Background: Glutathione S-transferases play a key role in the detoxification of persistent oxidative stress products which are one of several risks factors that may be associated with many types of disease processes such as cancer, diabetes, and hypertension. In the present study, we characterize the null genotypes of GSTM1 and GSTT1 in order to investigate the association between them and the risk of developing essential hypertension. Methods: We conducted a case-control study in Burkina Faso, including 245 subjects with essential hypertension as case and 269 control subjects with normal blood pressure. Presence of the GSTT1 and GSTM1 was determined using conventional multiplex polymerase chain reaction followed by gel electrophoresis analysis. Biochemical parameters were measured using chemistry analyzer CYANExpert 130. Results: Chi-squared test shows that GSTT1-null (OR = 1.82; p = 0.001) and GSTM1-active/GSTT1-null genotypes (OR = 2.33; p < 0.001) were significantly higher in cases than controls; the differences were not significant for GSTM1-null, GSTM1-null/GSTT1-active and GSTM1-null/GSTT1-null (p > 0.05). Multinomial logistic regression revealed that age ≥ 50 years, central obesity, family history of hypertension, obesity, alcohol intake and GSTT1 deletion were in decreasing order independent risk factors for essential hypertension. Analysis by gender, BMI and alcohol showed that association of GSTT1-null with risk of essential hypertension seems to be significant when BMI < 30 Kg/m2, in non-smokers and in alcohol users (all OR ≥ 1.77; p ≤ 0.008). Concerning GSTT1, GSTM1 and cardiovascular risk markers levels in hypertensive group, we found that subjects with GSTT1-null genotype had higher waist circumference and higher HDL cholesterol level than those with GSTT1-active (all p < 0.005), subjects with GSTM1-null genotype had lower triglyceride than those with GSTM1-active (p = 0.02) and subjects with the double deletion GSTM1-null/GSTT1-null had higher body mass index, higher waist circumference and higher HDL cholesterol than those with GSTM1-active/GSTT1-active genotype (all p = 0.01). Conclusion: Our results confirm that GSTT1-null genotype is significantly associated with risk of developing essential hypertension in Burkinabe, especially when BMI < 30 Kg/m2, in non-smokers and in alcohol users, and it showed that the double deletion GSTM1-null/GSTT1-null genotypes may influence body lipids repartition.
Keywords: Essential hypertension; GSTM1; GSTT1; Null genotypes; Burkina Faso
Herman Karim Sombié and Abel Pegdwendé Sorgho contributed equally to this work.
High blood pressure is the most common risk factor for cardiovascular mortality and morbidity worldwide and is thought to be responsible for just under 8 million deaths a year worldwide and nearly 100 million days of disability [[
There is considerable evidence that oxidative stress resulting from an imbalance between the generation of reactive oxygen species (ROS) and the body's antioxidant defense systems is involved in the pathophysiology of hypertension [[
Several cases-controls studies have reported that GSTT1 and/or GSTM1 null genotype were associated to the risk of developing hypertension in some populations, but rather the results are still controversial [[
The Internal Research Ethics Committee of CERBA/LABIOGENE and National Ethics Committee for Health Research of Burkina Faso approved the protocol of this case-control study. We recruited a total of 514 subjects, including 245 patients with essential hypertension and 269 subjects with normal blood pressure in the cardiology and general consultation departments at Saint Camille Hospital in Ouagadougou and at the Yalgado Ouédraogo University Hospital Center in Ouagadougou. All participants were from the central region of Burkina Faso and resided there.
Essential hypertension was determined by the cardiologist when no secondary cause of blood pressure elevation was present [[
When a subject met the above selection criteria, he was referred by the cardiologist to the principal investigator who was responsible for clearly explaining the study. Once his consent obtained in a free and transparent manner, using a standardized questionnaire completed throughout the study, data on anthropometric parameters, lifestyle, clinical and biological parameters were collected (see questionnaire in Supplementary file 3). The information collected mainly were age, sex, parents' ethnicity, occupation, weight, height, waist circumference, lifestyle, blood pressure, personal, family history, electrocardiogram data, cardiac echo-Doppler data and biochemical data.
