Association of APOA5 rs662799 and rs3135506 polymorphisms with arterial hypertension in Moroccan patients

  • Sanaa Ouatou1,

    Affiliated with

    • Maria Ajjemami1,

      Affiliated with

      • Hicham Charoute1,

        Affiliated with

        • Hajar Sefri1,

          Affiliated with

          • Noreddine Ghalim2,

            Affiliated with

            • Houria Rhaissi3,

              Affiliated with

              • Houda Benrahma1,

                Affiliated with

                • Abdelhamid Barakat1Email author and

                  Affiliated with

                  • Hassan Rouba1

                    Affiliated with

                    Contributed equally
                    Lipids in Health and Disease201413:60

                    DOI: 10.1186/1476-511X-13-60

                    Received: 24 December 2013

                    Accepted: 26 March 2014

                    Published: 1 April 2014

                    Abstract

                    Background

                    The goal of the study is to investigate the association between the APOA5 polymorphisms and haplotypes with Arterial Hypertension (AHT) in Moroccan patients.

                    Methods

                    The study was performed in 283 subjects, 149 patients with AHT and 134 controls. All subjects were genotyped for the APOA5 -1131 T > C (rs662799), 56C > G (rs3135506) and c.553G > T (rs2075291) polymorphisms.

                    Results

                    There was a strong association between -1131 T > C and 56C > G polymorphisms with AHT. The -1131 T > C and 56C > G polymorphisms were significantly associated with increased systolic blood pressure (SBP) and triglycerides (TG) levels. There were 4 haplotypes with a frequency higher than 5%, constructed from APOA5 polymorphisms, with the following order: -1131 T > C, 56C > G and c.553G > T. Haplotype H1 (TCG) was associated with decreased risk of AHT, whereas the haplotypes H2 (CCG) and H4 (CGG) were significantly associated with an increased risk of AHT. Carriers of H1 haplotype had a lower SBP and DBP and TG. In contrast, significant elevated SBP, DBP and TG were found in H4 haplotypes carriers.

                    Conclusions

                    Our data demonstrate for the first time that several common SNPs in the APOA5 gene and their haplotypes are closely associated with modifications of blood pressure and serum lipid parameters in the AHT patient.

                    Keywords

                    APOA5 gene Polymorphisms Haplotypes Arterial hypertension Morocco

                    Background

                    Arterial hypertension (AHT) is one of major public health problem in the world, it will affect more than 1.56 billion adults worldwide in 2025 [1]. Hypertensive individuals have higher risk to develop coronary artery disease (CAD), cerebrovascular disease and heart failure than normotensive persons [2]. Effectively, The American Heart Association reported that the hypertension and dyslipidemia are one of the main risk factor for development of CAD [3, 4]. AHT is a polygenic and multifactorial disease resulting from combination between genetics and environment factors.

                    Apolipoprotein A5 (APOA5) gene, related to the metabolism of triglycerides in several different ethnic groups [5]. It locates on chromosome 11q23, and forms a cluster with APOA4, APOC3 and APOA1 genes. In mice, invalidation and over-expression of APOA5 result in increased and decreased triglyceride levels, respectively by controlling the activity of lipoprotein lipase (LPL) [1]. APOA5 codes for a protein of 366 amino acids which function is to modulate intracellular hepatic VLDL synthesis. The most studied APOA5 SNP was the 56C > G (S19W, rs 3135506), because it is associated with increased TG levels [6, 7]. Indeed, numerous studies in different ethnic populations have shown significant associations between two minor APOA5 haplotypes, APOA5*2 and APOA5*3, and elevated plasma triglyceride levels [5, 8]. It is estimated that 53% of Hispanics, 35% of African-Americans, and 24% of Caucasians carry at least one of these two haplotypes [6], thus suggesting that these haplotypes are common risk factors for atherosclerosis.

                    In this study, we investigated the association of the APOA5 polymorphisms and haplotypes with AHT in Moroccan patients.

