Association of the ST3GAL4 rs11220462 polymorphism and serum lipid levels in the Mulao and Han populations

  • Quan-Zhen Lin1,

    Affiliated with

    • Rui-Xing Yin1Email author,

      Affiliated with

      • Tao Guo1,

        Affiliated with

        • Jian Wu1,

          Affiliated with

          • Jia-Qi Sun1,

            Affiliated with

            • Shao-Wen Shen1,

              Affiliated with

              • Guang-Yuan Shi1,

                Affiliated with

                • Jin-Zhen Wu1,

                  Affiliated with

                  • Cheng-Wu Liu2 and

                    Affiliated with

                    • Shang-Ling Pan2

                      Affiliated with

                      Lipids in Health and Disease201413:123

                      DOI: 10.1186/1476-511X-13-123

                      Received: 10 June 2014

                      Accepted: 24 July 2014

                      Published: 3 August 2014

                      Abstract

                      Background

                      A previous genome-wide association study has displayed the association of the ST3 beta-galactoside alpha-2,3-sialytransferase 4 (ST3GAL4) gene variant and lipid traits in the individuals of European ancestry, but the reproducibility of this association has not been detected in the Chinese population. The present study was undertaken to detect the association of ST3GAL4 rs11220462 single nucleotide polymorphism (SNP) and several environmental factors with serum lipid profiles in the Mulao and Han populations.

                      Methods

                      A total of 700 unrelated individuals of Mulao nationality and 694 subjects of Han nationality were randomly selected from our previous stratified randomized samples. Genotypes of the SNP were determined via polymerase chain reaction and restriction fragment length polymorphism in combination with gel electrophoresis, and then verified by direct sequencing.

                      Results

                      Serum apolipoprotein (Apo) B levels were higher and the ApoAI/ApoB ratio was lower in Mulao than in Han (P < 0.05-0.01). There were no significant differences in the genotypic and allelic frequencies of the ST3GAL4 rs11220462 SNP between the two ethnic groups or between males and females. The A allele carriers in both Mulao males and females had higher total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and ApoB levels than the A allele non-carriers (P < 0.05-0.01). The subjects with AA genotype in Han males but not in females had higher TC and triglyceride (TG) levels than the subjects with AG or GG genotype (P < 0.01 for each). Multiple linear regression analyses showed that the levels of TC, LDL-C and ApoB in Mulao females; TC and LDL-C in Mulao males; and TC in Han males were correlated with the genotypes (P < 0.05-0.001). Serum lipid parameters were also associated with several environmental factors in both ethnic groups (P < 0.05 -0.001).

                      Conclusions

                      The association of ST3GAL4 rs11220462 SNP and serum lipid levels was different between the Mulao and Han populations, suggesting that there may be a racial/ethnic-specific association, and/or sex-specific association between the ST3GAL4 rs11220462 SNP and serum lipid parameters in some ethnic groups.

                      Keywords

                      Lipids ST3 beta-galactoside alpha-2,3-sialytransferase 4 Single nucleotide polymorphism Environmental factors

                      Background

                      It is well known that coronary artery disease (CAD) is the leading cause of morbidity and mortality globally [1, 2]. Dyslipidemia, associated with elevating total cholesterol (TC) [3], triglyceride (TG) [4], low-density lipoprotein cholesterol (LDL-C) [5], and apolipoprotein (Apo) B [6], or low levels of high-density lipoprotein cholesterol (HDL-C) [7] and ApoAI [6], plays a significant role for CAD and is the main target for therapeutic intervention. The heritability estimates of the interindividual variations in serum lipid levels from both twin and family studies are in the range of 0.48-0.87 [811], suggesting a considerable genetic contribution. Therefore, the understanding of the association of genetic variants and serum lipid levels may provide new insights into target preventive intervention for CAD.

                      Over the years, candidate gene studies as well as genome-wide association studies (GWASs) have enabled the identification of thousands of single nucleotide polymorphisms (SNPs) associated with complex human diseases and traits. These studies based on large numbers of normolipidemic individuals indicated that considerable new SNPs had replicable modest associations with plasma concentrations of TC, TG, LDL-C, and HDL-C [1218]. The ST3 beta-galactoside alpha-2,3-sialytransferase 4 (ST3GAL4, as known as SIAT4) rs11220462 (http://​www.​ncbi.​nlm.​nih.​gov/​gene/​6484) is one of the identified SNPs in recent years. ST3GAL4 locates on chromosome 11q24.2 [15] and encodes a member of the glycosyltransferase 29 family, a group of enzymes involved in protein glycosylation. The products of ST3GAL4 were associated with increasing risk of cirrhosis, type 2 diabetes and cardiovascular disease by influencing liver enzyme concentrations [19]. Interestingly, ST3GAL4 has been correlated to endothelial dysfunction and the synthesis of E-selectin ligands, which were associated with metabolic syndrome and coronary artery calcification [20, 21]. In a previous GWAS, the SNP of ST3GAL4 rs11220462 has been associated with TC and LDL-C concentrations in European population [15]. But the reproducibility and repeatability of this association have not been carried out in the Chinese population so far. Therefore, it would be attractive to characterize the association between the rs11220462 SNP and serum lipid levels in the Chinese population.

                      China is a multiethnic country. Han nationality is the largest one of 56 ethnic groups, and Mulao nationality is the twenty-ninth largest (with population of 207,352 in 2000) among the 55 minorities. Ninety percent of them live in the Luocheng Mulao Autonomous Country, Guangxi Zhuang Autonomous Region, People’s Republic of China. The history of this minority can be traced back to the Jin Dynasty (AD 265–420). It is well known that the people of Mulao are the descendants of the ancient Baiyue tribe in south China. In a previous study, Xu et al. [22] showed that the genetic relationship between Mulao nationality and other minorities in Guangxi was much closer than that between Mulao and Han or Uighur nationality. To the best of our knowledge, the association between ST3GAL4 rs11220462 SNP and serum lipid levels has not been previously explored in the Chinese population. Therefore, the aim of the present study was to detect the association of ST3GAL4 rs11220462 SNP and several environmental factors with serum lipid traits in the Guangxi Mulao and Han populations.

