Ethical considerations
The work has been done according to Helsinki Declaration, and the study’s objectives and procedures were approved by the independent ethics committee at the Algerian National Agency for the Development of Health Research (ANDRS) (since renamed as the Thematic Agency of Research in Health Sciences, ATRSS). All participants provided written informed consent prior to enrolment.
Study population
Participants were recruited during the ISOR (InSulino-résistance à ORan) study, a population-based, cross-sectional study of a representative sample of 787 individuals (378 men and 409 women, mean age 44.1 ± 10.1 years) recruited in the city of Oran, Algeria, from 2007 to 2009 [13].
Data collection
Data were collected using a preconceived questionnaire on socioeconomic information, physical activity (The level of physical activity was defined in quartiles as “none,” “low,” “medium,” and “high” after summing exercise scores for sporting activities, walking, housework, and physical activity at work), tobacco use and alcohol intake, past medical history and family history, current medications, as well as anthropomorphic characteristics including height, weight, waist circumference, hip circumference, and blood pressure. Height and weight were measured while the subject was barefoot and lightly dressed. The BMI was calculated according to the Quetelet equation [14]. Systolic and diastolic blood pressure values (systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively) were measured on the right arm with the subject in the sitting position, using a standard mercury sphygmomanometer. Measurements were made before and after completion of the questionnaire, with an interval of at least 10 min. The mean value of the blood pressure readings was considered for analysis. Regarding tobacco use, participants were categorized as either smokers (i.e., individuals reporting at least one cigarette per day) or non-smokers. After a 12-h overnight fast, blood was collected aseptically via venipuncture in an EDTA tube for DNA extraction and subsequent molecular analysis, and in a heparin tube for biochemistry tests [13].
Metabolic syndrome diagnosis criteria
In this study, we have adopted the definition of metabolic syndrome according to the criteria of the “National Cholesterol Education Program - Adult Treatment Panel III” (NCEP ATPIII) [15]; the metabolic syndrome is diagnosed when a subject has three or more of the following risk factors:
- Abdominal obesity: waist circumference > 102/88 cm (men/women);
- Triglyceride level ≥ 150 mg/dL (1.69 mmol/L), fibrate treatment excluded;
- HDL cholesterol < 40/50 mg/dL (1.04/1.29 mmol/L) (men/women);
- Blood pressure ≥ 135/85 mmHg or treatment for hypertension;
- Fasting glucose ≥ 110 mg/dL (6.1 mmol/L), or treatment for diabetes.
Type 2 diabetes diagnosis criteria
The definition adopted in this study is that of the American Diabetes Association (ADA) [16]
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Fasting plasma glucose ≥ 7.0 mmol/L twice after 8 h of fasting
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Occasional blood glucose ≥ 11.1 mmol/L in the presence of symptoms of hyperglycemia (polyuria, polydipsia, unexplained weight loss)
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Diabetics declared under treatment including oral antidiabetic drugs and/or insulin
High blood pressure diagnosis criteria
Hypertension (HBP) has been defined according to the WHO criteria [17]: mean systolic blood pressure [SBP] greater than 140 mmHg and/or mean diastolic blood pressure [DBP] greater than 90 mmHg, and/or self-reported current treatment for hypertension with antihypertensive drugs.
Obesity diagnosis criteria
The body mass index (BMI) is calculated according to the Quetelet equation. A subject is considered obese if he has a BMI greater than or equal to 30 kg/m2 [14].
Biochemistry and molecular testing
A multichannel analyzer and dedicated kits (Humastar®, HUMAN Diagnostics, Wiesbaden, Germany) were used for the colorimetric, enzymatic measurement of cholesterol (kit: monotest cholesterol with cholesterol esterase, cholesterol oxidase and peroxidase), triglycerides (kit: peridochrom triglyceride with glycerol phosphate oxidase and peroxidase), and glucose (kit: glucose, glucose oxidase, and peroxidase). Plasma LDL cholesterol levels were calculated according to the Friedewald equation. High-density lipoprotein cholesterol levels were measured after sodium phosphotungstate/magnesium chloride precipitation of chylomicrons and VLDL and LDL cholesterol and then centrifugation. Plasma insulin levels were measured using a microparticle enzyme immune assay running on an AxSYM analyzer (Abbott Laboratories, Abbott Park, Illinois, USA).
Genomic DNA was extracted from white blood cells by using the Stratagene® kit (Agilent Technologies, Les Ulis, France), according to the manufacturer’s protocol. The APOE SNPs (rs429358, rs7412, rs439401, and rs4420638) were genotyped using KASPar technology (KBioscience, Hoddesdon, UK) with the following probes:
rs429358: [GACATGGAGGACGTG[C/T]GCGGCCGCCTGGTGC],
rs7412: [GATGACCTGCAGAAG[C/T]GCCTGGCAGTGTACC],
rs439401: [GCCGGCACTCTCTTC[C/T]CCTCCCACCCCCTCA],
rs4420638: [TGCTACAC TTTTCCT[A/G]GTGTGGTCTACCCGA].
The genotyping success rates ranged from 93 to 96% [13].
Statistical analysis
Statistical analysis was performed with SAS 9.1 software (SAS Institute Inc., Cary, NC, USA). The Hardy-Weinberg equilibrium was tested using a χ2 test with one degree of freedom (d.f.). Some of the biochemical traits (fasting glucose levels, triglycerides, and insulin levels) were not normally distributed; we therefore log-transformed these parameters to obtain normal data distributions. Intergroup comparisons of means were performed with a general linear model, and multivariate logistic regression analyses were used to calculate the odds ratios for MetS, type 2 diabetes (T2D), high blood pressure (HBP), and obesity (Obes). The confounding variables were age, gender, smoking status, and physical activity. After Bonferroni correction, only associations with an uncorrected p value below 0.017 were considered to be statistically significant (i.e., 0.05 divided by the number of polymorphisms considered).