                    Methods

                    Study population

                    Our study concerned 283 Moroccan adult volunteers (177 women and 106 men), 149 patients diagnosed as hypertensive by medical corps (systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg, and treated by antihypertensive drugs) and 134 controls (non smoking healthy persons with normal lipid levels and normal blood pressure, and women were non-pregnant and non-breast-feeding). At enrolment in the Medical Biology Center of Pasteur Institute of Morocco in Casablanca, they completed a health and lifestyle questionnaire including social demographics characteristics, medical history, medications intake and lifestyle factors as tobacco consumption, physical activity and alcohol intake. The clinical examination consisted of questionnaire and a physical examination: age, gender, geographical origin, family history, body mass index, smoking habits, the presence of hypertension, diabetes, hypercholesterolemia or cocaine use, and levels of physical activity and alcohol consumption performed at the Pasteur Institute in Casablanca. All subjects (patients and controls groups) included in this study were from different geographic and ethnic backgrounds from Morocco.

                    Ethics statement

                    We obtained written informed consent from each subject and the research protocol was approved by the committee on research ethics of Pasteur Institute of Morocco.

                    Blood pressure measurement

                    Arterial pressure was measured by the auscultatory method using a stethoscope and a sphygmomanometer. All measurements were performed by nurse in the Medical Biology Center of Pasteur Institute of Morocco in Casablanca.

                    Biochemical measurements

                    The blood was collected in two tubes (EDTA and dry tubes) from subject after 12 hours overnight fasting. The samples were centrifuged and stored at -20°C. Glycemia, total cholesterol (TC), Triglycerides (TG) and High-Density Lipoprotein Cholesterol (HDL-C) levels were determined using the VITROS (5.1 FS Chemistery System). Low-density Lipoprotein Cholesterol (LDL-C) level was calculated according to the Friedwald’s formula. All biochemical measurements were performed in the Biochemistry Laboratory of the Medical Biology Center in Pasteur Institute of Morocco.

                    Molecular analysis: genotyping APOA5 polymorphisms

                    Genomic DNA was extracted from peripheral leukocytes by standard methods including proteinase K digestion, followed by phenol–chloroform extraction and ethanol precipitation. The 56C > G and -1131 T > C polymorphisms were determined by PCR-RFLP analysis. All PCR were performed in a Biometra thermal cycler, using Taq Polymerase (Bioline). A fragment of 157 bp including the 56C > G polymorphism was amplified using two oligonucleotides, forward: 5′- GGC TCT TCT TTC AGG TGG GTCBTCCG -3′reverse: 5′- GCC TTT CCG TGC CTG GGT GGT -3′ [9]. The PCR conditions were an initial denaturing at 96°C for 5 min, followed by 30 cycles of 96°C for 30 s, 64°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 10 min. The PCR products were digested for 2 hours at 65°C with TaqI restriction enzyme: the C56 allele presents a TaqI restriction site which is suppressed in the 56G allele. Genotyping for -1131 T > C was performed with the following primers: Forward: 5′- CCC CAG GAA CTG GAG CGA AATT-3′, reverse 5′- TTC AAG CAG AGG GAA GCC TGTA-3′. The PCR conditions were an initial denaturing at 96°C for 5 min, followed by 32 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 10 min. The PCR products were digested with Mse I. A fragment of 138 bp of the c.553G > T polymorphism was amplified using two oligonucleotides. The PCR conditions were an initial denaturing at 96°C for 5 min, followed by 35 cycles of 96°C for 30 s, 63°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 10 min. The PCR products were digested for overnight at 37°C with HaeIII restriction enzyme. All molecular analyses were performed in the Human Genetic Laboratory in Pasteur Institute of Morocco.

                    Statistical analysis

                    Clinical and biochemical data were expressed as means ± standard deviation (SD). Student’s t test was applied for comparison of quantitative traits that follow a normal distribution. Otherwise, we used Manne-Whitney test. Chi-square test and logistic regression analysis were performed to test the association between Arterial Hypertension and APOA5 genotypes and haplotypes. Logistic regression analysis was adjusted by age and gender. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using STATA software, version 11.0. The P-values were corrected with the Bonferroni correction by multiplying with the number of comparisons. For haplotype frequencies estimation and comparison we used the PLINK software, version 1.07. All haplotypes with frequencies less than 5% were ignored in analysis. Linkage disequilibrium between each pair of APOA5 polymorphisms was estimated using Haploview software, version 4.2.