                      Results

                      General characteristics and serum lipid levels

                      The baseline characteristics and serum lipid levels of the Mulao and Han populations are presented in Table 1. The levels of weight, body mass index (BMI), the ratio of ApoAI to ApoB and the percentages of individuals who smoked cigarettes were lower in Mulao than in Han (P < 0.05-0.001), whereas the levels of ApoB were higher in Mulao than in Han (P < 0.05). There were no significant differences in the levels of age structure, body height, waist circumference, systolic blood pressure, diastolic blood pressure, pulse pressure, blood glucose, TC, TG, HDL-C, LDL-C, ApoAI; the percentages of subjects who consumed alcohol; and the ratio of male to female between the two ethnic groups (P > 0.05 for all).
                      Table 1

                      Comparison of demography, lifestyle and serum lipid levels between the Mulao and Han populations

                      Parameter

                      Han

                      Mulao

                      t(χ 2 )

                      P

                      Number

                      694

                      700

                        

                      Male/female

                      331/363

                      337/363

                      0.280

                      0.867

                      Age (years)

                      52.26 ± 15.02

                      52.56 ± 15.04

                      0.379

                      0.705

                      Height (cm)

                      154.93 ± 8.12

                      155.49 ± 8.07

                      1.291

                      0.197

                      Weight (kg)

                      54.02 ± 9.12

                      53.02 ± 9.40

                      −1.998

                      0.046

                      Body mass index (kg/m2)

                      22.48 ± 3.39

                      21.87 ± 3.08

                      −3.553

                      0.000

                      Waist circumference (cm)

                      75.62 ± 7.91

                      75.13 ± 8.67

                      −1.099

                      0.272

                      Cigarette smoking [n (%)]

                          

                       Nonsmoker

                      480 (69.2)

                      512 (73.1)

                        

                       ≤ 20 cigarettes/day

                      192 (27.6)

                      151 (21.6)

                      9.728

                      0.008

                       > 20 cigarettes/day

                      22 (3.2)

                      37 (5.3)

                        

                      Alcohol consumption [n (%)]

                          

                       Nondrinker

                      516 (74.3)

                      515 (73.6)

                        

                       ≤ 25g/day

                      81 (11.7)

                      70 (10.0)

                      2.305

                      0.316

                       > 25g/day

                      97 (14.0)

                      115 (16.4)

                        

                      Systolic blood pressure (mmHg)

                      130.51 ± 19.29

                      129.25 ± 21.52

                      −1.150

                      0.250

                      Diastolic blood pressure (mmHg)

                      82.14 ± 11.01

                      80.97 ± 11.89

                      1.912

                      0.056

                      Pulse pressure (mmHg)

                      48.37 ± 15.02

                      48.28 ± 15.82

                      −0.102

                      0.918

                      Blood glucose (mmol/L)

                      5.97 ± 1.58

                      5.97 ± 1.52

                      −0.034

                      0.973

                      Total cholesterol (mmol/L)

                      5.03 ± 0.94

                      4.98 ± 1.08

                      −0.931

                      0.352

                      Triglyceride (mmol/L)

                      1.08 (0.86)

                      1.07 (0.79)

                      −0.882

                      0.378

                      HDL-C (mmol/L)

                      1.75 ± 0.54

                      1.75 ± 0 .45

                      0.096

                      0.924

                      LDL-C (mmol/L)

                      2.90 ± 0.80

                      2.93 ± 0.83

                      0.088

                      0.930

                      Apolipoprotein (Apo) AI (g/L)

                      1.35 ± 0.60

                      1.32 ± 0.40

                      −1.815

                      0.070

                      ApoB (g/L)

                      0.85 ± 0.18

                      0.96 ± 0.54

                      5.196

                      0.000

                      ApoAI/ApoB

                      1.65 ± 0 .48

                      1.57 ± 0.74

                      −2.298

                      0.022

                      TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ApoAI, apolipoprotein AI; ApoB, apolipoprotein B. The quantitative variables were presented as mean ± standard deviation and their difference between the groups was determined by the t-test. The values of triglyceride were presented as median (interquartile range), and their difference between the groups was determined by the Wilcoxon-Mann–Whitney test. The difference in Percentages of cigarette smoking and alcohol consumption between the groups was determined by Chi-square -test.

                      Genotypic and allelic frequencies

                      The genotypic and allelic frequencies of the rs11220462 SNP in the both ethnic groups are shown in Table 2. The frequencies of G and A alleles were 64.5% and 35.5% in Mulao; and 61.7% and 38.3% in Han (P > 0.05); respectively. The frequencies of GG, AG and AA genotypes were 40.7%, 48.1% and 11.2% in Mulao, and 36.4%, 50.6% and 13.0% in Han (P > 0.05); respectively. There was no significant difference in the genotypic and allelic frequencies between males and females in the both ethnic groups (P > 0.05 for all).
                      Table 2

                      Comparison of the genotypic and allelic frequencies between the Mulao and Han populations [n (%)]

                      Group

                      n

                      Genotype

                      Allele

                      HWE

                        

                      GG

                      AG

                      AA

                      G

                      A

                      (P value)

                      Han

                      694

                      253 (36.4)

                      351 (50.6)

                      90 (13.0)

                      857 (61.7)

                      531 (38.3)

                      0.063

                      Mulao

                      700

                      281 (40.1)

                      341 (48.7)

                      78 (11.2)

                      903 (64.5)

                      497 (35.5)

                      0.092

                      χ 2

                      -

                      2.444

                      2.275

                      P

                      -

                      0.295

                      0.131

                      Han

                             

                      Male

                      331

                      119 (36.0)

                      169 (51.0)

                      43 (13.0)

                      407 (61.5)

                      255 (38.5)

                      0.156

                      Female

                      363

                      134 (36.9)

                      182 (50.1)

                      47 (13.0)

                      450 (62.0)

                      276 (38.0)

                      0.224

                      χ 2

                      -

                      0.073

                      0.037

                      P

                      -

                      0.964

                      0.847

                      Mulao

                             

                      Male

                      337

                      127 (37.6)

                      165 (49.0)

                      45 (13.4)

                      419 (62.2)

                      255 (37.8)

                      0.453

                      Female

                      363

                      154 (42.4)

                      176 (48.5)

                      33 (9.1)

                      484 (66.7)

                      242 (33.3)

                      0.083

                      χ 2

                      -

                      3.835

                      3.092

                      P

                      -

                      0.147

                      0.079

                      HWE, Hardy–Weinberg equilibrium. Difference in genotype or allele distribution between the groups was tested by the Chi-square test; a standard goodness-of-fit test was used to test the Hardy–Weinberg equilibrium.

                      Genotypes and serum lipid levels

                      As shown in Table 3, the levels of TC and LDL-C in Mulao but not in Han were different among the AA, AG and GG genotypes after adjusting age, sex, height, BMI, blood pressure, cigarette smoking, alcohol consumption and blood glucose (P < 0.01 for each); the A allele carriers had higher levels of TC and LDL-C than the A allele non-carriers. In the subgroup analyses, the levels of TC and TG in Han males but not in females were different among the genotypes (P < 0.01 for each), the subjects with AA genotype had higher TC and TG levels than the subjects with AG or GG genotype. For the Mulao population, the levels of TC, LDL-C and ApoB in both males and females were also different among the genotypes (P < 0.05-0.01), the A allele carriers had higher serum TC, LDL-C and ApoB levels than the A allele non-carriers.
                      Table 3

                      Comparison of serum lipid levels among the genotypes in the Mulao and Han populations

                      Genotype

                      n

                      TC

                      TG

                      HDL-C

                      LDL-C

                      ApoAI

                      ApoB

                      ApoAI/ApoB

                        

                      (mmol/L)

                      (mmol/L)

                      (mmol/L)

                      (mmol/L)

                      (g/L)

                      (g/L)

                       

                      Han

                              

                      GG

                      253

                      5.00 ± 0.96

                      1.08 (1.07)

                      1.71 ± 0.55

                      2.85 ± 0.78

                      1.36 ± 0.29

                      0.87 ± 0.19

                      1.64 ± 0.53

                      AG

                      351

                      5.05 ± 0.92

                      1.05 (0.74)

