Association between the PLTP rs4810479 SNP and Serum Lipid Traits in the Chinese Maonan and Han Populations

The association between the phospholipid transfer protein (PLTP) gene rs4810479 single-nucleotide polymorphism (SNP) and serum lipid levels is largely unknown. This investigation aimed to evaluate the relationship between the PLTP rs4810479 SNP, several environmental risk factors, and serum lipid parameters in the Chinese Maonan and Han nationalities. Polymerase chain reaction-restriction fragment length polymorphism, gel electrophoresis, and direct sequencing were employed to determine the PLTP rs4810479 genotypes in 633 Maonan and 646 Han participants. The frequencies of CC, CT, and TT genotypes and the C allele were different between Maonan and Han groups (29.07%, 53.08%, 17.85%, and 55.61% vs. 35.60%, 49.70%, 14.70%, and 60.45%, respectively, P < 0.05). The C allele carriers in the Maonan group had higher high-density lipoprotein cholesterol levels than the C allele noncarriers, but this finding was only found in Maonan males but not in females. The C allele carriers in Han males had lower total cholesterol and low-density lipoprotein cholesterol levels than the C allele noncarriers. Serum lipid profiles were also affected by several traditional cardiovascular risk factors in both populations. There might be an ethnic- and/or sex-specific association between the PLTP rs4810479 SNP and serum lipid traits.


Introduction
Cardiovascular disease (CVD) is one of the leading causes of disability and early death worldwide, accounting for about one-third of the global mortality rate [1]. e cost of CVD constitutes a major economic burden to the society [2]. Many studies have proven that serum or plasma triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) concentrations are independent risk factors for CVD [3][4][5].
It is well known that various genetic and environmental factors can lead to abnormalities of plasma lipids and lipoproteins [6][7][8]. Plasma lipid and lipoprotein concentrations are themselves highly heritable-estimates range from 40% to 60%. A number of genome-wide association studies (GWASes) have identified more than 95 genetic loci associated with plasma lipid phenotypes. One of the newly discovered loci is the phospholipid transfer protein (PLTP) gene [9][10][11][12].
PLTP (also called lipid transfer protein 2) is a member of lipid transfer/lipopolysaccharide-(LPS-) binding protein family.
is family includes PLTP, LPS-binding protein (LBP), bactericidal/permeability-increasing protein (BPI), and cholesterol ester transfer protein (CETP) [13][14][15]. ere are two molecular weights of PLTP, 55 kDa and 81 kDa. is may be due to different glycosylation [15]. PLTP is a monomeric and nonspecific lipid transfer protein, which can efficiently transfer free cholesterol, diacylglycerol, α-tocopherol, cerebroside, LPS, phospholipids, and sphingosine-1-phosphate [13][14][15]. ere are two forms of lipoproteinassociated plasma PLTP (high active one and low active one) which are associated with apolipoprotein (Apo) A1-and ApoE-containing lipoproteins, respectively [16]. However, the cause for the existence of active and inactive PLTP in plasma is unclear. It is quite possible that PLTP might have other activities except its lipid transfer function [15]. PLTP is produced in various types of cells and secreted into plasma. It is highly expressed in human tissues such as the ovary, thymus, placenta, lung [17], liver, and small intestine and in macrophages [18,19] and atherosclerotic lesions [19,20]. e gene encoding PLTP (PLTP) is located on human chromosome 20. Its cDNA has a length of 1750 base pairs, including an open reading frame of 1518 nucleotides and a 3′-untranslated region (UTR) of 184 nucleotides. e mature PLTP contains 476 amino acids and 6 N-glycosylation sites that allow it to change its molecular weight (55 or 81 kDa) after different degrees of glycosylation modification [17]. Several previous studies have found that PLTP is an emerging cardiac metabolic factor which exerts a vital part in the development of blood lipid metabolism and atherosclerosis [21,22]. PLTP is a main factor modulating the size and composition of high-density lipoprotein particles in the circulation and plays an important role in controlling plasma HDL-C levels [23]. PLTP deficiency in mice can lower total cholesterol (TC), HDL-C, and ApoA1 but increase TG levels significantly, impact the biological quality of high-density lipoprotein [24], and attenuate high-fat dietinduced insulin resistance and obesity [25]. Plasma PLTP activity (PLTPa) was significantly inversely correlated with carotid artery disease (CAAD), with a 9% decrease in odds of CAAD per 1 unit increase in PLTPa. Plasma TG levels, diabetes, statin use, and PLTP rs4810479 SNP were also associated with PLTPa significantly [26]. In human studies, both PLTP mass and PLTPa were associated with plasma lipid traits, glucose regulation, and atherosclerosis. Common variation at the PLTP structural locus region could explain about 30% of variation in PLTPa [27]. PLTP variants were associated with the PLTP mRNA level [28] and CVD risk [27,29].
Being an isolated and conservative minority in China, the population of Maonan nationality was 107,166 (ranked 37) according to the statistics of China's sixth national census in 2010. ey have own unique culture and life customs, such as intraethnic marriages, clothing, special lifestyle, and dietary structure. ese characteristics are distinct from those in the largest ethnic group, Han Chinese. erefore, we hypothesize that the genotype distribution and genetic traits of some lipid metabolism-related genes in the Maonan ethnic group may be different from those in the Han ethnic group. In the Chinese populations, there is no previous study to explore the association between the PLTP rs4810479 SNP and serum lipid levels. us, the aim of this study was to appraise the association between the PLTP rs4810479 SNP, several environmental risk factors, and serum lipid traits in the Maonan and Han nationalities.