                    Results

                    Characteristics of controls and patients

                    Clinical characteristics and lipid parameters of AHT patients and controls are presented in Table 1. As expected, age, triglyceridemia (TG), LDL, total cholesterol (TC), Glycemia (Gly), CT/HDL ratio, Sbp and Dbp were significantly different between AHT patients and controls. In addition, HDL/LDL ratio was significantly elevated in controls. There was no significant difference in body mass index (BMI) and HDL between both groups.
                    Table 1

                    Clinical and biochemical characteristics of controls and patients with AHT

                     

                    Controls (n = 134)

                    Patients (n = 149)

                    P-value

                    Age (years)

                    50.78 ± 12.08

                    59.76 ± 11.65

                    <0.0001

                    Sbp (mmgHg)

                    121.69 ± 6.77

                    153.09 ± 19.19

                    <0.0001

                    Dbp (mmgHg)

                    77.49 ± 7.97

                    85.97 ± 10.91

                    <0.0001

                    BMI (kg/m2)

                    26.49 ± 4.11

                    27.42 ± 4.63

                    0.0739

                    TG (g/L)

                    0.98 ± 0.31

                    1.65 ± 0.85

                    <0.0001

                    LDL (g/L)

                    1.15 ± 0.28

                    1.34 ± 0.45

                    <0.0001

                    HDL (g/L)

                    0.51 ± 0.13

                    0.50 ± 0.17

                    0.5042

                    Gly (g/L)

                    0.91 ± 0.14

                    1.23 ± 0.57

                    <0.0001

                    TC (g/L)

                    1.88 ± 0.28

                    2.12 ± 0.51

                    <0.0001

                    TC/HDL

                    3.90 ± 0.94

                    4.64 ± 1.61

                    <0.0001

                    HDL/LDL

                    0.48 ± 0.24

                    0.43 ± 0.32

                    0.0002

                    BMI: bodymass index, TC: Serum total cholesterol, TG: Triglycerides, HDL: High-Density Lipoprotein Cholesterol, LDL: Low-density Lipoprotein Cholesterol, Sbp systolic blood pressure, Dbp diastolic blood pressure.

                    Genotype frequency and linkage disequilibrium

                    The -1131 T > C, 56C > G SNPs had a strong association with AHT in all genetic models, the p-values remained significant after Bonferroni correction. The c.553G > T polymorphism had a significant association with AHT in co-dominant and dominant models, but this association was lost after Bonferroni correction (Table 2).
                    Table 2

                    Genotypic distribution of APOA5 polymorphisms and statistic comparison between AHT subjects and controls

                    SNP

                    Model

                    Controls

                    Patients

                    OR (95% CI)

                    P

                    Pc

                    -1131 T > C

                          
                     

                    Codominant

                         
                     

                    TT

                    128 (95.5%)

                    97 (65.1%)

                    1.00

                      
                     

                    TC

                    2 (1.5%)

                    20 (13.4%)

                    11.16 (2.46-50.69)

                    0.002

                    0.006 *

                     

                    CC

                    4 (3%)

                    32 (21.5%)

                    14.20 (4.50-44.77)

                    <0.0001

                    <0.0001 *

                     

                    Dominant

                         
                     

                    TT

                    128 (95.5%)

                    97 (65.1%)

                    1.00

                      
                     

                    TC/CC

                    6 (4.5%)

                    52 (34.9%)

                    13.20 (5.14-33.91)

                    <0.0001

                    <0.0001 *

                     

                    Recessive

                         
                     

                    TT/TC

                    130 (97%)

                    117 (78.5%)

                    1.00

                      
                     

                    CC

                    4 (3%)

                    32 (21.5%)

                    12.35 (3.91-39.05)

                    <0.0001

                    <0.0001 *

                    56C > G

                          
                     

                    Codominant

                         
                     

                    CC

                    117 (87.3%)

                    106 (71.1%)

