Paulo Caleb Junior Lima Santos

Ethnicity and Arterial Stiffness in Brazil

BACKGROUND The impact of increased central arterial stiffness as a predictor of morbidity and mortality, independently of other cardiovascular (CV) risk factors, has been established. The main aim of the present work was to investigate the association of ethnicity on arterial stiffness in different ethnic groups from the Brazilian population. METHODS A total of 1,427 individuals from the general population were randomly selected from the Vitoria City metropolitan area and 588 Amerindians from a native community in Brazil. The ethnicity of the general population was classified by a standard questionnaire as Caucasian descent, African descent, or Mulattos (considered racially mixed subjects). Pulse wave velocity (PWV) was measured with a noninvasive automatic device (Complior, Colson; Garges les Gonesses, France). RESULTS Hemodynamic data of PWV, systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) was higher in African descent individuals than in the other groups (P < 0.001). These results were still observed after adjustment for age and mean arterial pressure (P < 0.001). In addition, studying only normotensive individuals, PWV adjusted levels were higher in African descent individuals, and lower in Amerindians when compared with other ethnic groups (P < 0.01), showing, without the possible confounder effects of time and severity of hypertension or medication use, that PWV is associated with ethnicity in our population. CONCLUSION The study of different ethnic groups from a highly admixtured population was able to demonstrate an association between ethnicity and arterial stiffness.

Non-HFE hemochromatosis

Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis.

CYP2C9 and VKORC1 Polymorphisms Are Differently Distributed in the Brazilian Population According to Self-Declared Ethnicity or Genetic Ancestry

BACKGROUND Warfarin-dosing pharmacogenetic algorithms have presented different performances across ethnicities, and the impact in admixed populations is not fully known. AIMS To evaluate the CYP2C9 and VKORC1 polymorphisms and warfarin-predicted metabolic phenotypes according to both self-declared ethnicity and genetic ancestry in a Brazilian general population plus Amerindian groups. METHODS Two hundred twenty-two Amerindians (Tupinikin and Guarani) were enrolled and 1038 individuals from the Brazilian general population who were self-declared as White, Intermediate (Brown, Pardo in Portuguese), or Black. Samples of 274 Brazilian subjects from Sao Paulo were analyzed for genetic ancestry using an Affymetrix 6.0(®) genotyping platform. The CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), and VKORC1 g.-1639G>A (rs9923231) polymorphisms were genotyped in all studied individuals. RESULTS The allelic frequency for the VKORC1 polymorphism was differently distributed according to self-declared ethnicity: White (50.5%), Intermediate (46.0%), Black (39.3%), Tupinikin (40.1%), and Guarani (37.3%) (p<0.001), respectively. The frequency of intermediate plus poor metabolizers (IM+PM) was higher in White (28.3%) than in Intermediate (22.7%), Black (20.5%), Tupinikin (12.9%), and Guarani (5.3%), (p<0.001). For the samples with determined ancestry, subjects carrying the GG genotype for the VKORC1 had higher African ancestry and lower European ancestry (0.14±0.02 and 0.62±0.02) than in subjects carrying AA (0.05±0.01 and 0.73±0.03) (p=0.009 and 0.03, respectively). Subjects classified as IM+PM had lower African ancestry (0.08±0.01) than extensive metabolizers (0.12±0.01) (p=0.02). CONCLUSIONS The CYP2C9 and VKORC1 polymorphisms are differently distributed according to self-declared ethnicity or genetic ancestry in the Brazilian general population plus Amerindians. This information is an initial step toward clinical pharmacogenetic implementation, and it could be very useful in strategic planning aiming at an individual therapeutic approach and an adverse drug effect profile prediction in an admixed population.

Presence and type of low density lipoprotein receptor (LDLR) mutation influences the lipid profile and response to lipid-lowering therapy in Brazilian patients with heterozygous familial hypercholesterolemia.

OBJECTIVES Familial hypercholesterolemia (FH) is an autosomal dominant disease caused mainly by LDLR mutations. This study assessed the influence of the presence and type of LDLR mutation on lipid profile and the response to lipid-lowering therapy in Brazilian patients with heterozygous FH. METHODS For 14 ± 3 months, 156 patients with heterozygous FH receiving atorvastatin were followed. Coding sequences of the LDLR gene were bidirectionally sequenced, and the type of LDLR mutations were classified according to their probable functional class. RESULTS The frequencies of the types of LDLR mutations were: null-mutation (n = 40, 25.6%), defective-mutation (n = 59, 37.8%), and without an identified mutation (n = 57, 36.6%). Baseline total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were higher in patients carrying a null mutation (9.9 ± 1.9 mmol/L, 7.9 ± 1.7 mmol/L), compared to those with a defective (8.9 ± 2.2 mmol/L, 7.0 ± 2.0 mmol/L), or no mutation (7.9 ± 1.9 mmol/L, 5.8 ± 1.9 mmol/L) (p < 0.001). After treatment, the proportion of patients attaining an LDL-C<3.4 mmol/L was significantly different among groups: null (22.5%), defective (27.1%), and without mutations (47.4%) (p = 0.02). The presence of LDLR mutations was independently associated with higher odds of not achieving the LDL-C cut-off (OR 9.07, 95% CI 1.41-58.16, p = 0.02). CONCLUSIONS Our findings indicate that the presence and type of LDLR mutations influence lipid profile and response to lipid-lowering therapy in Brazilian patients with heterozygous FH. Thus, more intensive care with pharmacological therapeutics should be performed in patients who have a molecular analysis indicating the presence of a LDLR mutation.