Blood pressure measurements were performed using a manual aneroid sphygmomanometer and electronic cuffed sphygmomanometer by the cardiologist and the principal investigator. Blood pressure was taken on both arms in a sitting position after at least 20 min of rest. All measurements were made at least twice with a minimum of 5 min between two measurements. Blood pressure values were obtained by averaging the measurements.
Height and weight were measured and body mass index (BMI) was calculated by dividing the weight (Kilogram) by the square of the height (meters). BMI was used to determine obesity when BMI ≥ 30 kg/m
Waist circumference (WC) was determined by measuring the circumference of the abdomen when the subject has minimal breathing using a tape measure. Abdominal obesity was determined in men when WC is greater than 102 cm and in women when it is greater than 88 cm [[
Participants with a family history of hypertension were defined as those with at least one close family member hypertensive before the age of 60 years.
Each participant also had a venous blood sample of approximately 8 ml in two tubes (EDTA and tube without anticoagulant) for analysis. Sera from anticoagulant-free tubes were directly used for biochemical analyzes in the biochemistry laboratory (CYANExpert 130), and blood white cells from EDTA tubes were placed in cryotubes and stored at − 20 °C in the molecular biology laboratory until extraction of the DNA.
We used standard salt fractionation method as described by Miller and al. in 1988, to isolated genomic DNA from peripheral blood white cells [[
The presence (homozygous +/+ and heterozygous +/−) or absence (homozygous for deletion −/−) of the GSTM1 and GSTT1 genes has been determined according to the method described by Chen and al [[
Data analyses were performed by using Statistical Package for Social Sciences (SPSS Version 20.0) and Epi Info (Version 6.0).
Following values have been taken into account to determinate sample size using Epi Info Version 6.0: 95% of two-sided confidence level, 80% of power, odds ratio more than 1.7 ratio of controls to cases 1.1, the proportion of control group having null genotypes of GSTM1 and GSTT1 about 30%.
We expressed quantitative variables as mean ± standard deviation and comparison between groups was assessed with Student's t-test.
Genotypic frequencies were expressed as percentage and comparisons between cases and controls were done with the chi-squared test.
To research factors associated with risk of essential hypertension in our study and possible interactions between them, we performed a multinomial logistic regression analysis (forward stepwise method) by considering hypertensive status as a dependent variable and including the factors we thought were involved in the development of essential hypertension.
For all analyses, difference was statistically significant when p < 0.05.
The characteristics of the study population are given in Table 1. We included a case group of 245 subjects with a diagnosis of essential hypertension (111 males and 134 females; 50.14 ± 8.22 years old) and a control group of 269 normotensive individuals (129 males and 140 females; 48.69 ± 9.43 years old). Statistical analysis of the distribution by sex and means of age showed no significant differences between cases and controls (p > 0.05), indicating that there is homogeneity between groups.
