Introduction

Coronary artery disease (CAD) is the leading cause of morbidity and mortality in industrialized countries, and the prevalence of this disease is increasing rapidly in develo** countries [1]. Consistent and compelling evidence has demonstrated association between dyslipidemia and CAD incidence worldwide [2–4]. It is well-established that dyslipidemia is a complex trait caused by multiple environmental and genetic factors [5–7] and their interactions [12–46]. Common variants at these loci together explain < 10% of variation in each lipid trait [23, 24, 40]. Rare variants with large individual effects may also contribute to the heritability of lipid traits [40]. In addition, GWAS also discovered a number of novel loci that influence serum lipid phenotypes [15, 17, 21, 39]. One of these newly identified single nucleotide polymorphisms (SNPs) is rs16996148 SNP in the NCAN/CILP2/PBX4. rs16996148 is located on chromosome 19p13 in an intergenic region between CILP2 and PBX4, which encodes a cartilage intermediate layer protein and a putative transcription factor expressed in testis, respectively [47, 48]. Neurocan (NCAN) is a nervous system-specific proteoglycan involved in neuronal pattern formation, remodeling of neuronal networks and regulation of synaptic plasticity [49], with no obvious relation to LDL-C or TG concentrations. The roles of CILP2 and PBX4 in lipid metabolism are also unclear at this time [50, 51]. In Europeans, however, rs16996148 in NCAN/CILP2/PBX4 has been showed significant associations with LDL-C and TG concentrations [15, 17]. Tai et al. [51] also reported that rs16996148 SNP was significantly associated with LDL-C and HDL-C concentrations in the Malays under a recessive model of inheritance. Nevertheless, Nakayama et al. [50] did not observe significant associations between rs16996148 SNP and blood lipid profiles in the Japanese population. These results suggest that the ability of associations to generalize across other racial/ethnic populations varied greatly, some of these GWAS-identified variants may not be functional and are more likely to be in linkage disequilibrium with the functional variants.

China is a multiethnic country with 56 ethnic groups. Han nationality is the largest ethnic group, and Mulao nationality is one of the 55 minorities with population of 207,352 according to the fifth national census statistics of China in 2000. Ninety percent of them live in the Luocheng Mulao Autonomous County, Guangxi Zhuang Autonomous Region, People's Republic of China. The history of this minority can be traced back to the ** Dynasty (AD 265-420). In a previous study, Xu et al. [52] 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, however, the association of rs16996148 SNP and serum lipid levels has not been previously reported in the Chinese population. Therefore, the aim of the present study was to detect the association of rs16996148 SNP in the NCAN/CILP2/PBX4 and several environmental factors with serum lipid profiles in the Mulao and Han populations.

Materials and Methods

Participants

Participants in the present study included 712 individuals of Mulao nationality living in Luocheng Mulao Autonomous County, Guangxi Zhuang Autonomous Region, People's Republic of China. They were randomly selected from our previous stratified randomized cluster samples [53]. The ages of the participants ranged from 15 to 86 years, with an average age of 51.81 ± 14.76 years. There were 330 males (46.3%) and 382 females (53.7%). All participants were rural agricultural workers. During the same period, a total of 736 people of Han nationality who reside in the same villages were also randomly selected from our previous stratified randomized cluster samples. The average age of the subjects was 51.77 ± 14.96 years (range 15 to 86). There were 308 men (41.8%) and 428 women (58.2%). All of them were also rural agricultural workers. All 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 or 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 were not taking medications known to affect serum lipid levels (lipid-lowering drugs such as statins or fibrates, beta-blockers, diuretics, or hormones). The experimental design was approved by the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University. All participants in this study provided written informed consent.

Epidemiological survey

The survey was carried out using internationally standardized methods [54]. All participants underwent a complete history, physical examination, and laboratory assessment of cardiovascular risk factors, including cigarette smoking, family history of myocardial infarction, blood pressure, presence of diabetes mellitus. Information on demographics, socioeconomic status, and lifestyle factors was collected with standardized questionnaires. The alcohol information 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 categorized into groups of cigarettes per day: ≤ 20 and > 20. At the physical examination, several parameters were measured. 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 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.

