The interaction of common polymorphisms of the lipoprotein lipase and PON1 genes with cardiovascular risk factors in the Phoenix community. The cardiovascular risk factor profile in teenagers and young Asian Indian adults in Phoenix Community, Durban.
Genetic patterns in the metabolic syndrome 1. Lipoprotein Lipase polymorphisms
Conclusion and recommendations
Limitations
LIST OF TABLES
Univariate and multivariate models of biochemistry, anthropometry and echocardiography for the detection of MS (IDF) Logistic regression for independent predictors of diastolic abnormalities.
CDL Chronic diseases of lifestyle CVD Cardiovascular disease DHF Diastolic Heart Failure DM Type 2 Diabetes Mellitus DNA Deoxyribose nucleic acid DT Delay time. VAT Visceral adipose tissue VHD Valvular heart disease WC Waist circumference WHO World Health Organization WT Wild-type.
ABSTRACT
There was a high prevalence of CV risk factors in this sample; especially increased waist circumference (79%), obesity (64%), insulin resistance (58%) and hypertension (50%) across the age groups. The high prevalence of CV risk factors and MS in this community has reached epidemic proportions.
CHAPTER ONE
Introduction and the Literature review
Problem identification and motivation for this study
The prevalence of cardiovascular disease (CVD) in developing countries is reaching epidemic proportions (Yusuf et al., 2001). These factors all contribute to the development of CVD (Koh et al., 2005), including coronary artery disease (CAD) and stroke, and have been associated with high cardiovascular mortality and morbidity (de Las Fuentes, 2007).
Aims and objectives
The Literature Review
- The Epidemiology of Cardiovascular disease
- Cardiovascular risk in South Asian Indians
- Cardiovascular risk in South Africans
- The Metabolic Syndrome
- THE PATHOPHYSIOLOGY OF THE METABOLIC SYNDROME
- THE PREVALENCE OF THE METABOLIC SYNDROME
- THE METABOLIC SYNDROME IN SOUTH ASIAN INDIANS
- THE METABOLIC SYNDROME IN SOUTH AFRICANS
- The use of echocardiography as a diagnostic tool for early detection of cardiac structural and functional abnormalities
- The Metabolic syndrome and cardiac structure
- Echocardiography and SEAT thickness
- Single Nucleotide Polymorphisms and the Metabolic syndrome
- LIPOPROTEIN LIPASE
- THE HUMAN PARAOXONASE ( PON) AND THE PON-1 GENE
The metabolic syndrome (MS) has been described as a heterogeneous (Zimmet et al., 2001) grouping of several risk factors (hypertension, dyslipidemia, glucose intolerance, abdominal obesity). LPL variants have also been associated with obesity in South Indian Asians (Radha et al., 2007).
Rationale
Outline of the thesis
64 gender, anthropometry and certain lifestyle habits and the presence or absence of metabolic syndrome or its individual components. This includes an in-depth discussion of each of the investigated gene polymorphisms and their intermediate expressions in this study. The results follow (chapter 3), which include the prevalence of risk factors, the prevalence of metabolic syndrome and echocardiographic changes, and finally the genetic analysis of the studied PON-1 and lipoprotein lipase polymorphisms.
The discussion and conclusion are found in chapter 4 and focus on the molecular mechanisms for the development of the metabolic syndrome, as well as the possible mechanisms underlying cardiac structural changes.
CHAPTER TWO
Materials and Method
Study design
- Study population and location
- Classification of normal controls
- Study period
- Sampling strategy
- SAMPLING
This was an analytic cross-sectional study in which a random sample of participants from the Phoenix community was selected and subdivided into two categories based on the presence or absence of the metabolic syndrome (outcome). Although the incidence and prevalence of Type 2 Diabetes Mellitus (DM) have been studied on limited occasions in the Indian population, there has been no recent community-based evaluation of the residents of the Phoenix community. 67 the transition in society's socio-economic status has detrimentally contributed to the acceleration of atherosclerotic disease.