General Characteristics of the study population
Parameters Total, 514 (100%) Cases, 245 (100%) Controls, 269 (100%) Gender (M/F) 240/274 111/134 129/140 0.59 Age (years) 49.38 ± 8.90 50.14 ± 8.22 48.69 ± 9.43 0.06 SBP (mmHg) 139.27 ± 30.32 166.24 ± 19.92 114.71 ± 11.35 < 0.001* DBP (mmHg) 83.77 ± 16.56 97.28 ± 12.09 71.47 ± 8.55 < 0.001* BMI (Kg/m2) 25.98 ± 6.19 28.35 ± 6.40 23.82 ± 5.11 < 0.001* WC (cm) 90.28 ± 12.21 96.28 ± 12.26 84.81 ± 9.25 < 0.001* Fasting Blood Glucose (mM) 5.15 ± 1.81 5.69 ± 1.56 3.75 ± 1.69 < 0.001* HDL-c (mM) 1.31 ± 0.60 1.40 ± 0.63 1.07 ± 0.42 0.001* LDL-c (mM) 2.90 ± 1.12 3.02 ± 1.09 2.61 ± 1.14 0.04* Total Cholesterol (mM) 4.90 ± 1.29 5.10 ± 1.18 4.38 ± 1.44 0.001* Triglycerides (mM) 1.18 ± 0.75 1.25 ± 0.77 1.00 ± 0.65 0.51 Creatinine (μM) 98.40 ± 23.82 96.80 ± 25.54 106.10 ± 10.38 0.26 Calcium (mM) 2.54 ± 1.04 2.51 ± 1.09 2.68 ± 0.83 0.63 Magnesium (mM) 0.79 ± 0.29 0.81 ± 0.31 0. 69 ± 0.11 0.22 Sodium (mM) 140.46 ± 5.47 139.98 ± 5.38 142.54 ± 5.62 0.16 Potassium (mM) 4.16 ± 0.82 4.01 ± 0.71 4.80 ± 1.00 0.03* Chlorine (mM) 102.78 ± 5.99 102.24 ± 5.64 105.17 ± 7.14 0.14
Values are reported as means ± standard deviation for continuous variables; Statistical analysis (Cases versus controls) by t test or chi-square; *: significant difference between groups (p < 0.05); MD Means difference, CI Confidence interval, SBP Systolic blood pressure, DBP Diastolic blood pressure, WC Waist circumference, HDL-c High density lipoprotein cholesterol, LDL-c Low density lipoprotein cholesterol, mM Millimolar, μM Micromolar
We found that means of body mass index, waist circumference, serum levels of blood sugar, Total cholesterol, LDL Cholesterol, HDL cholesterol were higher in hypertensive compared to normotensive group and differences were significant (all p < 0.05). These results suggest that many of our patients with essential hypertension are overweight or obese, and that many may also have diabetes and / or hypercholesterolemia, although it is difficult for us to confirm this based on our unique biochemical assay.
We also found that serum levels of Potassium was higher in normotensive group compared to hypertensive (p = 0.03), but there was no significant difference in level of Triglycerides, creatinine, Calcium, Sodium, Magnesium and Chlorine between the two groups (all p > 0.05).
The Table 2 shows the distribution of GSTM1 and GSTT1 variants in the study population. A total of 514 subjects (245 cases and 269 controls) were genotyped for the deletion genotype of two GST isoforms. In the general study population, we found that the frequency of GSTM1-active and GSTT1-active were 71.60 and 37.94% respectively; those of GSTM1-null and GSTT1-null were 28.40 and 62.06% respectively. Based on the ratio controls to cases, odds ratio, and frequencies of GSTM1-null and GSTT1-null, the genetic power calculator indicated that the sample size is large enough to perform a case-control analysis with 85% power for GSTT1, those of GSTM1 is less than 40%.
Distribution of genotypic frequencies for GSTM1 and GSTT1 in the study population
Genotypes Total 514 (100%) Cases 245 (100%) Controls 269 (100%) OR CI 95% 368 (71.60) 183 (74.69) 185 (68.77) 1.00 146 (28.40) 62 (25.30) 84 (31.23) 0.74 0.50–1.09 0.14 194 (37.74) 75 (30.61) 119 (44.24) 1.00 320 (62.26) 170 (69.39) 150 (55.76) 1.79 1.25–2.58 0.001* 125 (24.32) 45 (18.37) 80 (29.74) 1.00 69 (13.42) 30 (12.25) 39 (14.50) 1.36 0.75–2.49 0.35 243 (47.28) 138 (56.32) 105 (39.03) 2.33 1.50–3.65 < 0.001* 77 (14.98) 32 (13.06) 45 (16.73) 1.26 0.71–2.26 0.45
Analysis by chi-square to obtain odds ratio values (OR) and confidence interval; (+): active; (−): null; CI Confidence interval, OR Odds ratio; #: reference; *: significant difference between groups (p < 0.05)
When we compared these frequencies between cases and controls, we found that subjects with GSTT1-null genotype (69.39% versus 55.39%; OR = 1.82; p = 0.001) and GSTM1-active/GSTT1-null genotype (56.32% versus 39.03%; OR = 2.33; p < 0.001) were more present in case group than controls and difference between the two group was significant. But we didn't find a significant difference between cases and controls concerning GSTM1-null genotype (25.30% versus 31.23%; OR = 0.74; p = 0.14) and the double deletion GSTM1-null/GSTT1-null (13.06% versus 16.73%; OR = 1.26; p = 0.45).
