Introduction

Human and animal model studies have confirmed that abnormal lipid metabolism leads to kinds of diseases, especially altered lipoprotein levels are crucial risk factors for atherosclerosis [1]. A number of studies have established that elevated concentrations of plasma high-density lipoprotein cholesterol (HDL-C) and lower low-density lipoprotein cholesterol (LDL-C) levels reduce the risk for coronary heart disease (CHD) [2–5], but the causality of this inverse association is still incompletely known. There are multiple mechanisms by which HDL-C can be cardioprotective and LDL-C inducing atherogenesis. A widely accepted view is that HDL-C is atheroprotective because of its role in reverse cholesterol transport, a metabolic pathway whereby excess cholesterol in peripheral tissues is transported to the liver for preventing detrimentally accumulation in body [6, 7]. It is estimated that up to 60% of the interindividual variation in plasma HDL-C and LDL-C levels is due to genetic variation [8–11], and that the major portion of this variation is polygenic attributable to sequence variation in various loci. It would be interesting to assess the possible association between particular gene variants and serum lipid profiles in the general population.

The ATP-binding cassette transporter A1 (ABCA1) is a member of the large ATP binding cassette transporters family, which comprises proteins translocating a wide variety of substrates across cell membranes utilising ATP [12]. It has been suggested that ABCA1 participates in the efflux of free cholesterol from peripheral cells, including macrophage-derived foam cells, and contributes to the formation of mature HDL by facilitating the lipidation of circulating nascent apolipoprotein (Apo) AI particles with free cholesterol at the plasma membrane [13–15]. Loss-of-function mutations in the ABCA1 gene cause Tangier disease, a rare genetic disorder characterized by near absence of HDL and accumulation of lipids within cells in various tissues including the blood vessel wall [16–19]. ABCA1 mutation carriers have markedly higher incidence of CHD compared with non-carriers [20]. In addition, in families of Tangier disease patients, onset of CHD is significantly earlier in mutation carriers than in noncarriers [21, 22]. These results suggest that functional deficiency of ABCA1 could also induce an atherogenic decrease in HDL-C levels, incriminating the ABCA1 gene as a candidate for atherosclerotic complications in the general population [23–25]. A number of single nucleotide polymorphisms (SNPs) have been identified both in the coding and promoter regions of the ABCA1 gene http://www.ncbi.nlm.nih.gov/SNP/. However, for most of them there are no robust data about their functional relevance. A common variant of V825I in the ABCA1 gene is a missense SNP in the exon 17 that locates in the middle part of the protein corresponding to sixth transmembrane α-helix with mutation of G TC→A TC. The ABCA1 V825I polymorphism has been found to be associated with modifications of serum HDL-C levels in some studies [26–29] but not in others [30–33]. Thus, it would be interesting to evaluate whether common genetic variations in the gene constitute a major source of interindividual variability in serum lipid levels and CHD susceptibility in the different ethinc populations.

Han is the largest ethnic group and Yao is the eleventh largest minority among the 55 minority groups in China according to the population size. Bai Ku Yao (White-trouser Yao, all of men wear white knee-length knickerbockers) is an isolated branch of the Yao minority with the population size about 30000. Because of isolation from the other ethnic groups, the special customs and cultures including their clothing, intra-ethnic marriages, dietary habits, and life style are still completely preserved to the present day. In several previous epidemiological studies, we showed that several serum lipid parameters were lower in Bai Ku Yao than in Han Chinese from the same region [34, 35]. This ethnic difference in serum lipid profiles remains unknown. We hypothesized that some genetic factors may be different between the two ethnic groups. Therefore, the aim of the present study was to detect the association of V825I polymorphism in the ABCA1 gene and several environmental factors with serum lipid phenotypes in the Guangxi Bai Ku Yao and Han populations.

Materials and methods

Study population

A total of 677 participants of Bai Ku Yao and 646 subjects of Han were randomly selected from our previous randomized cluster samples [34, 35]. All of them come from both Lihu and Baxu villages in Nandan County, Guangxi Zhuang Autonomous Region, People's Republic of China. They were undergone routine physical examination and appropriate laboratory tests. Ages of the Bai Ku Yao subjects ranged from 15 to 80 years, the average age was 39.74 ± 16.01 years old. There were 324 males (47.86%) and 353 females (52.14%). The mean age of the Han subjects was 41.29 ± 16.39 years (range 15 to 80). There were 315 men (48.76%) and 331 women (51.24%). Both the Han and Bai Ku Yao subjects had no evidence of any chronic illness, including hepatic, renal, or thyroid. The participants with a history of heart attack or myocardial infarction, stroke, congestive heart failure, diabetes or fasting blood glucose ≥ 7.0 mmol/L determined by glucose meter were also excluded. The participants were not taking medications known to affect serum lipid levels (lipid-lowering drugs such as statins or fibrates, beta-blockers, diuretics, or hormones). The present study was approved by the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University. Informed consent was obtained from all participants.