                      1.77 ± 0.60

                      2.95 ± 0.81

                      1.35 ± 0.23

                      0.85 ± 0.18

                      1.65 ± 0.44

                      AA

                      90

                      5.10 ± 0.99

                      1.08 (1.09)

                      1.73 ± 0.43

                      2.96 ± 0.81

                      1.36 ± 0.30

                      0.85 ± 0.19

                      1.68 ± 0.50

                      F

                      -

                      0.519

                      4.963

                      1.041

                      0.309

                      0.121

                      0.391

                      0.437

                      P

                      -

                      0.595

                      0.084

                      0.353

                      0.734

                      0.886

                      0.676

                      0.646

                      Mulao

                              

                      GG

                      286

                      4.80 ± 1.18

                      1.04 (0.84)

                      1.73 ± 0.41

                      2.82 ± 0.82

                      1.31 ± 0.41

                      0.89 ± 0.25

                      1.57 ± 0.70

                      AG

                      344

                      5.09 ± 1.01

                      1.10 (0.73)

                      1.77 ± 0.49

                      2.98 ± 0.83

                      1.33 ± 0.39

                      0.90 ± 0.53

                      1.58 ± 0.79

                      AA

                      76

                      5.17 ± 0.89

                      1.15 (0.77)

                      1.74 ± 0.41

                      3.09 ± 0.80

                      1.30 ± 0.42

                      0.98 ± 0.61

                      1.53 ± 0.64

                      F

                      -

                      7.378

                      1.784

                      0.910

                      5.405

                      0.056

                      0.587

                      0.086

                      P

                      -

                      0.001

                      0.410

                      0.403

                      0.005

                      0.946

                      0.556

                      0.918

                      Han/male

                              

                      GG

                      120

                      5.00 ± 0.97

                      1.32 (1.18)

                      1.65 ± 0.42

                      2.90 ± 0.73

                      1.36 ± 0.32

                      0.91 ± 0.19

                      1.57 ± 0.58

                      AG

                      182

                      5.20 ± 0.73

                      1.09 (0.72)

                      1.73 ± 0.35

                      3.02 ± 0.68

                      1.37 ± 0.23

                      0.90 ± 0.15

                      1.57 ± 0.35

                      AA

                      29

                      5.41 ± 0.95

                      1.36 (0.47)

                      1.70 ± 0.69

                      3.04 ± 0.86

                      1.41 ± 0.39

                      0.89 ± 0.17

                      1.66 ± 0.58

                      F

                      -

                      7.255

                      12.933

                      1.734

                      1.422

                      2.147

                      0.736

                      1.304

                      P

                      -

                      0.001

                      0.002

                      0.178

                      0.243

                      0.118

                      0.480

                      0.273

                      Han/female

                              

                      GG

                      134

                      4.99 ± 0.96

                      0.96 (0.38)

                      1.80 ± 0.43

                      2.88 ± 0.83

                      1.35 ± 0.25

                      0.83 ± 0.17

                      1.70 ± 0.48

                      AG

                      182

                      4.90 ± 1.05

                      0.98 (0.79)

                      1.81 ± 0.76

                      2.88 ± 0.91

                      1.32 ± 0.23

                      0.81 ± 0.20

                      1.73 ± 0.51

                      AA

                      47

                      4.80 ± 0.94

                      1.07 (0.59)

                      1.71 ± 0.38

                      2.88 ± 0.77

                      1.32 ± 0.18

                      0.81 ± 0.20

                      1.70 ± 0.40

                      F

                      -

                      1.215

                      3.999

                      0.648

                      0.113

                      0.305

                      1.166

                      0.973

                      P

                      -

                      0.298

                      0.135

                      0.524

                      0.894

                      0.737

                      0.313

                      0.379

                      Mulao/male

                              

                      GG

                      130

                      4.77 ± 1.17

                      1.04 (1.05)

                      1.75 ± 0.44

                      2.72 ± 0.82

                      1.31 ± 0.46

                      0.88 ± 0.17

                      1.59 ± 0.68

                      AG

                      167

                      5.04 ± 0.85

                      1.17 (1.05)

                      1.75 ± 0.57

                      2.96 ± 0.73

                      1.36 ± 0.39

                      0.90 ± 0.66

                      1.52 ± 0.69

                      AA

                      43

                      5.08 ± 0.84

                      1.15 (0.77)

                      1.66 ± 0.37

                      2.97 ± 0.81

                      1.23 ± 0.42

                      0.91 ± 0.47

                      1.42 ± 0.51

                      F

                      -

                      5.108

                      2.476

                      0.839

                      3.230

                      1.478

                      3.182

                      1.166

                      P

                      -

                      0.007

                      0.290

                      0.433

                      0.041

                      0.230

                      0.043

                      0.313

                      Mulao/female

                              

                      GG

                      156

                      4.82 ± 1.18

                      1.04 (0.64)

                      1.72 ± 0.39

                      2.90 ± 0.82

                      1.32 ± 0.37

                      0.88 ± 0.35

                      1.56 ± 0.71

                      AG

                      177

                      5.03 ± 1.14

                      1.01 (0.66)

                      1.78 ± 0.41

                      3.00 ± 0.91

                      1.30 ± 0.38

                      0.93 ± 0.33

                      1.64 ± 0.87

                      AA

                      33

                      5.40 ± 0.92

                      1.04 (0.73)

                      1.84 ± 0.44

                      3.29 ± 0.89

                      1.39 ± 0.41

                      1.04 ± 0.70

                      1.68 ± 0.76

                      F

                      -

                      4.376

                      0.393

                      1.251

                      3.097

                      0.891

                      3.678

                      0.289

                      P

                      -

                      0.023

                      0.822

                      0.287

                      0.049

                      0.411

                      0.026

                      0.749

                      TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ApoAI, Apo lipoprotein AI; ApoB, Apo lipoprotein B. The association of genotypes and serum lipid parameters was tested by analysis of covariance (co-variables include sex, age, body mass index, blood pressure, alcohol consumption, cigarette smoking and blood glucose). The value of TG was presented as median (interquartile range), and their difference among the genotypes was determined the Wilcoxon-Mann–Whitney test.