                    1.00

                      
                     

                    CG

                    15 (11.2%)

                    21 (14.1%)

                    1.54 (0.72-3.29)

                    0.269

                    NS

                     

                    GG

                    2 (1.5%)

                    22 (14.8%)

                    13.75 (3.01-62.75)

                    0.001

                    0.003 *

                     

                    Dominant

                         
                     

                    CC

                    117 (87.3%)

                    106 (71.1%)

                    1.00

                      
                     

                    CG/GG

                    17 (12.7%)

                    43 (28.9%)

                    2.90 (1.49-5.63)

                    0.002

                    0.006 *

                     

                    Recessive

                         
                     

                    CC/CG

                    132 (98.5%)

                    127 (85.2%)

                    1.00

                      
                     

                    GG

                    2 (1.5%)

                    22 (14.8%)

                    12.77 (2.82-57.73)

                    0.001

                    0.003 *

                    c.553G > T

                          
                     

                    Codominant

                         
                     

                    GG

                    130 (97%)

                    132 (88.6%)

                    1.00

                      
                     

                    GT

                    3 (2.2%)

                    15 (10.1%)

                    4.72 (1.28-17.35)

                    0.020

                    NS

                     

                    TT

                    1 (0.8%)

                    2 (1.3%)

                    1.64 (0.14-19.78)

                    0.695

                    NS

                     

                    Dominant

                         
                     

                    GG

                    130 (97%)

                    132 (88.6%)

                    1.00

                      
                     

                    GT/TT

                    4 (3%)

                    17 (11.4%)

                    3.92 (1.23-12.43)

                    0.02

                    NS

                     

                    Recessive

                         
                     

                    GG/GT

                    133 (99.2%)

                    147 (98.7%)

                    1.00

                      
                     

                    TT

                    1 (0.8%)

                    2 (1.3%)

                    1.47 (0.12-17.70)

                    0.763

                    NS

                    NS: Not Significant.

                    Pc: P value corrected for multiple comparisons (Bonferroni correction).

                    *P values remains significant after Bonferroni correction.

                    To determine the extent of linkage disequilibrium (LD) among the three polymorphisms, standardized LD coefficient D’ was calculated for all pairs of polymorphisms. Figure 1 shows that with the exception of -1131 T > C and 56C > G polymorphisms which were in strong linkage disequilibrium (D’ = 50), other polymorphisms were not in linkage disequilibrium (Figure 1).
                    http://static-content.springer.com/image/art%3A10.1186%2F1476-511X-13-60/MediaObjects/12944_2013_Article_1044_Fig1_HTML.jpg
                    Figure 1

                    The linkage disequilibrium (LD) between the three APOA5 SNPs.

                    Comparisons of clinical and biochemical parameters between APOA5 genotypes

                    We compared biological and clinical traits between APOA5 genotypes under dominant model for all patients and controls combined. The APOA5 -1131 T > C polymorphism was significantly associated with increased levels of systolic blood pressure, diastolic blood pressure and triglycerides (all P values <0.0001). We found significantly elevated systolic blood pressure (P = 0.0001) and triglycerides levels (P < 0.0001) in association with APOA5 56C > G polymorphism. No Significant association was observed for APOA5 c.553G > T polymorphism (Table 3).
                    Table 3

                    Association between APOA5 genotype variants and clinical and biochemical parameters

                     

                    APOA5 -1131 T > C

                    APOA5 56C > G

                    APOA5 c.553G > T

                    TT

                    TC + CC

                    P-value

                    CC

                    CG + GG

                    P-value

                    GG

                    GT + TT

                    P-value

                    Patient (number)

                    97

                    52

                     

                    106

                    43

                     

                    132

                    17

                     

                    Controls (number)

                    128

                    6

                     

                    117

                    17

                     

                    130

                    4

                     

                    Age (years)

                    54.94 ± 12.62

                    57.72 ± 12.68

                    0.1353

                    55.13 ± 12.62

                    56.90 ± 12.82

                    0.3386

                    55.24 ± 12.84

                    58.81 ± 9.87

                    0.215

                    Sbp (mmgHg)

