Association between APOL 1 Risk Genotypes and Left Ventricular Hypertrophy among Sub-Saharan Africans in Trypanosoma Brucei Gambiense Endemic Rural Area

Purpose. The relationship between APOL1 variants and cardiovascular disease remains controversial, thus, this study assessed the association between APOL1 high-risk genotypes and left ventricular hypertrophy (LVH) among sub-Saharan African in T.b. gambiense endemic area. Methodology. We enrolled 238 subjects living in the region of Masimanimba, an endemic area of T.b.gambiense HAT. We evaluated the association between LVH on echocardiography and the status of APOL1 genes in participants with or without HAT. APOL1 high-risk genotype (HRG) was defined as the presence of two risk variants (G1/G1, G2/G2, or G1/G2), and a low-risk genotype (LRG) with the presence of 0 or 1 single variant. Student’s and Pearson's Chi 2 tests or Fisher’s exact test were used to compare means and proportions. The Wilcoxon/Mann–Whitney test was used to compare medians. A multivariate logistic regression model was used to identify independent determinants of LVH. Odds ratios were provided with their 95% confidence intervals (Cis). Statistical significance was set at p < 0.05, based on 2-tailed test.


Association between APOL 1 Risk Genotypes and Left Ventricular Hypertrophy among Sub-Saharan Africans in
Trypanosoma Brucei Gambiense Endemic Rural Area Introduction Previous reports have established the relationship between APOL1 risk variants and CKD in African Americans (AA) and sub-Saharan Africans (1-10).However, this is not the case for subclinical and clinical cardiovascular diseases (CVD) for which the data are contradictory.Indeed, some studies demonstrated a positive correlation, while others observed no significant correlation (11)(12)(13)(14).This disparity would be linked to the difference in methodological approaches and the genetic heritage of AA who constitute a mixed population having approximately 80% of African ancestry and 20% of European ancestry (11,14).The lack of available data on APOL1 variants and CVD in sub-Saharan Africa requires continued research to clarify this relationship where human African trypanosomiasis (HAT) is endemic.Sub-Saharan Africans would not have the same susceptibility to CVD as AA, knowing that these variants do not confer protection against Tb gambiense which is prevalent in West and Central Africa (2)(3)(4).Therefore, we aimed to investigate the association between APOL1 risk variants and left ventricular hypertrophy (LVH) in a sample of the Bantu population in a Tb gambiense endemic area, in Central Africa.

Study Design and participant sampling
We conducted this cross-sectional study from April 2019 to April 2021 in an HAT-endemic rural region of Masimanimba (Democratic Republic of Congo (DRC)), where the inhabitants belong to the Bantu ethnic group.A total of 238 individuals aged 15 years or older were enrolled using a multi-stage sampling strategy.They were accrued from two Health Zones in the region: Masimanimba and Mosango.Ten Health areas (six in Masimanimba and four in Mosango) were randomly selected.The enrolment included only subjects justifying a stay of at least one year in the region.Pregnant women were excluded from this study.

Clinical measurements
Sociodemographic data (age, sex, ethnicity, and occupation) and history of cardiovascular risk factors (hypertension, diabetes, tobacco and alcohol use, and low vegetable and fruit consumption) were obtained through interviews.Anthropometric variables (height, weight, waist circumference, and body mass index), blood pressure, and heart rate were measured during the physical examination.Brachial and ankle blood pressure was measured using an automated sphygmomanometer (Omron M2 Basic (HEM-7120-E), Kyoto, Japan) with an appropriate size secured on each arm and ankle in turn after the subject had rested in the supine position for at least 5 min.The average of three measurements at 1 min intervals was used in the analysis.Pulse pressure (PP) is the difference between systolic blood pressure (SBP) and diastolic blood pressure (DBP).The ankle-brachial index (ABI) was calculated as the ratio of the highest systolic blood pressure at the two ankles to the highest systolic blood pressure at the two arms.