                      Risk factors for serum lipid parameters

                      The risk factors for serum lipid parameters in Mulao and Han are shown in Table 4. The levels of TC and LDL-C in Mulao but not in Han were correlated with the genotypes (P < 0.05 for each). Serum TC and LDL-C levels in both Mulao males and females, ApoB levels in Mulao females, and TC levels in Han males were correlated with genotypes (P < 0.05 for all, Table 5); respectively. Serum lipid parameters were also correlated with several environmental factors such as age, gender, height, weight, BMI, waist circumference, alcohol consumption, cigarette smoking, blood pressure and blood glucose in both ethnic groups (P < 0.05-0.001; Tables 4 and 5).
                      Table 4

                      Relative risk factors for serum lipid parameters in the Mulao and Han populations

                      Lipid parameter

                      Risk factor

                      Unstandardized coefficient

                      Standard error

                      Standardized coefficient

                      t

                      P

                      Han/Mulao

                            

                      TC

                      Age

                      0.010

                      0.002

                      0.168

                      5.242

                      0.000

                      Genotype

                      −0.133

                      0.040

                      −0.087

                      −3.359

                      0.001

                       

                      Alcohol consumption

                      0.080

                      0.036

                      0.058

                      2.232

                      0.026

                       

                      Systolic blood pressure

                      0.004

                      0.001

                      0.083

                      2.867

                      0.004

                      Body mass index

                      0.046

                      0.008

                      0.147

                      5.530

                      0.000

                      TG

                      Waist circumference

                      0.029

                      0.006

                      0.196

                      4.695

                      0.000

                      Alcohol consumption

                      0.185

                      0.044

                      0.113

                      4.171

                      0.000

                      Blood glucose

                      0.068

                      0.020

                      0.086

                      3.375

                      0.001

                      Diastolic blood pressure

                      0.010

                      0.003

                      0.097

                      3.681

                      0.000

                      Weight

                      0.095

                      0.025

                      0.723

                      3.796

                      0.000

                       

                      Height

                      −0.066

                      0.018

                      −0.442

                      −3.646

                      0.000

                      HDL-C

                      Waist circumference

                      −0.007

                      0.002

                      −0.109

                      −2.695

                      0.007

                      Alcohol consumption

                      0.104

                      0.019

                      0.154

                      5.586

                      0.000

                      Weight

                      −0.009

                      0.002

                      −0.175

                      −4.127

                      0.000

                      LDL-C

                      Body mass index

                      0.042

                      0.007

                      0.169

                      6.330

                      0.000

                      Age

                      0.008

                      0.002

                      0.152

                      5.350

                      0.000

                      Genotype

                      −0.083

                      0.032

                      −0.068

                      −2.611

                      0.009

                      Alcohol consumption

                      −0.081

                      0.029

                      −0.074

                      −2.804

                      0.005

                      Systolic blood pressure

                      0.002

                      0.001

                      0.059

                      2.044

                      0.041

                      ApoAI

                      Ethnic group

                      0.040

                      0.017

                      0.059

                      2.265

                      0.024

                       

                      Gender

                      0.059

                      0.021

                      0.087

                      2.774

                      0.006

                      Age

                      0.001

                      0.001

                      0.052

                      2.007

                      0.045

                       

                      Alcohol consumption

                      0.130

                      0.014

                      0.284

                      9.210

                      0.000

                      Waist circumference

                      −0.005

                      0.001

                      −0.125

                      −4.675

                      0.000

                      ApoB

                      Ethnic group

                      −0.122

                      0.021

                      −0.149

                      −5.772

                      0.000

                      Body mass index

                      0.010

                      0.005

                      0.079

                      2.168

                      0.030

                      Waist circumference

                      0.007

                      0.002

                      0.134

                      3.695

                      0.000

                       

                      Pulse pressure

                      0.003

                      0.001

                      0.117

                      4.464

                      0.000

                       

                      Blood glucose

                      0.019

                      0.007

                      0.07

                      2.657

                      0.008

                      ApoAI/ApoB

                      Ethnic group

                      0.103

                      0.032

                      0.083

                      3.256

                      0.001

                       

                      Gender

                      0.202

                      0.044

                      0.162

                      4.553

                      0.000

                       

                      Waist circumference

                      −0.013

                      0.003

                      −0.173

                      −4.086

                      0.000

                       

                      Blood glucose

                      −0.031

                      0.011

                      −0.077

                      −2.957

                      0.003

                      Alcohol consumption

                      0.132

                      0.026

                      0.156

                      5.125

                      0.000

                      Height

                      0.014

                      0.003

                      0.178

                      4.929

                      0.000

                      Weight

                      −0.010

                      0.003

                      −0.145

                      −2.874

                      0.004

                      Han

                            

                      TC

                      Age

                      0.009

                      0.003

                      0.150

                      3.613

                      0.000

                      Waist circumference

                      0.022

                      0.005

                      0.182

                      4.702

                      0.000

                      Systolic blood pressure

                      0.005

                      0.002

                      0.110

                      2.642

                      0.008

                      Alcohol consumption

                      0.136

                      0.051

                      0.104

                      2.653

                      0.008

                      Height

                      −0.011

                      0.005

                      −0.098

                      −2.401

                      0.017

                      TG

                      Waist circumference

                      0.056

                      0.006

                      0.329

                      9.030

                      0.000

                      Blood glucose

                      0.121

                      0.030

                      0.141

                      4.076

                      0.000

                      Alcohol consumption

                      0.240

                      0.065

                      0.129

                      3.697

                      0.001

                      Diastolic blood pressure

                      0.010

                      0.004

                      0.086

                      2.422

                      0.016

                      HDL-C

                      Alcohol consumption

                      0.118

                      0.033

                      0.158

                      3.583

                      0.000

                      Waist circumference

                      −0.013

                      0.003

                      −0.195

                      −5.094

                      0.000

                       

                      Gender

                      0.222

                      0.055

                      0.206

                      4.067

                      0.000

                       

                      Cigarette smoking

                      0.142

                      0.047

                      0.141

                      3.022

                      0.003

                       

                      Age

                      0.003

                      0.001

                      0.080

                      2.178

                      0.030

                      LDL-C

                      Body mass index

                      0.044

                      0.009

                      0.185

                      5.085

                      0.000

                      Age

                      0.012

                      0.002

                      0.233

                      6.394

                      0.000

                      ApoAI

                      Gender

                      0.065

                      0.026

                      0.125

                      2.515

                      0.012

                      Alcohol consumption

                      0.142

                      0.015

                      0.394

                      9.480

                      0.000

                       

                      Weight

                      −0.007

                      0.001

                      −0.250

                      −6.288

                      0.000

                       

                      Cigarette smoking

                      0.092

                      0.021

                      0.189

                      4.308

                      0.000

                      ApoB

                      Waist circumference

                      0.006

                      0.001

                      0.254

                      4.621

                      0.000

                       

                      Systolic blood pressure

                      0.001

                      0.000

                      0.136

                      3.800

                      0.000

                      Body mass index

                      0.007

                      0.003

                      0.120

                      2.207

                      0.028

                      Blood glucose

                      0.014

                      0.004

                      0.118

                      3.411

                      0.001

                      Gender

                      −0.060

                      0.016

                      −0.162

                      −3.712

                      0.000

                       

                      Height

                      −0.002

                      0.001

                      −0.105

                      −2.146

                      0.032

                      ApoAI/ApoB

                      Body mass index

                      −0.030

                      0.007

                      −0.214

                      −4.592

                      0.000

                      Alcohol consumption

                      0.134

                      0.028

                      0.199

                      4.848

                      0.000

                      Waist circumference

                      −0.011

                      0.003

                      −0.183

                      −3.850

                      0.000

                      Gender

                      0.258

                      0.046

                      0.267

                      5.641

                      0.000

                      Cigarette smoking

                      0.170

                      0.039

                      0.189

                      4.322

                      0.000

                      Mulao

                            