Epidemiological survey

The epidemiological survey was carried out under internationally standardized methods by following a general protocol [36]. Common information on demographics, socioeconomic status, and lifestyle factors of the participants was collected by answering all the detailed questions of a standardized questionnaire. The data of alcohol consumption included questions about the number of liangs (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. Smoking status was divided into groups of cigarettes per day: <20 and ≥ 20. Several parameters such as height, weight, and waist circumference were measured. Sitting blood pressure was measured three times with the use of a mercury sphygmomanometer after a 5-min 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 by the fifth Korotkoff sound. Body weight, to the nearest 50 grams, was measured using a portable balance scale. Subjects were weighed without shoes and in a minimum of clothing. Height was measured, to the nearest 0.5 cm, using a portable steel measuring device. From these two measurements body mass index (BMI, kg/m2) was calculated.

Biochemical analyses

Venous blood sample (8 mL) was obtained from each subject between 8 and 11 AM, after 12 hours of fasting. A part of the sample (3 mL) was collected into glass tube and used to determine serum lipid levels. The remaining of the sample (5 mL) was transferred to tubes 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 total cholesterol (TC), triglyceride (TG), HDL-C, and LDL-C in samples were detected by enzymatic methods with commercially available kits, Tcho-1, TG-LH (RANDOX Laboratories Ltd., Ardmore, Diamond Road, Crumlin Co. Antrim, United Kingdom, BT29 4QY), Cholestest N HDL, and Cholestest LDL (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), respectively. 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.

DNA amplification and genetic analysis

Genomic DNA from peripheral blood leukocytes was extracted using the phenol-chloroform method [Full size image

Genotypic and allelic frequencies

Table 2 gives the genotypic and allelic frequencies of V825I polymorphism in the ABCA1 gene. The frequency of G and A alleles was 57.4% and 42.6% in Bai Ku Yao, and 57.7% and 42.3% in Han (P > 0.05); respectively. The frequency of GG, GA and AA genotypes was 33.7%, 47.4% and 18.9% in Bai Ku Yao, and 33.4%, 48.6% and 18.0% in Han (P > 0.05); respectively. There was no significant difference in the genotypic and allelic frequencies between males and females in both ethnic groups.

Table 2 Genotypic and allelic frequencies of the ABCA1 V825I polymorphism between the Bai Ku Yao and Han populations [n (%)]
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The nucleotide sequence of V825I polymorphism

The results were shown as GG, GA and AA genotypes by PCR-RFLP, the GG, GA and AA genotypes were also confirmed by sequencing (Figure 3); respectively.

Figure 3
figure 3

A part of the nucleotide sequence of the ABCA1 V825I polymorphism. (A) GG genotype; (B) GA genotype; (C) AA genotype.

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Genotypes and serum lipid levels

As shown in Table 3, the levels of TC in Bai Ku Yao but not in Han was different among the GG, GA and AA genotypes (P < 0.05), the subjects with AA genotype had higher serum TC levels than the subjects with GG and GA genotypes.

Table 3 Genotypic frequencies of the ABCA1 V825I polymorphism and serum lipid levels between the Bai Ku Yao and Han populations
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The levels of HDL-C and ApoAI in Han but not in Bai Ku Yao was different among the GG, GA and AA genotypes (P < 0.05), the subjects with AA genotype had lower serum HDL-C and ApoAI levels than the subjects with GG and GA genotypes, but these findings were restricted to males but not females.

Relative factors for serum lipid parameters

Multivariate linear regression analysis showed that the levels of TC in Bai Ku Yao and HDL-C and ApoAI in Han were correlated with genotypes (P < 0.05 for all; Table 4). When the multivariate linear regression analysis was performed according to sex in both ethnic groups; respectively, we found that the levels of HDL-C and ApoAI in Han were correlated with genotypes in males but not in females (P < 0.05 for each, Table 5). Serum lipid parameters were also correlated with sex, age, BMI, alcohol consumption, cigarette smoking, and blood pressure in both ethnic groups (Tables 4 and 5).

Table 4 Correlative factors for serum lipid parameters between the Bai Ku Yao and Han populations
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Table 5 Correlative factors for serum lipid parameters between males and females in both ethnic groups
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