Paraclinical and genetic variables assessment
Laboratory variables (serum creatinine, glucose, total cholesterol, HDL and LDL-cholesterol, uric acid, C-reactive protein (CRP), and triglycerides) were measured with a Cobas C 111 analyzer using an enzymatic colorimetric method at the National Institute for Biomedical Research (NIBR).Estimated glomerular filtration rate (eGFR) was calculated using the MDRD (Modification of Diet in Renal Disease) formula: The albumin/creatinine ratio in freshly voided morning urine samples was evaluated.CKD was an eGFR < 60mL/min/1.73 m 2 and/or a urinary albumin/creatinine ratio ≥ 30 mg/g persistent for ≥ 3 months (15).HAT was diagnosed using serological [card agglutination test for trypanosomiasis (CATT)] and parasitological tests [mini anion exchange column (mAECT)].
DNA was extracted from whole blood samples according to the Maxwell method, following the manufacturer's instructions at the Genetics Laboratory of the National Institute for Biomedical Research (INRB).The extracted DNA was transferred to the Laboratory of Development and Regeneration at Katholieke University, Leuven (KU Leuven, Belgium) for storage and genotyping.APOL1 genotyping was performed for two renal risk alleles: G1 (coding variants rs73885319A>G [p.Ser342Gly] and rs60910145G>T [p.Ile384Met]) and G2 (6-bp deletion, rs71785313).Exon 7 (883 bp) of APOL1 was amplified using the gene-specific primer pairs (Fw50-GTCACTGAGCCAATCTCAGC-30 and Rv50-CATATCTCTCCTGGTGGCTG-30).Polymerase chain reaction experiments were performed on genomic DNA using GoTaq Green DNA Polymerase (Promega Corporation, Fitchburg, Wisconsin) and consisted of 35 cycles at an annealing temperature of 55 o C. Alkaline phosphatase and exonuclease exoSAP IT (Affymetrix, Santa Clara, CA) were used for polymerase chain reaction purification.Sanger sequencing was performed on an ABI 3100XL High-Throughput DNA Sequencer (Applied Biosystems, Foster City, CA, USA).APOL1 HRG was defined as the presence of two risk alleles (G1/G1, G2/G2, or G1/G2), and LRG was defined as the presence of zero or one risk allele.

Echocardiographic measurements
Detailed two-dimensional transthoracic echocardiography was performed using an EDAN echocardiograph (D-20537, Hamburg, Germany) outfitted with a 3.5 MHz transducer.Left ventricle measurements were performed according to the 2015 American Society of Echocardiography and European Association of Cardiovascular Imaging updated guidelines for cardiac chamber quantification (16).Two-dimensional guided M-mode echocardiography was performed in the parasternal long-axis view.The interventricular septum (IVS) thickness in diastole (IVSd) in mm, left ventricular posterior wall (PW) thickness in diastole (LVPWd) in mm, and left ventricular end-diastolic diameter (LVEDd) in mm were measured at end-diastole just below the mitral valve leaflets.ECG was simultaneously recorded at the same time to correlate the measurements with the cardiac cycle.Diastolic wall thickness was measured at the onset of the QRS complex.Left ventricular mass (LVM) was calculated according to the American Society of Echocardiography simplified cubed equation linear method using the following equation: LVM (grams) = 0.8 × 1.04 × [(LVEDd + IVSd + LVPWd) 3 − (LVEDd) 3 ] + 0.6 g.LVM was indexed to the BSA and height.LVM was considered high when ≥ 115 g/m 2 or ≥ 48 g/m 2 in males and ≥ 95 g/m 2 or ≥ 44 g/m 2 in females.The relative wall thickness (RWT) of the left ventricle (LV) was calculated as (2 × LVPWd)/LVEDd.LV geometric patterns were defined as follows: normal geometry (normal LVM and RWT ≤ 0.43), concentric remodeling (normal LVM and RWT > 0.43), and concentric hypertrophy (high LVM and RWT > 0.43), and eccentric hypertrophy (High LVM and RWT ≤ 0.43).To assess LV systolic function, ejection fraction (stroke volume/diastolic volume × 100), percentage of LV systolic shortening (%) ([diastolic diameter − systolic diameter]/diastolic diameter × 100], and cardiac output (stroke volume × heart rate) were calculated.The images were stored for subsequent validation by two cardiologists.
Height (in meters) was measured using a flexible tape measure with the participants in an upright position without shoes.Body weight was measured using a digital scale (EKS, Germany), and body mass index (BMI) was calculated as weight (in kilograms) divided by the square of height (in meters).Overweight was defined as BMI of 25 to 29.9 kg/m 2 and obesity as BMI of 30 kg/m 2 (20).A tape measure was used to measure the waist circumference at the top of the hip bone.Central obesity was defined as waist circumference ≥ 94 cm in men and ≥ 80 cm in women (20).High cardiometabolic risk was defined as a waist-to-height ratio ≥ 0.5 (21) HAT-infected participants were positive for both serological and parasitological tests and a CATT plasma dilution ≥ 1/8, parasitology negative; non-infected participants had negative or positive CATT plasma dilution < 1/8.Diabetes mellitus was a fasting serum glucose ≥ 126 mg/dl or use of antidiabetic medication (20).Dyslipidemia was defined as total cholesterol level > 1.9 g/L, triglycerides > 1.5 g/L, and HDLcholesterol < 0.4 g/l for males and < 0.5 g/l for females) (22).Metabolic syndrome was defined according to IDF/AHA/ NHLBI (2009) by the presence of 3 out of the following 5 criteria: waist circumference ≥ 94 cm in males and ≥ 80 cm in females, -SBP ≥ 130 mmHg and/or DBP ≥ 85 mmHg or use of antihypertensive drug treatment, serum triglycerides ≥ 1.5 g/L, HDL-C < 0.40 g/L for males and < 50 g/L for females, fasting serum glucose ≥ 100 mg/dl or use of antidiabetic medication (20).C-reactive protein level > 6 mg/L was considered an inflammatory marker (23).Anemia was defined as hemoglobin (Hb) level of < 12.0 g/dl in women and < 13.0 g/dl in men (24).Elevated uric acid was considered when blood levels were > 6 mg/dl or 360 µmol/L (25)