Subjects.
e study populations were stochastically chosen from our earlier stratified random specimens. e detailed inclusion and exclusion criteria have been described in a previous report [30]. In brief, all selected people were basically healthy and had no evidence of any chronic illness such as cardiac, hepatic, renal, or thyroid diseases. e participants who had a history of heart attack or myocardial infarction, stroke, congestive heart failure, and diabetes were excluded. ey did not use medications known to affect serum lipid levels such as lipid-lowering drugs (statins or fibrates), β-blockers, diuretics, or hormones. e present study included 633 unrelated Maonan participants (251 males, 39.65%, and 382 females, 60.35%) and 646 unrelated Han subjects (268 males, 41.49%, and 378 females, 58.51%) [30]. e participants aged from 22 to 92 years (mean: 55.92 ± 14.30 years in Maonan and 54.50 ± 14.50 years in Han groups). e age structure and sex ratio between the two populations were matched. Basic information and health status of all participants refer to our previous study [31].
is research project was approved by the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University (no. Lunshen-2014-KY-Guoji-001, March 7, 2014). All participants provided written informed consent before the study.

Epidemiological Survey.
e survey was conducted using an internationally standardized method [32]. A standardized questionnaire was used to gather the information related to demographic statistics, socioeconomic status, and life style factors. Drinking and smoking were grouped according to daily consumption (0, ≤25, and >25 and 0, ≤20, and >20, respectively). Several parameters such as weight, body mass index (BMI), height, waist circumference, and blood pressure were also obtained.

Biochemical Measurements.
After 12 hours of fasting, a cubital vein blood sample of 5 ml was obtained from all participants. Biochemical measurements including TC, TG, HDL-C, LDL-C, ApoA1, ApoB, and blood glucose were performed as previously described [33,34].

DNA Amplification and
Genotyping. Genomic DNA of the samples was extracted by the phenol-chloroform method [34]. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was utilized to determine the genotypes of the PLTP rs4810479 SNP. e forward and reverse primer pairs for PCR amplification were 5′-ATCCTCCGATCTTGGCTTCC-3′ and 5′-CCAGGTA-GAGGGAACAGCAA-3′, respectively. e specific reaction condition was 5 min pretreatment at 95°C, denaturation at 95°C for 30 s, annealing at 59°C for 30 s, followed by extension for 40 s at 72°C for 33 cycles, and finally a 7 min extension at 72°C. e restriction enzyme was KpnI. After electrophoresis on 2.0% agarose gel containing 0.5 μg/ml of ethidium bromide, the results were obtained under ultraviolet light.
e PCR products of six samples were also confirmed by direct sequencing using ABI Prism 3100 (Applied Biosystems) in Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., China. e normal reference values of serum lipid parameters including TG, TC, LDL-C, HDL-C, ApoA1, ApoB concentrations, and ApoA1/ApoB ratio as well as the diagnostic criteria of hyperlipidemia, hypertension, and type 2 diabetes, and normal weight, overweight, and obesity have been described in detail in our several previous research studies [30,31,[33][34][35].