The Table 3 presents multinomial logistic regression for essential hypertension risk factors in our study population to obtain adjusted odds ratio values and confidence intervals. We found that advanced age (≥ 50 years; OR = 5.33; p < 0.001), central obesity (OR = 4.80; p < 0.001), family history of hypertension (OR = 4.61; p < 0.001), obesity (OR = 3.95; p = 0.001), alcohol intake (OR = 2.16; p < 0.001) and GSTT1 deletion (OR = 1.81; p = 0.001) were in decreasing order independent risk factors for developing essential hypertension in our general study population.
Multinomial logistic regression for risk analysis of essential hypertension
Factors OR CI 95% Obesity 3.95 2.48–6.29 0.001* Central obesity 4.80 3.23–7.14 < 0.001* Alcohol intake 2.16 1.5–3.1 < 0.001* Smoking 1.24 0.49–3.16 0.8 Sex M 0.89 0.63–1.27 0.59 Family history of HTA 4.61 3.17–6.69 < 0.001* Age ≥ 50 years 5.33 3.61–7.86 < 0.001* 1.81 1.26–2.60 0.001*
*Significant difference between groups (p < 0.05); CI, Confidence interval, OR Odds ratio
Considering BMI, alcohol and smoking difference in essential hypertension, we further stratified genotyping results by BMI, smoking and alcohol status (Table 4). Interestingly results showed that association between GSTT1-null and essential hypertension seems to be significant when BMI < 30 Kg/m
BMI, age, and sex stratified analysis of association between GSTM1 and GSTT1 variants with essential hypertension
Genes Parameters groups Variants ( OR CI (95%) active BMI < 30 Kg/m2 Cases 38 124 – Controls 72 167 0.71 0.45–1.12 0.17 BMI ≥ 30 Kg/m2 Cases 24 59 – Controls 12 18 0.61 0.25–1.45 0.36 BMI < 30 Kg/m2 Cases 113 49 – Controls 135 104 1.77 1.16–2.70 0.008* BMI ≥ 30 Kg/m2 Cases 57 26 – Controls 15 15 2.19 0.93–5.14 0.07 Smoking YES Cases 15 50 – Controls 13 48 1.10 0.47–2.57 0.83 Smoking NO Cases 47 133 – Controls 71 137 0.68 0.43–1.05 0.09 Smoking YES Cases 46 19 – Controls 37 24 1.57 0.74–3.29 0.26 Smoking NO Cases 124 56 – Controls 113 95 1.86 1.22–2.82 0.003* Alcohol intake YES Cases 26 92 – Controls 27 52 0.47 0.25–0.88 0.07 Alcohol intake NO Cases 36 91 – Controls 57 133 0.99 0.60–1.63 0.80 Alcohol intake YES Cases 86 32 – Controls 45 38 2.26 1.25–4.10 0.007* Alcohol intake NO Cases 84 43 – Controls 105 81 1.50 0.94–2.40 0.09
Analysis by chi-square to obtain odds ratio values (OR) and confidence interval; CI Confidence interval, OR Odds ratio; *: significant difference between groups (p < 0.05)
In addition, concerning GSTT1, GSTM1 and cardiovascular risk markers levels in hypertensive group we compared the average of cardiovascular risk markers between the GSTM1 and GSTT1 null and active genotypes. We found that hypertensive individuals with GSTM1-null genotype had a lower average triglyceride than GSTM1-active genotypes; GSTT1-null genotype had a higher average waist circumference and HDL cholesterol than GSTT1-active genotype and the double deletions GSTM1-null/GSTT1-null have higher body mass index, higher waist circumference and higher HDL cholesterol than GSTM1-active/GSTT1-active (data not shown). Supplementary Table 1.