                    133.67 ± 19.71

                    155.86 ± 18.94

                    <0.0001**

                    135.32 ± 20.27

                    148.98 ± 22.62

                    0.0001**

                    137.82 ± 21.15

                    143.24 ± 25.40

                    0.2495

                    Dbp (mmgHg)

                    80.57 ± 10.60

                    87.33 ± 8.24

                    <0.0001**

                    81.27 ± 9.43

                    84.48 ± 13.59

                    0.0355*

                    82.13 ± 10.33

                    79.81 ± 12.60

                    0.3319

                    BMI (kg/m2)

                    26.82 ± 4.21

                    27.58 ± 5.11

                    0.2428

                    26.79 ± 4.21

                    27.68 ± 5.06

                    0.1676

                    27.09 ± 4.41

                    25.63 ± 4.30

                    0.1446

                    TG (g/L)

                    1.23 ± 0.63

                    1.76 ± 0.93

                    <0.0001**

                    1.23 ± 0.64

                    1.75 ± 0.91

                    <0.0001**

                    1.32 ± 0.74

                    1.53 ± 0.65

                    0.0564

                    LDL (g/L)

                    1.22 ± 0.37

                    1.37 ± 0.44

                    0.0101*

                    1.24 ± 0.38

                    1.31 ± 0.43

                    0.1889

                    1.25 ± 0.39

                    1.30 ± 0.43

                    0.5751

                    HDL (g/L)

                    0.51 ± 0.17

                    0.47 ± 0.11

                    0.0631

                    0.51 ± 0.16

                    0.47 ± 0.12

                    0.0746

                    0.50 ± 0.16

                    0.53 ± 0.16

                    0.3976

                    Gly (g/L)

                    1.06 ± 0.46

                    1.13 ± 0.43

                    0.0582

                    1.09 ± 0.48

                    1.04 ± 0.29

                    0.9913

                    1.06 ± 0.43

                    1.33 ± 0.65

                    0.0668

                    TC (g/L)

                    1.97 ± 0.41

                    2.15 ± 0.52

                    0.0045*

                    1.98 ± 0.41

                    2.10 ± 0.51

                    0.0627

                    2.00 ± 0.44

                    2.13 ± 0.42

                    0.1688

                    *P values not significant after Bonferroni correction.

                    **P values remains significant after Bonferroni correction.

                    APOA5 haplotype analysis

                    To examine the combined effect of three variants (in the order of - 1131 T > C, 56C > G and c.553G > T polymorphisms of APOA5) in the APOA5 gene. There were 4 haplotypes identified in the APOA5gene in our population, with frequencies greater than 5% (Table 4). Results of the logistic regression analysis suggested that the haplotype H1 (APOA5*1) has a protective effect against AHT. Two haplotypes confers significant susceptibility to AHT; haplotype H2 (APOA5*2) and haplotype H4. No significant association was observed between the haplotype H3 (APOA*3) and AHT. The frequencies of the four haplotypes are listed in Table 4. Analysis of the interaction between haplotypes, Serum lipid parameters and environment factors showed that the H1 haplotype was associated with lower systolic blood pressure (SBP), diastolic blood pressure (DBP) and total plasma (TG). The H2 haplotype was significantly associated with increased SBP. Carriers of the H4 haplotype had a significant increased SBP, DBP and TG. No significant difference was detected between carriers and non-carriers of H3 haplotype. Age, BMI, LDL, HDL and glycemia were also analyzed but no significant difference was found in these parameters among the four haplotypes (Table 5).
                    Table 4

                    Association analysis of haplotypes derived from polymorphic sites using genotype data

                    Haplotype

                    -1131 T > C

                    56C > G

                    c.553G > T

                    Frequency

                    OR (95% CI)

                    P

                    Pc

                    Controls

                    Cases

                    H1

                    T

                    C

                    G

                    0.8970

                    0.6543

                    0.338 (0.226-0.504)

                    <0.0001

                    <0.0001

                    H2

                    C

                    C

                    G

                    0.0307

                    0.1307

                    3.07 (1.49-6.31)

                    0.0005

                    0.002

                    H3

                    T

                    G

                    G

                    0.0649

                    0.0625

                    0.809 (0.395-1.66)

                    0.563

                    NS

                    H4

                    C

                    G

                    G

                    0.0073

                    0.1525

                    12.46 (2.93-53 )

                    <0.0001

                    <0.0001

                    NS: Not Significant.