Statistical Analysis.
e number of study samples in this research was estimated using Quanto software. All of the statistical analyses were accomplished using SPSS software (version 23.0). Normally distributed quantitative variables were expressed as mean ± standard deviation (nonnormally distributed serum TG levels were expressed as median and quartiles). Direct counting and standard goodness-of-fit test were used to determine the allele frequency and verify the Hardy-Weinberg equilibrium (HWE), respectively. e genotype distribution was tested by chi-square test, and the general features between the two ethnic groups were analyzed by unpaired t-test. e association between genotype and serum lipid parameters was assessed by the covariance analysis (ANCOVA), in which age, gender, blood pressure, BMI, cigarette smoking, and alcohol consumption were used as covariates. Stepwise modeling of multiple linear regression analyses was used to determine the relevant risk factors for serum lipid parameters in the Maonan, Han, male, and female (CC/CT genotypes � 1 and TT genotype � 2), respectively. Bilateral P value <0.05 was considered statistically significant.

General Features and Serum Lipid Profiles.
e features and serum lipid levels are presented in Table 1. e values of gender ratio, age structure, BMI, weight, and height; the percentages of cigarette smoking and alcohol intake; the levels of blood glucose, TC, LDL-C, ApoA1, and ApoB; and the ratio of ApoA1/ApoB were not different between the Maonan and Han populations (P > 0.05 for all). However, the levels of waist circumference, pulse pressure, diastolic and systolic blood pressures, and serum TG were higher, whereas the levels of HDL-C were lower in Maonan than in Han ethnic groups (P < 0.05 − 0.001).

Genotyping and Genotypes.
After electrophoresis of the PCR product, the products of 609 bp nucleotide sequences were observed in all samples ( Figure 1). e bands of the three genotypes are presented in Figure 2: CT genotype (286, 323, and 609 bp), CC genotype (286 and 323 bp), and TT genotype (609 bp). e genotypes were distinguished by the presence of the enzyme restriction site (C allele) or absence (T allele). e results of direct sequencing of the samples are shown in Figure 3. Table 2, there were significant differences in the frequencies of CC, CT, and TT genotypes and C allele between the Maonan and Han populations (29.07%, 53.08%, 17.85%, and 55.61% vs. 35.60%, 49.70%, 14.70%, and 60.45%, respectively, P < 0.05). However, the genotype and allele frequencies of the rs4810479 SNP in both ethnic groups were not significantly different between men and women (P > 0.05 for all). Tables 3 and 4, serum HDL-C concentrations in the Maonan group were significantly different among the three genotypes (P < 0.05), and serum HDL-C concentrations were higher in the C allele carriers than the C allele noncarriers, but this finding was only restricted to males but not females. Lower TC and LDL-C concentrations in Han males were also observed in the C allele carriers than the C allele noncarriers (P < 0.05 for all).

Relevant Factors for Serum Lipid Parameters.
Multiple linear regression analyses showed that serum HDL-C and ApoA1 concentrations in the Maonan group were correlated with the PLTP rs4810479 genotypes (P < 0.05; Table 5). Serum HDL-C concentrations in Maonan males, HDL-C and ApoA1 concentrations in Maonan females, and TC and LDL-C concentrations in Han males were associated with the genotypes (P < 0.05; Table 6). In addition to the PLTP rs4810479 genotypes, serum lipid traits in the participants were also influenced by several risk factors such as gender, age, waist circumference, BMI, pulse pressure, diastolic blood pressure, systolic blood pressure, fasting blood glucose, alcohol consumption, and cigarette smoking (P < 0.05 for all; Tables 5 and 6).