There is increasing interest in the role of Glutathione S-transferase polymorphism as a contributory factor to oxidative stress and consequently to cellular damage and physiological anomalies such us cancers, diabetes, cardiovascular disorders [[
Graph: Fig. 1 GSTM1 and GSTT1 genes deletion frequency in African countries [[
In our study we analyzed also the relationship between Glutathione S-transferase M1 and T1 genes deletion and their connection with essential hypertension and any complications that may have accompanied this disease in Burkina Faso. Association between GSTM1 and GSTT1 polymorphisms and cardiovascular disorders has long been studied. Some studies have shown that GSTM1 [[
Summary of previous studies examining GSTM1 and GSTT1 polymorphisms and hypertension risk
Country Population (Cases/controls) Ethnicity Genotyping method association with the risk of hypertension Years First author/ references Portugal Hypertension/congestive heart failure (94/207) Caucasian PCR 2007 Marinho [ Egypt General population (40/40) African Multiplex PCR 2009 Bessa [ Italy Older subjects (255/99) Caucasian PCR 2009 Capoluongo [ India Tea garden workers (223/236) Asian Multiplex PCR 2011 Borah [ Italy General population (193/210) Caucasian Multiplex PCR 2011 Polimanti [ Korea General population (227/130) Asian PCR 2011 Han [ United Arab Emirates General population (30/33) Asian Multiplex PCR none 2012 Hussain [ Korea Lead-exposed workers (258/497) Asian Multiplex PCR 2012 Lee [ Slovenia hypertension/type 2 diabetes (1015) Caucasian Multiplex PCR 2014 Petrovic [ India General population (138/116) Asian Multiplex PCR 2015 Abbas [
Essential hypertension is a complex disease, with many risk factors [[
In addition, we also compared in hypertensive group, means of certain cardiovascular risk markers between null and active genotypes of GSTM1 and GSTT1 genes. We found significantly lower triglyceride in GSTM1-null genotype compared to GSTM1-active; we also observed in GSTT1-null subjects, significantly higher waist circumference and serum HDL cholesterol compared to GSTT1-active genotype. The double deletion GSTM1-null/GSTT1-null was associated with body mass index, waist circumference and serum HDL cholesterol increasing. These results suggest a probable association of Glutathione S-transferase M1 and T1 genes deletion with body fat and serum cholesterol level. There is not a lot of study about these association, while Almoshabek and al. reported in young age Saudis that frequencies of GSTM1-active/GSTT1-null (OR = 2.70; p < 0.001) and GSTM1-null/GSTT1-null (OR = 2.43, p = 0.018) were significantly higher in overweight/obese as compared to normal weight [[
In conclusion, our study suggests the significant association between GSTT1-null genotype and the risk of developing essential hypertension in Burkinabe population, therefore the important role of oxidative stress in the development of essential hypertension. The study also suggests that the double deletion GSTT1-null/GSTM1-null may affect body lipids repartition in hypertensive. However large-scale study will be necessary to fully comprehend the role of GSTM1 and GSTT1 variant in the development of essential hypertension.
This work was supported by West African Economic and Monetary Union (WAEMU) through the "Programme d'appui et de développement des centres d'excellence régionaux" (PACERII) and "Centre national de l'Information, de l'Orientation Scolaire et Professionnelle, et des Bourses" (CIOSPB) especially for researcher life stipend. Financial support for reagents and consumables was provided by Italian Episcopal Conference (CEI). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
The authors wish to thank all participants in this study. A deep gratitude to all the staff of Saint Camille Hospital of Ouagadougou (HOSCO) and Biomolecular Research Center Pietro Annigoni (CERBA) for technical support.