                      TC

                      Age

                      0.010

                      0.003

                      0.142

                      3.874

                      0.000

                      Body mass index

                      0.053

                      0.013

                      0.150

                      4.087

                      0.000

                      Genotype

                      −0.207

                      0.060

                      −0.126

                      −3.432

                      0.001

                      TG

                      Waist circumference

                      0.021

                      0.006

                      0.171

                      3.296

                      0.001

                      Alcohol consumption

                      0.136

                      0.051

                      0.095

                      2.669

                      0.008

                      Body mass index

                      0.055

                      0.018

                      0.158

                      3.059

                      0.002

                       

                      Diastolic blood pressure

                      0.010

                      0.003

                      0.107

                      2.888

                      0.004

                      HDL-C

                      Body mass index

                      −0.041

                      0.005

                      −0.277

                      −7.641

                      0.000

                      Alcohol consumption

                      0.101

                      0.026

                      0.169

                      3.876

                      0.000

                       

                      Gender

                      0.084

                      0.039

                      0.093

                      2.126

                      0.034

                      LDL-C

                      Body mass index

                      0.046

                      0.010

                      0.170

                      4.610

                      0.000

                      Age

                      0.007

                      0.002

                      0.127

                      3.467

                      0.001

                      Genotype

                      −0.142

                      0.046

                      −0.113

                      −3.060

                      0.002

                       

                      Gender

                      0.149

                      0.061

                      0.090

                      2.440

                      0.015

                      ApoAI

                      Alcohol consumption

                      0.115

                      0.024

                      0.219

                      4.887

                      0.000

                      Waist circumference

                      −0.005

                      0.002

                      −0.100

                      −2.632

                      0.009

                       

                      Gender

                      0.073

                      0.036

                      0.091

                      1.995

                      0.046

                      ApoB

                      Waist circumference

                      0.010

                      0.002

                      0.162

                      4.367

                      0.000

                      Pulse pressure

                      0.005

                      0.001

                      0.140

                      3.782

                      0.000

                      ApoAI/ApoB

                      Waist circumference

                      −0.018

                      0.003

                      −0.217

                      −5.846

                      0.000

                      Blood glucose

                      −0.049

                      0.018

                      −0.102

                      −2.761

                      0.006

                      Alcohol consumption

                      0.083

                      0.036

                      0.085

                      2.301

                      0.022

                      TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ApoAI, Apolipoprotein AI; ApoB, Apolipoprotein B. The correlative factors were determined by multivariable linear regression analysis with stepwise modeling.

                      Table 5

                      Relative risk factors for serum lipid parameters in males and females of the Mulao and Han populations

                      Lipid parameter

                      Risk factor

                      Unstandardized coefficient

                      Standard error

                      Standardized coefficient

                      t

                      P

                      Han/male

                            

                      TC

                      Diastolic blood pressure

                      0.019

                      0.004

                      0.241

                      4.461

                      0.000

                      Genotype

                      −0.292

                      0.072

                      −0.219

                      −4.049

                      0.000

                       

                      Alcohol consumption

                      0.113

                      0.056

                      0.109

                      2.024

                      0.044

                      TG

                      Waist circumference

                      0.074

                      0.010

                      0.387

                      7.655

                      0.000

                      Blood glucose

                      0.131

                      0.045

                      0.148

                      2.924

                      0.004

                      Alcohol consumption

                      0.278

                      0.092

                      0.152

                      3.017

                      0.003

                      HDL-C

                      Waist circumference

                      −0.018

                      0.003

                      −0.335

                      −6.398

                      0.000

                      Alcohol consumption

                      0.119

                      0.027

                      0.227

                      4.398

                      0.000

                      Cigarette smoking

                      0.142

                      0.039

                      0.192

                      3.681

                      0.000

                      Diastolic blood pressure

                      0.006

                      0.002

                      0.167

                      3.164

                      0.002

                      LDL-C

                      Body mass index

                      0.031

                      0.010

                      0.163

                      2.967

                      0.003

                      Cigarette smoking

                      −0.187

                      0.068

                      −0.152

                      −2.763

                      0.006

                       

                      Age

                      0.005

                      0.003

                      0.113

                      2.063

                      0.040

                      ApoAI

                      Alcohol consumption

                      0.157

                      0.017

                      0.453

                      9.328

                      0.000

                      Weight

                      −0.009

                      0.002

                      −0.290

                      −5.951

                      0.000

                      Cigarette smoking

                      0.090

                      0.024

                      0.183

                      3.783

                      0.000

                      Diastolic blood pressure

                      0.003

                      0.001

                      0.128

                      2.644

                      0.009

                      ApoB

                      Body mass index

                      0.013

                      0.003

                      0.292

                      4.625

                      0.000

                      Waist circumference

                      0.004

                      0.001

                      0.190

                      2.986

                      0.003

                      Blood glucose

                      0.013

                      0.005

                      0.136

                      2.691

                      0.008

                      ApoAI/ApoB

                      Alcohol consumption

                      0.143

                      0.028

                      0.253

                      5.140

                      0.000

                      Weight

                      −0.055

                      0.013

                      −1.045

                      −4.357

                      0.000

                      Cigarette smoking

                      0.130

                      0.039

                      0.162

                      3.360

                      0.001

                      Height

                      0.040

                      0.012

                      0.645

                      3.442

                      0.001

                      Systolic blood pressure

                      0.004

                      0.001

                      0.142

                      2.862

                      0.004

                      Han/female

                            

                      TC

                      Age

                      0.019

                      0.004

                      0.272

                      5.099

                      0.000

                       

                      Waist circumference

                      0.026

                      0.007

                      0.186

                      3.716

                      0.000

                       

                      Height

                      −0.025

                      0.009

                      −0.151

                      −2.862

                      0.004

                      TG

                      Waist circumference

                      0.042

                      0.008

                      0.268

                      5.104

                      0.000

                      Blood glucose

                      0.098

                      0.040

                      0.123

                      2.427

                      0.016

                       

                      Diastolic blood pressure

                      0.013

                      0.005

                      0.121

                      2.312

                      0.021

                      HDL-C

                      Waist circumference

                      −0.010

                      0.005

                      −0.118

                      −2.208

                      0.028

                      LDL-C

                      Age

                      0.017

                      0.003

                      0.285

                      5.288

                      0.000

                       

                      Waist circumference

                      0.026

                      0.006

                      0.217

                      4.374

                      0.000

                       

                      Height

                      −0.018

                      0.007

                      −0.129

                      −2.447

                      0.015

                      Alcohol consumption

                      −0.409

                      0.179

                      −0.113

                      −2.279

                      0.023

                      ApoAI

                      Body mass index

                      −0.011

                      0.004

                      −0.141

                      −2.628

                      0.009

                      Cigarette smoking

                      0.187

                      0.068

                      0.145

                      2.760

                      0.006

                       

                      Diastolic blood pressure

                      −0.003

                      0.001

                      −0.131

                      −2.431

                      0.016

                      ApoB

                      Waist circumference

                      0.009

                      0.001

                      0.336

                      7.006

                      0.000

                       

                      Pulse pressure

                      0.003

                      0.001

                      0.194

                      3.746

                      0.000

                       