Statistical analysis
Data analysis was performed using SPSS for Windows software version 21.Student's and Pearson's Chi 2 tests or Fisher's exact test were used to compare means and proportions, where appropriate.The Wilcoxon/Mann-Whitney test was used to compare medians.A multivariate logistic regression model was used to identify independent determinants of left ventricular Hypertrophy (LVH).Odds ratios were provided with their 95% confidence intervals (Cis).
Statistical significance was set at p < 0.05, based on 2-tailed test.A chi-squared test was used to test the deviation from Hardy-Weinberg equilibrium.

Distribution of Cardiovascular risk factors and APOL1 risk genotypes by LVH status
The distribution of cardiovascular risk factors in participants with and without LVH is shown in Table 2. Participants with LVH demonstrated high frequency of hypertension (20% vs. 9.2%, p = 0.019), WHR ≥ 0.5 (34.7% vs. 20.9%,p = 0.018) and CKD (53.3% vs. 39.3%;p = 0.029), while an abnormal ABI was noted in participants without LVH (33.7% vs. 20%, p = 0.021).The other variables did not differ between the two groups.
APOL1 sequence analysis showed that four of 30 (13.3%) participants with LVH and six of 94 (6.4%) participants without LVH carried HRG; the difference between the two groups was not significant (p = 0.253).Participants in both groups carried G1G1, G2G2, and G1G2 genotypes.

Determinants of LVH in the study population
As reported in

Discussion
This study aimed to investigate the possible association between APOL1 risk variants with LVH in a sample of the Bantu population in a T. b. gambiense endemic area.Salient points in the results were a similar prevalence of LVH in HTA-infected and-uninfected participants.The presence of hypertension, albuminuria, and CKD, but not APOL1 HRG, was associated with LVH.