DR Prakaschandra was responsible for most of the data collection, including performing the echocardiographic examination and genetic analysis (nucleic acid extraction, allele-specific polymerase chain reaction, sequencing and analysis).
Statistical planning
- Sample size
Only one member of each household was included in the study to improve generalizability to the Phoenix population. In order to obtain sufficient numbers in all strata without enrolling more than one individual in the household, the field worker followed a prescribed guideline (Appendix 1) when visiting a randomly selected household.
Data collection methods and tools
- Recruitment process and administration of the STEPS Questionnaire
- Questionnaire parameters
Demographic information included age, gender, education, income, number of people living in the household activity profiles, and dietary patterns. Other information included a history of DM, hypertension, or an immediate family history of cardiovascular risk factors.
The Clinical Examination
- Blood pressure and heart rate
- The diagnosis of systemic hypertension and pre-hypertension
- Anthropometry measurements
- HEIGHT
- WEIGHT
- BODY MASS INDEX
Systemic hypertension was defined according to Joint National Committee VII (JNC VII) criteria as a BP ≥140/≥90 mmHg and/or current antihypertensive treatment (Chobanian et al., 2003) or a self-report of previously diagnosed hypertension. This was measured to the nearest 0.1 cm using a metal tape measure attached to a wall and a flat headboard perpendicular to the wall to ensure an accurate reading. Subjects were measured without shoes, heels against the wall and the angle at eye level with the external auditory meatus.
This was determined to the nearest 0.5 kg on a balance scale, with the subject wearing light clothing and no shoes.
Biochemical measurements
- Definition of Impaired fasting glucose concentration, prediabetes, insulin resistance and Type 2 Diabetes Mellitus
- THE DIAGNOSIS OF INSULIN RESISTANCE
- DEFINITION OF DYSLIPIDAEMIA
74 The glucose concentration after the 2-hour glucose tolerance test is in the range of 7.8mmol/l – 11.0mmol/l. Subjects with IFG and IGT, without a prior history of DM, were classified as those in the prediabetic stage, according to the guidelines from the Report of the International Expert Committee on the role of A1C analysis in the diagnosis of DM (2009). Subjects who had no previous history of DM but had a fasting plasma glucose concentration ≥7.0 mmol/L (Genuth et al; 2003), a 2-hour fasting glucose concentration of ≥11.1 mmol/l and/ or current medical therapy with an oral hypoglycemic agent and/or insulin, were classified, according to the established American Diabetes Association (2006) criteria for defining DM, as diabetic.
Although the HOMA-IR index is inferior to the clamping technique in terms of accuracy, the utility of this index makes it possible to study a large number of subjects in a fasting state, with a single.
The diagnosis of the Metabolic Syndrome
- Diagnosis using the NCEP ATP III criteria
- Diagnosis using IDF criteria
- Diagnosis using the “Harmonized” criteria
76 a composite of the ATP III and IDF criteria: this was aptly called the 'Harmonizing criteria'. Therefore, we decided to use the three common definitions of MS in our study, as the previous definitions have been modified or replaced. The presence of metabolic syndrome as determined by the NCEP APTIII criteria was defined as 3 or more of the following 5 risk factors in Table 2-1. 77 hypertension or DM were included, regardless of the values obtained during the clinical examination or biochemistry.
This definition is identical to the NCEP ATP III definition, except that ethnicity-specific cutoffs (IDF waist circumference) for waist circumference measurements are included.
Echocardiography
- Two-dimensional and M-Mode measurements
- Doppler Echocardiography
- CALCULATION OF THE LEFT VENTRICULAR END DIASTOLIC PRESSURE USING TDI
- Definition of diastolic abnormalities and diastolic dysfunction
- Sub-epicardial adipose tissue thickness measurement
- Intra-observer variability
81 The left ventricular (LV) internal dimensions and the thickness of the posterior and septal wall were measured according to published guidelines (Schiller et al., 1989). Trans-mitral inflow velocities were used to detect and quantify diastolic dysfunction and were obtained using Pulsed-wave (PW) Doppler in the apical 4-chamber view with the sample volume placed between the tips of the mitral valve leaflets (Quinones et al ., 2002). They also recommended that a measurement of the LAVI be used with the cut-off point, according to the Heart Failure and Echocardiography Societies of the European Society of Cardiology (Paulus et al., 2007), with a LAVI > 40 ml/m2 as indicating LV diastolic dysfunction.