                      Height

                      −0.005

                      0.002

                      −0.150

                      −3.021

                      0.003

                      Blood glucose

                      0.015

                      0.007

                      0.112

                      2.265

                      0.024

                      ApoAI/ApoB

                      Waist circumference

                      −0.018

                      0.003

                      −0.277

                      −5.576

                      0.000

                       

                      Systolic blood pressure

                      −0.006

                      0.001

                      −0.239

                      −4.698

                      0.000

                       

                      Cigarette smoking

                      0.399

                      0.125

                      0.154

                      3.194

                      0.002

                       

                      Height

                      0.009

                      0.004

                      0.118

                      2.371

                      0.018

                      Mulao/male

                            

                      TC

                      Body mass index

                      0.043

                      0.018

                      0.133

                      2.442

                      0.015

                       

                      Genotype

                      −0.161

                      0.081

                      −0.108

                      −1.976

                      0.049

                      TG

                      Waist circumference

                      0.025

                      0.011

                      0.171

                      2.280

                      0.023

                      Diastolic blood pressure

                      0.015

                      0.006

                      0.139

                      2.588

                      0.010

                       

                      Body mass index

                      0.085

                      0.032

                      0.200

                      2.626

                      0.009

                      HDL-C

                      Body mass index

                      −0.050

                      0.008

                      −0.309

                      −5.993

                      0.000

                      Alcohol consumption

                      0.110

                      0.029

                      0.195

                      3.775

                      0.000

                      LDL-C

                      Genotype

                      −0.153

                      0.063

                      −0.133

                      −2.435

                      0.015

                      ApoAI

                      Alcohol consumption

                      0.121

                      0.025

                      0.253

                      4.786

                      0.000

                      Waist circumference

                      −0.006

                      0.002

                      −0.134

                      −2.526

                      0.012

                      ApoB

                      Pulse pressure

                      0.008

                      0.002

                      0.219

                      3.986

                      0.000

                      Age

                      −0.004

                      0.002

                      −0.117

                      −2.145

                      0.033

                       

                      Body mass index

                      0.022

                      0.010

                      0.124

                      2.323

                      0.021

                      ApoAI/ApoB

                      Waist circumference

                      −0.017

                      0.004

                      −0.230

                      −4.361

                      0.000

                       

                      Alcohol consumption

                      0.136

                      0.040

                      0.180

                      3.411

                      0.001

                      Mulao/female

                            

                      TC

                      Age

                      0.014

                      0.004

                      0.185

                      3.652

                      0.000

                      Genotype

                      −0.261

                      0.091

                      −0.145

                      −2.862

                      0.004

                      Body mass index

                      0.060

                      0.019

                      0.160

                      3.168

                      0.002

                      TG

                      Waist circumference

                      0.024

                      0.005

                      0.232

                      4.529

                      0.000

                      HDL-C

                      Body mass index

                      −0.033

                      0.007

                      −0.248

                      −4.861

                      0.000

                      LDL-C

                      Age

                      0.012

                      0.003

                      0.213

                      4.285

                      0.000

                       

                      Body mass index

                      0.068

                      0.014

                      0.236

                      4.747

                      0.000

                       

                      Genotype

                      −0.261

                      0.091

                      −0.145

                      −2.862

                      0.004

                      ApoB

                      Age

                      0.004

                      0.002

                      0.127

                      2.470

                      0.014

                       

                      Waist circumference

                      0.013

                      0.003

                      0.189

                      3.740

                      0.000

                       

                      Blood glucose

                      0.056

                      0.020

                      0.143

                      2.769

                      0.006

                      Genotype

                      0.098

                      0.042

                      0.117

                      2.339

                      0.020

                      ApoAI/ApoB

                      Waist circumference

                      −0.019

                      0.005

                      −0.191

                      −3.733

                      0.000

                       

                      Age

                      −0.008

                      0.003

                      −0.159

                      −3.114

                      0.002

                      TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ApoAI, Apolipoprotein AI; ApoB, Apolipoprotein B. The correlative factors were determined by multivariable linear regression analysis with stepwise modeling.

                      Discussion

                      In the current study, we showed that the levels of ApoB were higher whereas the ratio of ApoAI to ApoB was lower in Mulao than in Han (P < 0.05 for each). There were no significant differences in the levels of TC, TG, HDL-C, LDL-C and ApoAI between the two ethnic groups (P > 0.05 for all). In contrast with developed countries, China has experienced a considerable increase in the prevalence of CAD over the past several decades [23]. Evidence of the relationship between dyslipidemia and CAD has been established worldwide. It is well recognized that dyslipidemia is a multifactorial and complicated origin which combined by genetic factors with environmental factors including demographics, diet, alcohol consumption, cigarette smoking, obesity, exercise, hypertension [24]. Thus, prediction of the risk for dyslipidemia on the basis of genetic variants would be beneficial for the personalized prevention of CAD. Recent GWASs and meta-analysis on European population have identified many genes previously implicated in lipid regulation. However, validation of these loci on different global populations is still unclear.

                      Mulao nationality is one of the 11 minorities in Guangxi Zhuang Autonomous Region, which is a genetic feature distinctive nationality. Strict intra-ethnic marriages have been performed in this population and they have unique lifestyle as well as diet from time immemorial. Therefore, we surmised that the hereditary characteristic and some lipid-related gene polymorphisms in the Mulao population may be different from those in Han nationality.

                      The genotypic and allelic frequencies of ST3GAL4 rs11220462 SNP in diverse racial/ethnic groups have not been reported. The data in the International HapMap Project’s data-base have showed that the frequencies of A allele and AA, AG genotypes were 42.0%, 15.9% and 52.3% in Han Chinese in Beijing; 30.7%, 9.1% and 43.2% in Japanese; and 15.8%, 1.7% and 0% in Yoruba; respectively. In the present study, we showed that the frequencies of A allele and AA, AG genotypes were 35.5%, 11.2% and 48.7% in Mulao; and 38.3%, 13.0% and 50.6% in Han; respectively. There were no conspicuous differences in the genotypic and allelic frequencies of the rs11220462 SNP between the Mulao and Han populations, or between males and females in the both ethnic groups. As compared with the data in the International HapMap Project’s data-base, we found that the frequencies of the A allele and AA, AG genotypes in our study populations were lower than those in Han Chinese from Beijing, which may be caused by different sample sizes and regions.

                      The potential association between the rs11220462 SNP and serum lipid levels in humans has been detected in a previous GWAS. Teslovich et al. [15] showed that the rs11220462 SNP was significant associated with LDL-C concentration (P = 1.20 × 10−15) in population of European descent. In our present study, we found that the levels of TC and LDL-C were different among the AA, AG and GG genotypes of the rs11220462 SNP in Mulao, the A allele carriers had higher serum TC, LDL-C and ApoB levels than the A allele non-carriers in the both males and females. For Han nationality, the levels of TC and TG were also different among the three genotypes in males but not in females, the subjects with AA genotype had higher TC and TG levels than the subjects with AG or GG genotype. These findings suggest that the association of rs11220462 SNP and serum lipid levels is different between the two ethnic groups, and there may be a sex-specific association of rs11220462 SNP and serum lipid parameters in the Han population. These differences may also be related to the variations in examined populations, including physical condition, environmental factors such as diet or under medication. Therefore, the association of the ST3GAL4 rs11220462 SNP and serum lipid levels needs to be further studied with larger sample size.