Determination of serum lipid levels

Venous blood samples were collected after an overnight (at least 12 hours) fast. A part of the sample (2 mL) was collected into glass tubes and allowed to clot at room temperature, and used to determine serum lipid levels. Another part of the sample (3 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. Serum TC, TG, HDL-C, and LDL-C levels in the samples were measured according to standard enzymatic methods. Serum ApoAI and ApoB levels were detected by the immunoturbidimetric immunoassay. 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 [6, 7].

DNA preparation and genoty**

Genomic DNA was isolated from peripheral blood leukocytes using the phenol-chloroform method [DNA sequencing

Six samples (GG, GT and TT genotypes in two; respectively) detected by the PCR-RFLP were also confirmed by direct sequencing. The PCR products were purified by low melting point gel electrophoresis and phenol extraction, and then the DNA sequences were analyzed in Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., 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 [53]. The individuals with TC > 5.17 mmol/L and/or TG > 1.70 mmol/L were defined as hyperlipidemic [6, 7, 53]. Hypertension was diagnosed according to the criteria of 1999 World Health Organization-International Society of Hypertension Guidelines for the management of hypertension [55, 56]. 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, and > 28 kg/m2; respectively [Full size image

Nucleotide sequences

The results were shown as GG, GT and TT genotypes of the rs16996148 SNP by PCR-RFLP, the genotypes were also confirmed by sequencing (Figure 3); respectively.

Figure 3
figure 3

A part of the nucleotide sequence of rs16996148 SNP. (A) GG genotype; (B) GT genotype; (C) TT genotype.

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Genotypic and allelic frequencies

The genotypic and allelic frequencies of rs16996148 SNP in the both ethnic groups are shown in Table 2. The G and T allele frequencies of rs16996148 SNP were 87.2% and 12.8% in Mulao, and 89.9% and 10.1% in Han (P < 0.05); respectively. The frequencies of GG, GT and TT genotypes were 76.0%, 22.5% and 1.5% in Mulao, and 81.2%, 17.4% and 1.4% in Han (P < 0.05); respectively. There were no significant differences in the genotypic and allelic frequencies between males and females in both groups.

Table 2 The genotypic and allelic frequencies of rs16996148 SNP the Mulao and Han populations [n (%)]
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Genotypes and serum lipid levels

As shown in Table 3, the levels of HDL-C in Mulao were different among the three genotypes (P < 0.05), the T allele carriers had higher serum HDL-C levels than the T allele noncarriers. When serum lipid parameters in Mulao were analyzed according to sex, we found that the levels of HDL-C, ApoAI, and the ratio of ApoAI to ApoB in males but not in females were different between the GG and GT/TT genotypes (P < 0.01 for all), the subjects with GT/TT genotypes had higher serum levels of HDL-C, ApoAI, and the ratio of ApoAI to ApoB than the subjects with GG genotype.

Table 3 The genotypes of rs16996148 SNP and serum lipid levels in the Mulao and Han populations
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In the Han population, the levels of TC, TG, LDL-C and ApoAI among the three genotypes, or the levels of TC, LDL-C, ApoAI, and ApoB between the GG and GT/TT genotypes were different (P < 0.05-0.001), the T allele carriers had higher serum TC, TG, LDL-C, ApoAI and ApoB levels than the T allele noncarriers. When serum lipid parameters in Han were stratified according to sex, we showed that the levels of TC, TG, LDL-C, ApoAI, and ApoB in males but not in females were different between the GG and GT/TT genotypes (P < 0.05-0.001), the subjects with GT/TT genotypes had higher serum levels of TC, TG, LDL-C, ApoAI, and ApoB than the subjects with GG genotype.

Risk factors for serum lipid parameters

The correlation between the genotypes of rs16996148 SNP and serum lipid parameters in Mulao and Han is shown in Table 4. The levels of HDL-C, ApoAI, and the ratio of ApoAI to ApoB in Mulao were correlated with the genotypes in males (P < 0.05-0.01) but not in females. The levels of TC, TG, HDL-C, LDL-C, ApoAI and ApoB in Han were associated with the genotypes in males (P < 0.05-0.001) but not in females.

Table 4 Correlation between the genotypes of rs16996148 SNP and serum lipid parameters in the Mulao and Han populations
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Serum lipid parameters were also correlated with several environment factors such as age, gender, alcohol consumption, cigarette smoking, blood pressure, blood glucose, and BMI in both ethnic groups (P < 0.05-0.001; Table 5).

Table 5 Correlation between environmental factors and serum lipid parameters in the Mulao and Han populations
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