                    Pc: P value corrected for multiple comparisons (Bonferroni correction).

                    Table 5

                    Association between APOA5 haplotypes and clinical and biochemical parameters

                     

                    Haplotype H1 (TCG)

                    Haplotype H2 (CCG)

                    Haplotype H3 (TGG)

                    Haplotype H4 (CGG)

                     

                    Present

                    Not present

                    P value

                    Present

                    Not present

                    P value

                    Present

                    Not present

                    P value

                    Present

                    Not present

                    P value

                    Age (years)

                    55.10 ± 12.36

                    57.52 ± 14.00

                    0.2276

                    56.97 ± 13.12

                    55.33 ± 12.62

                    0.4976

                    57.03 ± 14.38

                    55.33 ± 12.46

                    0.4865

                    57.50 ± 12.69

                    55.25 ± 12.66

                    0.3458

                    Sbp (mmgHg)

                    134.79 ± 19.84

                    155.00 ± 21.55

                    <0.0001**

                    152.55 ± 17.19

                    136.46 ± 21.33

                    <0.0001**

                    139.10 ± 21.19

                    138.11 ± 21.56

                    0.7795

                    157.56 ± 21.21

                    135.75 ± 20.27

                    <0.0001**

                    Dbp (mmgHg)

                    81.03 ± 10.41

                    86.48 ± 9.88

                    0.001**

                    86.35 ± 8.47

                    81.41 ± 10.62

                    0.0132*

                    81.40 ± 16.88

                    82.02 ± 9.52

                    0.7606

                    87.88 ± 8.59

                    81.20 ± 10.50

                    0.0006**

                    BMI (kg/m2)

                    26.85 ± 4.30

                    27.64 ± 4.90

                    0.2596

                    26.50 ± 4.67

                    27.04 ± 4.38

                    0.5181

                    27.11 ± 4.88

                    26.97 ± 4.36

                    0.8684

                    28.12 ± 5.56

                    26.83 ± 4.23

                    0.1195

                    TG (g/L)

                    1.24 ± 0.65

                    1.81 ± 0.92

                    <0.0001**

                    1.38 ± 0.69

                    1.33 ± 0.74

                    0.497

                    1.61 ± 1.00

                    1.31 ± 0.69

                    0.1055

                    1.99 ± 0.98

                    1.25 ± 0.65

                    <0.0001**

                    LDL(g/L)

                    1.23 ± 0.37

                    1.36 ± 0.47

                    0.0402*

                    1.42 ± 0.46

                    1.23 ± 0.38

                    0.0107*

                    1.24 ± 0.43

                    1.25 ± 0.39

                    0.8132

                    1.36 ± 0.45

                    1.24 ± 0.38

                    0.0883

                    HDL(g/L)

                    0.51 ± 0.16

                    0.45 ± 0.10

                    0.0061*

                    0.48 ± 0.10

                    0.50 ± 0.16

                    0.4389

                    0.47 ± 0.14

                    0.51 ± 0.16

                    0.2057

                    0.47 ± 0.12

                    0.51 ± 0.16

                    0.1585

                    Gly (g/L)

                    1.07 ± 0.46

                    1.12 ± 0.39

                    0.2406

                    1.16 ± 0.49

                    1.07 ± 0.44

                    0.1619

                    0.99 ± 0.21

                    1.09 ± 0.47

                    0.6938

                    1.08 ± 0.33

                    1.08 ± 0.46

                    0.5571

                    TC (g/L)

                    1.98 ± 0.41

                    2.15 ± 0.54

                    0.0122*

                    2.15 ± 0.53

                    1.99 ± 0.42

                    0.0604

                    2.00 ± 0.48

                    2.01 ± 0.43

                    0.8825

                    2.18 ± 0.54

                    1.98 ± 0.42

                    0.0162*

                    *P values not significant after Bonferroni correction.