                      There was no previous study to report the direct effect of ST3GAL4 rs11220462 SNP on serum lipid levels. We suspected that there may be several potential mechanisms to explain the association between the rs11220462 SNP and serum lipid levels. Firstly, ST3GAL4 encodes a member of the glycosyltransferase 29 family which was associated with increasing risk of type 2 diabetes and cardiovascular disease [19]. Secondly, Adamska et al.[21] reported that the ST3GAL4 plays a key role in the synthesis of E-selectin ligands which is associated with metabolic syndrome and coronary artery calcification. Thirdly, a recent GWAS has identified several intronic SNPs including rs11220462 associated with lipid levels [15]. About 5-10% of genomic variants found in familial hypercholesterolemia are located in the introns of the candidate genes and are classified as splicing mutations [25]. More and more researcher found that intron is involved in mRNA transcription and translation regulation. Finally, microRNAs (miRNAs), a family of small non-coding RNA molecules, appear to regulate animal lipid metabolism and preadipocyte conversion to form lipid-assimilating adipocytes [26]. Recently, genetic model of beef cattle was used to analyze the association between genomic context characteristics of miRNAs (38.7% located in intron) with their function in adipose tissue [27]. The findings suggested that miRNAs play a role in the regulation of bovine adipogenesis and fat metabolism. Taken together, the rs11220462 SNP may act in an intronic variation to influence the ST3GAL4-associated regulatory network or specific miRNAs regulating adipogenesis. However, the biological function and detailed role of ST3GAL4 rs11220462 SNP in lipid metabolism remain unclear and need to be further explored.

                      It is well realized that environmental factors such as dietary patterns, lifestyle and physical inactivity are all strongly related with serum lipid levels [24]. In the present study, multivariate linear regression analysis also showed that age, sex, BMI, alcohol consumption, cigarette smoking, and blood pressure were involved in determining serum lipid parameters in both ethnic groups. These data suggest that the environmental factors also play an important role in determining serum lipid levels in our study populations. Differences in serum lipid levels between the two ethnic groups could be related to factors such as differences in the genetic background, dietary patterns and lifestyle and their interactions. Although rice and corn are the staple food in the both ethnic groups, the people of Mulao nationality prefer cold foods along with acidic and spicy dishes. Therefore bean soy sauce and pickle vegetables become members of their most popular dishes. Meanwhile, they enjoy eating animal offals which contain abundant saturated fatty acid. For nearly 50 years it has been widely accepted that high-fat diets, particularly those contain rich saturated fatty acids, can raise the serum cholesterol concentrations as well as the risk of suffering cardiovascular disease [28]. Diet alone could be responsible for up to 2.5% of the variability on serum lipid levels [29, 30]. A meta-analysis stated that diet modification is a key strategy for prevention and regression of CAD, and for every 1% decrease in energy consumed as dietary saturated fatty acid, TC decreased by 0.056 mmol/L and LDL-C by 0.05 mmol/L [31]. In addition, we also found that the percentages of smokers were higher in Mulao than in Han nationalities (P <0.05). Over the years, the relationship between cigarette smoking and dyslipidemia has been gotten in focus by more and more investigators. A recent study in smoking males (ages from 25 to 35 years old) has found that a significant increase in TC and LDL-C in tobacco users [32, 33]. Another recent meta-analysis also reported that smoking increased TG by 0.15 mmol/L, and decreased HDL-C by 0.09 mmol/L with every 20 cigarettes smoked [34]. Therefore, the results of exposure to different lifestyle and environmental factors probably further modify the association of genetic variations and serum lipid levels in our study populations.

                      There are several major strengths in our study. First, the study is an investigation of a representative random sample of the Mulao population, which retains its regional and special customs in China and may be a useful subgroup for population genetic studies. Second, the sample size of the study is moderate with 700 subjects of Mulao and 694 subjects of Han Chinese. Third, a recent GWAS has reported the association between the rs11220462 SNP and serum lipid levels and our present study is the first replication of GWAS signals in the Chinese population to provide significant evidence for the association of the rs11220462 SNP with lipid traits. To interpret the findings, however, several potential limitations in our study should be acknowledged. First, all of the lifestyle, diet pattern and physical activities are the important factors for lipid regulation and prevalence of CAD, however, the cross-sectional study design limits the ability to determine the effects of them during the statistical analysis since they were self-reported and difficult to classify. Second, although we have explored the association of ST3GAL4 rs11220462 SNP and serum lipid levels, there are still many unmeasured environmental and genetic factors such as other ST3GAL4 SNPs and the interactions of gene-gene and/or gene-environment. Finally, the ST3GAL4 rs11220462 SNP is an intronic variation which does not lead to a change in the amino acid sequence of the ST3GAL4. Thus, the effect of the rs11220462 SNP on lipid metabolism may be limited. To confirm our findings, further in-depth studies on the biological actions of ST3GAL4 rs11220462 variation and the interactions of gene-environment are necessary.

                      Conclusions

                      The present study showed that the genotypic and allelic frequencies of ST3GAL4 rs11220462 SNP were not different between the Mulao and Han populations, or between males and females in the both ethnic groups. But the association of the rs11220462 SNP and serum lipid levels is different between the two ethnic groups. The A allele carriers in the both Mulao males and females had higher TC, LDL-C and ApoB levels than the A allele non-carriers. The subjects with AA genotype in Han males but not in females had higher TC and TG levels than the subjects with AG or GG genotype. These findings suggest that there may be a racial/ethnic-specific association, and/or sex-specific association between the ST3GAL4 rs11220462 SNP and serum lipid parameters in some ethnic groups.

                      Methods and materials

                      Participants

                      Participants in the present study included 700 individuals of Mulao nationality who live in Luocheng Mulao Autonomous County, Guangxi Zhuang Autonomous Region, People’s Republic of China. There were 337 males (48.1%) and 363 females (51.9%). All of them were randomly selected from our previous stratified randomized samples [35]. The ages of the participants ranged from 15 to 93 years, with an average age of 52.56 ± 15.04 years. All of them were rural agricultural workers. In the meantime, a total of 694 Han nationality who reside in the same villages were also randomly selected from our previous stratified randomized samples. The average age of the subjects was 52.26 ± 15.02 years, which ranged from 15 to 84 years. There were 331 men (47.7%) and 363 women (52.3%). All of them were also rural agricultural workers. The whole study subjects were essentially healthy and had no evidence of any chronic illness, including hepatic, renal, or thyroid. The participants with a history of heart attack of myocardial infarction, stroke, congestive heart failure, diabetes or fasting blood glucose ≥ 7.0 mmol/L determined by glucose meter were excluded from the analyses. The participants did not take medications known to affect serum lipid levels (lipid-lowering drugs such as statins or fibrates, beta-blockers, diuretics, or hormones). Ethical approval for this study was obtained from the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University. Written informed consent was provided by all participants.