Epicardial fat thickness was measured in the right ventricular free wall in the parasternal long and short axis as suggested by Iacobellis et al (2003) [Figure 2.3].
The Genetic analysis
- Sample collection
- Selection of genes and SNPs for analysis
- DNA isolation
- DNA QUANTIFICATION
- Genotyping
- RATIONALE FOR CHOICE OF THE GENOTYPING METHOD
- PRINCIPLES FOR HOMOGENOUS REAL-TIME SNP GENOTYPING USING HYBRIDIZATION PROBES
- PRINCIPLES FOR AND MELTING CURVE ANALYSIS
- REAL-TIME POLYMERASE CHAIN REACTION AND MELTING CURVE ANALYSIS The real-time polymerase Chain reaction (PCR) was performed according to the
- NEGATIVE CONTROLS
- Gel electrophoresis
- Genotyping by sequencing
- PURIFICATION OF PCR PRODUCTS
- SEQUENCE REACTION
- PURIFICATION OF SEQUENCING PRODUCTS (PLATE CLEAN-UP)
A summary of the primer and probes used for the PCR amplification and melting curve analysis is shown in Table 2.5 below. The multiwell plate was then sealed, centrifuged, and then transferred to the plate holder of the Roche Light Cycler 480 Instrument, and the real-time PCR reaction was initiated. Real-time observation of amplification was possible as fluorescence was measured at the end of the annealing period for each cycle.
Purification of the sequencing products was performed on the same day as the sequencing reaction.
Statistical analysis
Receiver operator characteristic (ROC) curve analysis was used to determine the discriminating capacity of biochemical, anthropometric measurements, genotypes and echocardiography parameters in determining the presence of MS. The area under the curve (AUC) was used to determine the predictive effectiveness of each of these variables. The analysis was performed without prior knowledge of the grouping of subjects into those with and without MS. Gene counts were used to estimate genotype and allele frequencies and were expressed as percentages of the total.
Nonparametric variables were expressed as mean ranks, and the Kruskal–Wallis test was used to determine significance.
Ethical considerations
Median values for MS components as well as echocardiography variables were compared between the three genotype groups.
CHAPTER THREE Results
Demographic data
The study involved 1428 randomly selected subjects from the suburb of Phoenix, which is the situation in the EThekwini Municipality of KwaZulu-Natal. The sample was dominated by women, who made up 72% of the sample, while the male component made up 28%. The highest number of subjects within the sample was in the age group 45-54 years (Table 3-1).
Prevalence of cardiovascular risk factors
- Family History of Cardiovascular risk factors
- Anthropometric parameters
- Lifestyle and behavioural characteristics
- Clinical observations
- Biochemical parameters
- BLOOD GLUCOSE AND INSULIN
- Overall risk factor profile
- Correlation between cardiovascular risk factors
- The age distribution of cardiovascular risk factors
A positive family history for CV risk factors (obesity, type 2 diabetes [DM], hypertension, myocardial infarction/stroke, and heart failure [CHF]) was recorded in all subjects. A significant increase in the prevalence of hypertension was observed from the first age group (6%) to the 5th (71%). There was a total number of 455 (32%) subjects who were classified as diabetic, with the highest prevalence (49%) observed in the 55-64 age group.
There were approximately 3% of subjects in the 55-65 age group who also had 4 risk factors.