Impact of diabetes mellitus on arterial stiffness in a representative sample of an urban Brazilian population

BackgroundIndependent of other cardiovascular (CV) risk factors, increased arterial stiffness has been established as a predictor of morbidity and mortality. The main aim of this study was to investigate the impact of diabetes on arterial stiffness in a representative sample of an urban Brazilian population plus Amerindians.MethodsA total of 1,415 individuals from the general population were randomly selected plus 588 Amerindians from a native community in Brazil. In addition, a sub-sample of 380 individuals from the general population had 5-year follow-up data. Pulse wave velocity (PWV) was measured with a non-invasive automatic device (Complior, Colson; Garges les Gonesses, France) and increased arterial stiffness was defined as PWV ≥ 12 m/s.ResultsIn the overall group, diabetic individuals had higher frequencies of increased arterial stiffness and hypertension. They also had higher values of PWV, body mass index, total cholesterol, triglycerides, systolic and diastolic blood pressures compared to non-diabetic individuals (p < 0.01). In an analysis stratified by hypertension, PWV values and increased arterial stiffness frequency were higher in diabetic individuals in both groups (hypertensive and non-hypertensive) (p < 0.05). Furthermore, higher risk for increased arterial stiffness was observed in the diabetic individuals from the overall group (OR = 2.27; CI = 1.47-3.52, p < 0.001) and from the hypertensive group (OR = 2.70; CI = 1.58-4.75, p < 0.001), adjusted for covariates. Regarding the ethnic stratification, diabetic individuals from Amerindian, White, and Mulatto (mixed-race) groups had higher PWV values and a greater frequency of increased arterial stiffness compared to non-diabetic individuals. Both diabetic and non-diabetic individuals had higher PWV values after 5 years. There was no significant difference in the 5-year PWV progression in diabetic compared to non-diabetic individuals.ConclusionsThese results confirm, in a sample of Brazilian population, that the presence of diabetes is associated with increased arterial stiffness and it may contribute in part to increased cardiovascular risk in diabetic patients.

Genotyping of the hemochromatosis HFE p.H63D and p.C282Y mutations by high-resolution melting with the Rotor-Gene 6000® instrument

Abstract Background: The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods. Methods: Genotypes for the HFE p.C282Y (c.G845A; rs1800562) and p.H63D (c.C187G, rs1799945) mutations were assessed by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor-Gene 6000® instrument. Validation studies were conducted in samples bi-directionally sequenced. Results: The melting assay was developed in a unique procedure and to ensure the result in approximately 112 min (31 min for sample preparation and 81 min for the PCR-HRM step). Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80–86°C. For the HFE p.H63D, genotypes were also easily distinguished in the region of 76–82°C, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. In validation, genotypes were 100% concordant between methods. Conclusions: Our genotyping assay with the Rotor-Gene 6000® instrument applies to the laboratorial routine with several advantages, especially in large-scale demand. The main advantages were the non-dependence on gel electrophoresis and on mutagenic reagents for visualization of fragments, reduction of the chances for contamination due to sample preparation, the lack of use of probe-based methods and cost-effectiveness.

Development of a pharmacogenetic-based warfarin dosing algorithm and its performance in Brazilian patients: highlighting the importance of population-specific calibration

BACKGROUND The main aims of the present study were to develop a pharmacogenetic-based warfarin dosing algorithm and to validate it in a highly admixed population. MATERIALS & METHODS We included two patient cohorts treated with warfarin (first cohort, n = 832; and second cohort, n = 133). RESULTS Our algorithm achieved a determination coefficient of 40% including the variables age, gender, weight, height, self-declared race, amiodarone use, enzyme inducers use, VKORC1 genotypes and predicted phenotypes according to CYP2C9 polymorphisms. CONCLUSION Data suggest that our developed algorithm is more accurate than the IWPC algorithm when the application is focused on patients from the Brazilian population. Population-specific derivation and/or calibration of warfarin dosing algorithms may lead to improved performance compared with general use dosing algorithms currently available. Original submitted 26 November 2014; Revision submitted 9 April 2015.

Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population

NADPH oxidase p22phox subunit is responsible for the production of reactive oxygen species in the vascular tissue. The C242T polymorphism in the p22phox gene has been associated with diverse coronary artery disease phenotypes, but the findings about the protective or harmful effects of the T allele are still controversial. Our main aim was to assess the effect of p22phox C242T genotypes on arterial stiffness, a predictor of late morbidity and mortality, in individuals from the general population. We randomly selected 1,178 individuals from the general population of Vitoria City, Brazil. Genotypes for the C242T polymorphism were detected by PCR-RFLP, and pulse wave velocity (PWV) values were measured with a noninvasive automatic device Complior. p22phox and TNF-α gene expression were quantified by real-time PCR in human arterial mammary smooth muscle cells. In both the entire and nonhypertensive groups: individuals carrying the TT genotype had higher PWV values and higher risk for increased arterial stiffness [odds ratio (OR) 1.93, 95% confidence interval (CI) 1.27-2.92 and OR 1.78, 95% CI 1.07-2.95, respectively] compared with individuals carrying CC+CT genotypes, even after adjustment for covariates. No difference in the p22phox gene expression according C242T genotypes was observed. However, TNF-α gene expression was higher in cells from individual carrying the T allele, suggesting that this genetic marker is associated with functional phenotypes at the gene expression level. In conclusion, we suggest that p22phox C242T polymorphism is associated with arterial stiffness evaluated by PWV in the general population. This genetic association shed light on the understanding of the genetic modulation on vascular dysfunction mediated by NADPH oxidase.

Higher incidence of death in multi-vessel coronary artery disease patients associated with polymorphisms in chromosome 9p21

BackgroundWe investigated whether 9p21 polymorphisms are associated with cardiovascular events in a group of 611 patients enrolled in the Medical, Angioplasty or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function.MethodsThe participants of the MASS II were genotyped for 9p21 polymorphisms (rs10757274, rs2383206, rs10757278 and rs1333049). Survival curves were calculated with the Kaplan–Meier method and compared with the log-rank statistic. We assessed the relationship between baseline variables and the composite end-point of death, death from cardiac causes and myocardial infarction using a Cox proportional hazards survival model.ResultsWe observed significant differences between patients within each polymorphism genotype group for baseline characteristics. The frequency of diabetes was lower in patients carrying GG genotype for rs10757274, rs2383206 and rs10757278 (29.4%, 32.8%, 32.0%) compared to patients carrying AA or AG genotypes (49.1% and 39.2%, p = 0.01; 52.4% and 40.1%, p = 0.01; 47.8% and 37.9%, p = 0.04; respectively).Significant differences in genotype frequencies between double and triple vessel disease patients were observed for the rs10757274, rs10757278 and rs1333049. Finally, there was a higher incidence of overall mortality in patients with the GG genotype for rs2383206 compared to patients with AA and AG genotypes (19.5%, 11.9%, 11.0%, respectively; p = 0.04). Moreover, the rs2383206 was still significantly associated with a 1.75-fold increased risk of overall mortality (p = 0.02) even after adjustment of a Cox multivariate model for age, previous myocardial infarction, diabetes, smoking and type of coronary anatomy.ConclusionsOur data are in accordance to previous evidence that chromosome 9p21 genetic variation may constitute a genetic modulator in the cardiovascular system in different scenarios. In patients with established CAD, we observed an association between the rs2383206 and higher incidence of overall mortality and death from cardiac causes in patients with multi-vessel CAD.

ACTN3 R577X polymorphism and long-term survival in patients with chronic heart failure

BackgroundPrevious studies have shown the occurrence of actinin-3 deficiency in the presence of the R577X polymorphism in the ACTN3 gene. Our hypothesis is that this deficiency, by interfering with the function of skeletal muscle fiber, can result in a worse prognosis in patients with chronic heart failure.MethodsA prospective cohort study was conducted from 2002 to 2004. The eligibility criteria included diagnosis of chronic heart failure stage C from different etiologies. We excluded all patients with concomitant disease that could be related to poor prognosis. ACTN3 rs1815739 (R577X) polymorphism was detected by high resolution melting analysis. Survival curves were calculated with the Kaplan-Meier method and evaluated with the log-rank statistic. The relationship between the baseline variables and the composite end-point of all-cause death was assessed using a Cox proportional hazards survival model.ResultsA total of 463 patients were included in this study. The frequency of the ACTN3 577X variant allele was 39.0%. The LVEF mean was 45.6 ± 18.7% and the most common etiology of this study was hypertensive. After a follow-up of five years, 239 (51.6%) patients met the pre-defined endpoint. Survival curves showed higher mortality in patients carrying RX or XX genotypes compared with patients carrying RR genotype (p = 0.01).ConclusionR577X polymorphism in the ACTN3 gene was independently associated with worse survival in patients with chronic heart failure. Further studies are necessary to ensure its use as a marker of prognosis for this syndrome.