                      Epidemiological survey

                      The survey was carried out using internationally standardized methods [36]. All participants underwent a complete history, physical examination, and laboratory assessment of cardiovascular risk factors. Information on demographics, socioeconomic status, and lifestyle factors was collected with standardized questionnaires. Smoking status was categorized into groups of cigarettes per day: ≤ 20 and >20. The alcohol information included questions about the number of liang (about 50 g) of rice wine, corn wine, rum, beer, or liquor consumed during the preceding 12 months. Alcohol consumption was categorized into groups of grams of alcohol per day: ≤ 25 and > 25. Sitting blood pressure was measured three times with the use of a mercury sphygmomanometer after the subjects had a 5-minute rest, and the average of the three measurements was used for the level of blood pressure. Systolic blood pressure was determined by the first Korotkoff sound, and diastolic blood pressure was determined by the fifth Korotkoff sound. Body weight, to the nearest 50 grams, was measured by using a portable balance scale. Subjects were weighed without shoes and minimum of clothing. Height was measured, to the nearest 0.5 cm, using a portable measuring device. From these two measurements, BMI (kg/m2) was calculated.

                      Laboratory methods

                      A venous blood sample of 5 mL was drawn from all individuals after an overnight (at least 12 hours) fast. The sample was divided into two parts. One part of the sample (2 mL) was collected into glass tube and allowed to clot at room temperature and used to measure serum lipid levels. Another part of the sample (3 mL) was collected into glass tube with anticoagulate solution (4.80 g/L citric acid, 14.70 g/L glucose, and 13.20 g/L tri-sodium citrate) and used to extract DNA. The levels of serum TC, TG, HDL-C, and LDL-C in the samples were determined by enzymatic methods with commercially available kits (RANDOX Laboratories Ltd., Ardmore, Diamond Road, Crumlin Co. Antrim, United Kingdom, BT29 4QY; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). Serum ApoAI and ApoB levels were detected by the immunoturbidimetric immunoassay using a commercial kit (RANDOX Laboratories Ltd.). All determinations were performed with an autoanalyzer (Type 7170A; Hitachi Ltd., Tokyo, Japan) in the Clinical Science Experiment Center of the First Affiliated Hospital, Guangxi Medical University [24].

                      DNA preparation and genotyping

                      Genomic DNA was isolated from peripheral blood leukocytes using the phenol-chloroform method [35]. The extracted DNA was stored at −20°C until analysis. Genotyping of the rs11220462 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). PCR amplification was performed using 5′-CGATGCTATCCGATGAAC-3′ and 5′-TCACTGTAACCTCCACCTC-3′ (Sangon, Shanghai, People’s Republic of China) as the forward and reverse primer pairs; respectively. Each amplification reaction was performed in a total volume of 25 mL reaction mixture, including 100 ng (2 μL) of genomic DNA, 0.75 μL of each primer (10 μmol/L), 12.5 μL 2 × Taq PCR MasterMix (constituent: 0.1 U Taq polymerase/μL, 500 μM dNTP each, 20 mM Tris–HCl, pH 8.3, 100 mM KCL, 3 mM MgCl2, and stabilizers), and 9.0 μL nuclease-free water. After initial denaturizing at 94°C for 5 min, the reaction mixture was subjected to 32 cycles of denaturation at 94°C at 45 s, annealing at 57°C for 35 s and extension 35 s at 72°C, followed by a final 8 min extension at 72°C. After electrophoresis on a 2% agarose gel with 0.5 μg/mL ethidium-bromide, the amplification products were visualized under ultraviolet light. Then the Sat I restriction enzyme of 2.0 U was added directly to the reaction system consisting of PCR products (5 μL), 9 μL of nuclease-free water and 1 μL of 10 × buffer solution and digested at 37°C for 8 hours. After restriction enzyme digestion of the amplified DNA, genotypes were identified by electrophoresis on 2.0% agarose gel and visualized with ethidium-bromide staining ultraviolet illumination. The PCR produced a 437-bp fragment including one Sat I recognition site for the G allele (Additional file 1: Figure S1). The G allele can be cleaved whereas the A allele cannot be digested (Additional file 2: Figure S2). Therefore, the GG genotype is homozygote for the presence of the site (band at 340- and 97-bp), AG genotype is heterozygote for the absence and resence of the site (bands at 437-, 340- and 97-bp), and AA genotype is homozygote for the absence of the site (bands at 437-bp). Genotypes were scored by an experienced reader blinded to epidemiological data and serum lipid levels. The genotypes detected by PCR-RFLP were also confirmed randomly by direct sequencing (Additional file 3: Figure S3, Sangon Biotech Co., Ltd., Shanghai, People’s Republic of China).

                      Diagnostic criteria

                      The normal values of serum TC, TG, HDL-C, LDL-C, ApoAI, ApoB levels and the ratio of ApoAI to ApoB in our Clinical Science Experiment Center were 3.10-5.17, 0.56-1.70, 1.16-1.42, 2.70-3.10 mmol/L, 1.20-1.60, 0.80-1.05 g/L, and 1.00-2.50; respectively [37]. The individuals with TC > 5.17 mmol/L, and/or TG > 1.70 mmol/L were defined as hyperlipidemic [35]. Hypertension was diagnosed according to the criteria of 1999 World Health Organization-International Society of Hypertension Guidelines for the management of hypertension. Hypertension was defined as an average systolic blood pressure (SBP) ≥ 140 mmHg, and/or an average diastolic blood pressure (DBP) ≥ 90 mmHg, and/or self-reported current treatment for hypertension with antihypertensive medication [37]. The diagnostic criteria of overweight and obesity were according to the Cooperative Meta-analysis Group of China Obesity Task Force. Normal weight, overweight and obesity were defined as a BMI < 24, 24–28, > 28 kg/m2; respectively [38].

                      Statistical analysis

                      The statistical analyses were done with the statistical software package SPSS16.0 (SPSS Inc., Chicago, Illinois). Quantitative variables were expressed as mean ± standard deviation (serum TG levels were presented as medians and interquartile ranges). Qualitative variables were expressed as percentages. The difference in general characteristics between two ethnic groups was tested by the Student’s unpaired t-test. Allele frequency was determined via direct counting, and the standard goodness-of-fit test was used to test the Handy-Weinberg equilibrium. Difference in genotype distribution between the groups was obtained using the chi-square test. The association of genotypes and serum lipid parameters was tested by analysis of covariance (ANOVA). Age, sex, BMI, blood pressure, alcohol consumption, cigarette smoking and blood glucose were included in the statistical models as covariates. Multiple linear regression analyses adjusted for age, sex, BMI, blood pressure, alcohol consumption, cigarette smoking and blood glucose were also performed to assess the association of serum lipid levels with genotypes (AA = 0, AG = 1, GG = 2) and several environment factors. A P value of less than 0.05 was considered statistically significant.

                      Declarations

                      Acknowledgment

                      This study was supported by the National Natural Science Foundation of China (no. 30960130).

                      Authors’ Affiliations

                      (1)
                      Department of Cardiology, Institute of Cardiovascular Diseases, the First Affiliated Hospital, Guangxi Medical University
                      (2)
                      Department of Pathophysiology, School of Premedical Sciences, Guangxi Medical University

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