                    **P values remains significant after Bonferroni correction.

                    Discussion

                    Many evidences were reported showing that apolipoprotein A1/C3/A4/A5 gene cluster is associated with premature coronary artery disease [10] and serum lipid levels [10]. Recent findings indicate that APOA5 could also influence cholesterol homeostasis and probably could play a role in hypertriglyceridemia associated with diabetes and inflammation [11].

                    The APOA5 polymorphisms were also identified to be implicated in regulation of blood pressure and in the development of hypertension in Japanese population [12].

                    Our data demonstrate that the 56C > G SNP has a significant influence on blood pressure and triglyceride levels. This variant had not previously been investigated in Moroccan populations, although several different studies of other populations are available, supporting the importance of our study. The frequency of the 56G rare allele detected in Moroccan patients (22%) is higher than that of European populations. Indeed, studies carried out with North Americans and Europeans found that the 56G allele frequency is around 6% [6, 7, 13], whereas its frequency in North Americans of Hispanic ancestry is around 15% [14]. Ruiz- Narváez registered a value of 10.2% for the 56G allele in Costa Rica, while other populations exhibited lower frequencies in the same study (Caucasians 6%, African-Americans 7%) [15]. In addition to these studies, in 2003, Lai et al. investigated the frequency of the same polymorphism in people resident in Singapore [8]. The Chinese, Malay and Indian populations living in this region exhibited extremely low frequencies of the 56G polymorphism (0.1%, 1% and 3%, respectively). The 56G allele frequency varies between 0.1% in a Chinese population [8] and 15.8% in Hispanic males [16].

                    APOA5 gene codes for an apolipoprotein involved in the regulation of LPL activity [17, 18]. ApoAV protein could facilitate the interaction between TG rich lipoproteins and proteoglycan-bound LPL. Transgenic mice models also suggested that apoAV might inhibit VLDL hepatic production but these findings were not confirmed in lipoprotein kinetic studies [19].

                    Although the association of 56C > G polymorphism with lipid profile was already shown in various healthy populations [20], their impact on AHT dyslipidemia remains undocumented. In another study, this polymorphism was associated with higher TG and lower HDLc in diabetic patients, but only for Indian-Asian carriers [21]. There was no report of the impact of this polymorphism on history of dyslipidemia in AHT. Nevertheless, an increased 56G allele frequency was previously reported in non diabetic patients with severe hypertriglyceridemia [22, 23]. The association between the 56G allele and increased triglycerides has already been documented in other populations, such as the North American and Northern Irish populations [24, 25]. The results of the present study confirm the relationship between APOA5 56C > G polymorphism and plasma triglycerides, as carriers of the 56G variant were associated strongly with triglyceride levels. In addition to elevated triglyceride levels, the APOA5 56G was associated with higher systolic blood pressure relative to 56C carriers. A direct effect of APOA5 on blood pressure regulation is unlikely. In contrast, there is experimental evidence to suggest that chronic hypertriglyceridemia leads to endothelium dysfunction, which is associated with an impaired response to vasodilator stimulation [26] and a subsequent decrease in nitric oxide availability phenomena, which may result in increased blood pressure.

                    We found a significant difference between patients with AHT and controls regarding the frequency of 56C > G and -1131 T > C genotypes in the additive, dominant and recessive models.

                    Several studies demonstrated that the presence of polymorphisms in the A1-C3-A4 cluster and other gene loci determines the variability of the postprandial lipoprotein response [27, 28]. Recently a gene coding for APOA5 was identified in this cluster, and this is emerging as a main candidate gene for modulating TG metabolism in humans [6]. Two polymorphisms,-1131 T > C and 56C > G, have been extensively studied and are independently associated with higher TG levels [6, 28, 29]. Previous studies have shown that plasma TG concentrations were 69% higher in CC subjects than TT subjects with the-1131 T > C polymorphism [8, 30] and 20–30% higher in CG than CC subjects with the 56C > G polymorphism [6, 31]. Moreno et al. demonstrated that carriers of the -1131C allele (-1131 T > C) displayed a higher plasma TG concentration [32]. However, association studies using haplotypes should increase our ability to detect true associations and interactions.