Study concept and design: JKK, HM and JS. Sampling and Laboratory analysis: APS, HKS, SY, DS, ITK, PB, IN, ETHDA and JKK. Statistical analysis and interpretation of data: HKS, APS, ATY, DT. Drafting of the manuscript: FWD, HKS, APS, BMN, AKO and JS. Critical revision of the manuscript for important intellectual content: AKO, HKS, DT, FWD, BMN, HM, JKK, PZ and JS. Administrative, technical, and material support: FWD, ATY, JKK and JS. Study supervision: JKK, HM, PZ and JS. The Corresponding Author declare that the manuscript has been read and approved by all named authors and that the order of authors listed in the manuscript has been approved by all of us.
The dataset generated in this study is available from NCBI Nucleotide under the accession number LC517160.1.
The present study has been approved by the National Ethics Committee for Health Research of Burkina Faso.CERS20186065, 6 June 2018, retrospectively registered. Free and written consent was obtained from all participants of this study. The anonymity and confidentiality of the patients were respected as stated in the IRB (Institutional Review Board) protocol.
Not Applicable.
The authors declare that they have no competing interests.
Graph: Additional file 1: Fig. S1. Locations of GSTM1 , GSTT1 and β-globin genes and corresponding bands in electrophoresis gel. This file shows locations of GSTM1 , GSTT1 and β-globin genes on chromosomes and corresponding bands. The number 1 through 19 represents individual sample and M represents Molecular weight marker. The strategy to identify presence or absence of GSTM1 or GSTT1 was as followed: to validate a PCR product (corresponding to a sample), we must have a band corresponding to β-globin and presence or absence of GSTM1 or GSTT1 was indicated respectively by the presence or absence of bands corresponding for each gene.
Graph: Additional file 2: Table S1. Distribution of cardiovascular risk markers according to GSTM1 and GSTT1 variants in hypertensive group. This table show and compare average of cardiovascular risk markers such as BMI, WC, serum level of blood sugar, TC, HDL-c, LDL-c and Triglycerides according to GSTM1 and GSTT1 variants in hypertensive group.
Graph: Additional file 3 Data collection. Information sheet and questionnaire. This file shows the fact sheets used to explain the study during the recruitment of the participants and the questionnaire which served for the data collection.
• BMI
- Body mass index
• CERBA
- Pietro annigoni biomolecular research center
• DBP
- Diastolic blood pressure
• EDTA
- Ethylenediaminetetraacetic
- GSTA
- Glutathione S-transferases alpha
- GSTK
- Glutathione S-transferases kappa
- GSTM1
- Glutathione S-transferases mu 1
- GSTO
- Glutathione S-transferases omega
- GSTP
- Glutathione S-transferases pi
- GSTS
- Glutathione S-transferases sigma
- GSTT1
- Glutathione S-transferases theta 1
- GSTZ
- Glutathione S-transferases zeta
• HDL-c
- High-density lipoprotein cholesterol
- LABIOGENE
- Laboratory of molecular biology and genetics
• LDL-c
- Low-density lipoprotein cholesterol
• MD
- Means difference
• PCR
- Polymerase chain reaction
• ROS
- Reactive Oxygen Species
• SBP
- Systolic blood pressure
• SD
- Standard deviation
• SPSS
- Statistical package for the social sciences
• TC
- Total cholesterol
• WC
- Waist circumference
Supplementary information accompanies this paper at 10.1186/s12881-020-0990-9.
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By Herman Karim Sombié; Abel Pegdwendé Sorgho; Jonas Koudougou Kologo; Abdoul Karim Ouattara; Sakinata Yaméogo; Albert Théophane Yonli; Florencia Wendkuuni Djigma; Daméhan Tchelougou; Dogfounianalo Somda; Isabelle Touwendpoulimdé Kiendrébéogo; Prosper Bado; Bolni Marius Nagalo; Youssoufou Nagabila; Enagnon Tiémoko Herman Donald Adoko; Patrice Zabsonré; Hassanata Millogo and Jacques Simporé
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