Clinical and paraclinical characteristics
LVH was observed in one of three subjects.It increased with age and was similar in HTA-infected and uninfected participants.The two groups of participants exhibited a similar frequency of APOL1 HRG, probably because these variants do not confer protection against T.b. gambiense (26).Our results indicated higher blood pressure, pulse pressure and anthropometric measurements in subjects with LVH who carried an HRG than in those carrying an LRG.The observed association of age ≥ 38 years, hypertension, WHR > 0.5, and CKD with LVH is consistent with the literature, which reports that age and blood pressure (BP) are the main determinants of vascular remodeling (27).The stiffness of the large arterial trunks contributes to the elevation of pulse pressure, which is a factor in the development of LVH ( 28), as well as greater intima-media thickness of largecaliber arteries and arterioles (29,30).Remodeling of small arteries results in an increase in peripheral resistance and, thus, in mean arterial pressure.In turn, arterial stiffness increases BP and central pulse pressure, which are remodeling factors for LVH.High and uncontrolled BP leads to vascular structural and functional modifications associated with neuro-hormonal, molecular, and genetic factors that cause LVH.However, LVH also occurs in the general population of patients with coronary disease, obesity, and diabetes mellitus (31,32).
Although these factors are known to contribute to LVH development, a high prevalence of diabetes, prehypertension, hypertension, and anemia was observed in HAT-infected participants without LVH.Such an observation could be accounted for by the somewhat higher frequency of APOL1 HRG in HAT-infected individuals than in non-infected individuals.Our findings are consistent with previous reports that APOL1 HRG is associated with increased SBP, albuminuria, and CKD (11,(33)(34)(35)(36)(37)(38).One should bear in mind, however, that subclinical nephropathy can manifest as increased blood pressure (11,33,34,36), and CKD can be the cause of anemia.Finally, an association between serum level of APOL1 and insulin resistance (HOMA-IR) has been reported to underlie the occurrence of diabetes mellitus (39).

Determinants of LVH
Our results showed a similar prevalence of LVH in both HAT-infected and-uninfected subjects with a similar frequency of HRG.Individuals with LVH who carried the HRG had high u-ACR and decreased eGFR and risk of CKD, although APOL1 HRG was not associated with LVH.Age ≥ 38 years emerged as the only independent correlate of LVH in the T.b. gambiense endemic area.Several studies have established that APOL1 HRG is not associated with LVH in AA (33,34,40), although HRG carriage has been strongly associated with albuminuria and/or CKD (2,5,11), which are predictors of LVH (37,38,41).Subclinical CKD is a risk factor for CVD, particularly persons of African descent (42); it is known that albuminuria and reduced GFR independently increase the risk of subclinical and clinical cardiovascular events.Therefore, it is difficult to attribute increased cardiovascular risk only to the APOL1 G1/G2 risk alleles (43).The «Jackson Heart Study (JHS) », a prospective cohort study, showed a positive association between APOL1 HRG and incident CVD, including myocardial infarction, stroke, cardiac surgery, and arterial catheterization, but not LVH, despite the presence of diabetes in some individuals (34).In contrast, the «Systolic Blood Pressure Intervention Trial «SPRINT» (33), a randomized multicenter study, did not show any association between APOL1 HRG and the prevalence of CVD (including myocardial infarction; coronary revascularization; carotid endarterectomy or carotid stenting; peripheral arterial disease with revascularization; 50% stenosis of a coronary, carotid, or lower extremity artery; abdominal aortic aneurysm ≥ 5 cm with or without repair; coronary artery calcium score ≥ 400 Agatston units; anklebrachial index ≤ 0.90; and left ventricular hypertrophy).Some authors have identified diabetes and prior CKD as strong risk factors that may obscure any independent effects of APOL1 risk genotypes.Thus, APOL1 has neutral or protective effects in the presence of diabetes (44,45).
The present study is the first to investigate a possible association between APOL1 HRG and CVD in sub-Saharan African living in a rural area endemic to T. b. gambiense.However, owing to its cross-sectional design we could not ascertain the cause-and-effect nature of any observed relationship.Moreover, the relatively small sample size does not allow for generalization of the findings.

Conclusion
LVH is more prevalent in areas of DRC endemic for T.b. gambiense.The prevalence was similar in HAT-infected and uninfected participants, with a similar frequency of APOL1 HRG.Only age ≥ 38 years emerged as an independent factor for LVH in T. b. gambiense endemic areas.An assessment of cardiovascular risk is essential for individuals with LVH carrying APOL1 HRG in order to benefit from early and appropriate medical intervention.Therefore, a larger prospective follow-up survey is required to assess the incidence of LVH in individuals with APOL1 HRG variants.

Table 1 :
General Characteristics of the study population Data are expressed as mean ± SD or absolute (n) and relative (%) frequency HAT = human African trypanosomiasis LVH = left ventricular hypertrophy

Table 3 :
Distribution of cardiovascular risk factors by HAT infection status

Tableau 4 :
Clinical and paraclinical characteristics of participants by APOL1 genotype status.

Table 5 .
Determinants of LVH in the study population