                    The ApoA5 -1131C allele in our study population was similar to that in Chinese (29.9%) [3335], Singaporean (29.4%) [8], Malays (30.0%) [8], slightly lower than that in Japanese (34.0%) [36, 37], but much greater than that of whites (8.0%) [5], Hispanic Americans (13.0%-16.0%) [6, 38] or Tunisian (13.0%) [39]. The frequency of ApoA5 c.553G > T allele in this study is extremely low, and is in agreement with that of two previous studies in Chinese (3.97%) [40] and Chinese Taiwanese (4.2-7.2%) [41, 42]. The ApoA5 c.553 T allele has been reported to be absent in Caucasians [43]. The ApoA5 c.553TT homozygous was similarly detected in our study population in agreement with a previous study [40].

                    To the best of our knowledge, this study is the first to determine that different haplotypes of APOA5 gene modulate the systolic, diastolic blood pressure and lipid levels in AHT patients. Thus, our data show markedly higher systolic and diastolic blood pressure in subjects with the APOA5 H2 and APOA5 H4 haplotypes, which may explain the higher risk of coronary heart disease associated with the 56G and -1131C alleles [31, 44].

                    In our study, both the APOA5 H2 and APOA5 H4 haplotypes were significantly associated with increase in systolic blood pressure. In addition the H4 haplotype was associated with higher DBP and TG levels. In contrast the H1 haplotype showed significant association with lower SBP, DBP and total plasma TG. These results suggest that each of these haplotypes may be associated with different mechanisms that enhance the plasma lipid levels and the risk of atherogenesis.

                    All these data suggested the importance of APOA5 in the regulation of plasma triglyceride concentrations. Furthermore, pair wise linkage disequilibrium comparison performed in this study between APOA5 -1131 T < C and APOA5 56C > G demonstrated that they are linked, suggesting cooperation mechanisms for the associations with plasma lipoproteins and related traits. Metabolic syndrome (MetS) is a cluster of disorders which includes visceral obesity, dyslipidaemia, hyperglycaemia, and hypertension [45]. The association of APOA5 polymorphisms with increased risk of metabolic syndrome was showed in several studies [46]. The APOA5 gene plays an important role in regulating triglyceride levels. This regulation may contribute to the association between APOA5 and hypertension showed in this paper, but we cannot exclude other mechanisms.

                    Our study has same limitations; waist conference wasn’t measured to assess central obesity. In addition, the APOA5 -1131 T > C and c.553G > T polymorphisms were not in Hardy-Weinberg equilibrium.

                    In summary, we demonstrated that common variants of APOA5 gene are associated with AHT and contribute to the variation in human plasma TG. Therefore, APOA5 variant was a significant predictor for high triglyceride risk and the APOA5 haplotypes affected dyslipidemia appreciably among the Moroccan population.

                    Conclusion

                    In conclusion, we demonstrate for the first time that APOA5 polymorphisms and haplotypes have a strong influence on systolic and diastolic blood pressures and triglyceride levels in Moroccan patients with AHT. Our results showed that APOA5 56C > G and -1131 T > C polymorphisms play a major role in elevated risk of developing coronary artery disease due to its association with increased plasma TG.

                    Declarations

                    Acknowledgments

                    We thank all families for their cooperation. The authors would also like to thank Dr. Guy Lenaers for his contribution to this work. This work was supported by a grant from the European Commission FP7 Integrated Project MEDIGENE (FP7-279171-1).

                    Authors’ Affiliations

                    (1)
                    Laboratoire de Génétique Moléculaire et Humaine, Département de Recherche Scientifique, Institut Pasteur du Maroc
                    (2)
                    Laboratoire de Biochimie, Centre de Biologie Médicale, Institut Pasteur du Maroc
                    (3)
                    Laboratoire de Physiologie et Génétique Moléculaire, Faculté des sciences Ben M’Sik, Université Hassan II

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                    This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.

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