammatory markers and adipokines were analyzed in blood samples. ADIPOQ rs2241766 (45 T > G) and IL6 rs1800795 (-174G > C) polymorphisms were analyzed by real-time PCR and PCR-RFLP, respectively. RESULTS: Type 2 diabetes, hypertension, dyslipidemia and increased values of waist circumference, body fat, leptin, fibrinogen, IL-1ß, hsCRP and TNFα were related to obesity (p < 0.05). Multiple linear regression analysis showed a positive correlation between BMI and waist circumference, body fat, leptin, fibrinogen, PAI-1, IL-1ß, hsCRP and TNFα values (p < 0.001) but not with adiponectin. Obese group had altered metabolic status, resistance to leptin and insulin, and atherogenic and pro-inflammatory profiles. ADIPOQ and IL6 variants were not directely related to obesity, leptin resistance or alterations in cardiometabolic markers. Individuals carrying ADIPOQ 45G allele (TG + GG genotype) had higher IL-6, IL-1ß and TNFα levels than TT genotype carriers (p < 0.05). IL6 -174GG genotype was associated with increased IL-1ß levels (p = 0.033). CONCLUSION: Obesity is associated with leptin resistance, cardiometabolic alterations and a pro-inflammatory status. Our results are suggestive that ADIPOQ and IL6 polymorphisms contribute to cardiometabolic risk in obese individuals.