The Metabolic Syndrome
- The prevalence of the Metabolic Syndrome
- The Kappa statistic
- The frequency and clustering of MS components
- The frequency of individual MS components in males and females
- Clinical and metabolic criteria in relation to the glycaemic profile
123 Table 3-9: Sex-wise prevalence of the Metabolic Syndrome according to IDF, NCEP ATP III and Harmonized criteria. As a result of applying the IDF criteria, the largest number of subjects were identified with MS. Analysis of the frequency of the MS components between the sexes in Figure 3.6, using the IDF criteria, indicated that there was a significantly higher incidence of MS in women, compared to men (p < 0, 05); with women more likely to present with abdominal obesity (p < 0.05).
130 FIGURE 3.6: Gender distribution of Metabolic Syndrome components in subjects with MS using IDF criteria.
The Echocardiographic analysis
- Echocardiography and Cardiovascular risk factor profile
- Correlation co-efficients between echocardiography variables
- Diastolic abnormalities and dysfunction in the sample
- Echocardiography and the Metabolic Syndrome
- The determination of normal echocardiography values in the Phoenix population
In the presence of CV risk factors, mean left atrial (LA) dimension, left atrial volume index (LAVI), left ventricular mass (LVM), left ventricular mass index (LVMI) and SEAT thickness were significantly higher (p < 0.05). Similarly, transmitral flow rates (Em and Em/Am) were lower in subjects with CV risk factors. Significant differences were observed between those with and without CV risk factors in posterior wall thickness, LV mass and LVMI in subjects aged 25–64 years.
LVEDD, LVESD, and EF were within normal limits in both subjects with and without MS, with significant differences in LVEDD and EF observed between the two groups (Table 3-18).
Mean 95 th
- The analysis of gene polymorphisms .1 The Hardy-Weinberg Equilibrium
- LPL polymorphisms
- PON-1 polymorphisms
- Distribution of polymorphisms in subjects with/without Metabolic Syndrome
- The association between polymorphisms and obesity
The frequency of the homozygous wild-type (SS) genotype of the S447X polymorphism was found in 25% of subjects (Table 3-26). There was a much higher prevalence of the heterozygous variant, which was found in 73% of subjects with a small number (2%) presenting with the homozygous mutant genotype (Figure 3.8). The frequency of the homozygous wild-type (LL) genotype of the L55M polymorphism was 34% in this sample (Table 3-26), with a high prevalence of the heterozygous (LM) variant (64%).
The frequency of the presence of wild-types and mutations in obese subjects using WHO and Asian cut-offs: there was a significantly higher number of female subjects with the Q192R mutation.
CHAPTER FOUR
Discussion, Conclusion and Limitations
Cardiovascular risk factor profiles and Lifestyle patterns
- Smoking
- Hypertension
- Positive family history for cardiovascular risk factors
- Lipid profiles
- Summary of cardiovascular risk factors and clustering
The high prevalence of cardiovascular risk factors in urban Asian Indians has been well documented previously (Balarajen, 1991; Enas et al., 1992; Cappucio, 1997; Yusuf et al., 2001). Currently, there is a global increase in the prevalence of obesity (Rheeder, 2006; . Goedecke et al., 2005), especially in developing countries. Recent estimates from the South African Demographics and Health Survey published in 2002 (Puoane et al., 2002).
The same was evident when comparing with the prevalence of DM among Asian Indians (17.4%) in the United States as reported by Misra et al (2010).
The Metabolic Syndrome
- The prevalence of the Metabolic Syndrome
- The distribution and relationship of individual MS components
Currently, there are still disagreements about the diagnostic criteria of MS and debate about whether MS is a true syndrome or a mixture of several phenotypes (Alberti et al., 2009). The increase in incidence with advanced age was most evident in women (Figures 3-4), and has also been reported in women of other ethnicities (Ford et al., 2004; Ford 2005). This pattern was also recently described by Misra et al. 2010), describing this as the characteristic Asian Indian male pattern, where dysmetabolism is observed early in life.
McNeill et al., (2005) described a risk gradient for the development of CVD associated with an increasing number of MS components.