Discussion
e current study revealed that the Maonan ethnic group had higher TG and lower HDL-C concentrations than the Han ethnic group (P < 0.001 for each). ere were no significant differences in the TC, LDL-C, ApoA1, and ApoB concentrations and the ApoA1/ApoB ratio between the Maonan and Han populations (P > 0.05 for all). It is common knowledge that dyslipidemia is one of the major changeable cardiovascular risk factors and is a major predictor of CVD mortality [1]. e difference in serum lipid profiles between the two populations may be due to distinct environmental, genetic factors and their interactions. Maonan nationality is one of 55 minorities in China. Being a mountain ethnic group, Maonan has its own unique history, custom, and culture, such as intraethnic marriages, specific clothing, inimitable lifestyle, and dietary habits. Most Maonan people are engaged in agricultural production, supplemented by animal husbandry, aquaculture, and other sideline industries. Rice and corn are their staple food, and pumpkin, sweet potato, and millet are the complementary foods. e preference for acidic food is the greatest feature of their diet culture. ey have unique eating habits and lifestyles compared to other ethnic groups. Maonan ethnic group advocates intraethnic marriages. eir marriages are mostly arranged by parents. ese results suggest that the genetic traits of some genes related to lipid metabolism may be different between the Maonan and Han ethnic groups.
According to the results of the International 1000 Genomes database (https://www.ncbi.nlm.nih.gov/variation/ tools/1000genomes/), we knew that the rs4810479C allele frequency was 26    In the present study, we found that the Maonan ethnic group had lower rs4810479C allele frequency than the Han ethnic group (55.61% vs. 60.45%, P < 0.05). e genotype distribution of the PLTP rs4810479 SNP in the present study was also different between the two ethnic groups (P < 0.05), but there was no significant difference in the genotype and allele frequencies between males and females in both populations. ese findings suggest that the PLTP rs4810479 SNP may have a racial/ethnic specificity. e association between the PLTP rs4810479 SNP and serum lipid concentrations in different racial/ethnic groups is still largely unclear. In a previous GWAS, Musunuru et al.  Genetics Research 5 [36] prompted that the PLTP rs4810479 SNP was associated with HDL-C concentrations in European populations. In the present study, we noted that the PLTP rs4810479 SNP was significantly associated with several serum lipid phenotypes in the Maonan and Han populations. Subgroup analyses of serum lipid profiles according to sex showed that the C allele carriers had higher HDL-C concentrations in Maonan males and lower TC and LDL-C concentrations in Han males than the C allele noncarriers. ese results indicate that there might be a race-and/or sex-specific association between the PLTP rs4810479 SNP and serum lipid traits in our study ethnic groups.
It is well known that serum lipid concentrations are also affected by many environmental risk factors such as population features, life style, diet structure, and physical inactivity [37]. In the current study, we also found that serum Table 4: Comparison of the genotypes and serum lipid levels between males and females in the Maonan and Han populations.      ey are also good at making various complementary foods with rice. ey like to eat spicy and acidic foods that contain a lot of oil and salt. e intake of a large amount of carbohydrate, oil, and salt can increase the waist circumference and blood pressure in the Maonan people. Several studies have shown that long-term high-salt diet is an important risk factor to affect blood pressure levels [38,39]. A meta-analysis showed that reduced sodium intake can lower blood pressure levels in people with or without hypertension [40]. In addition, the people of Maonan also like to eat pork, beef, and/or animal offals in a hot pot which is rich in saturated fatty acids. Many previous studies have shown that diet alone can explain the variation in blood lipid levels [41]. Longterm high-saturated fat diets are strongly associated with obesity, hypertension, dyslipidemia, and atherosclerosis [42,43]. erefore, different environmental risk factors such as unhealthy lifestyle and diet structure may further alter the association between genetic variation and blood lipid concentrations in our research populations.

Ethnic/genotype n TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) ApoA1 (g/L) ApoB (g/L) ApoA1/ApoB
Our work may have some limitations. First, we could not exclude the influence of diet and other environmental risk factors in the statistical analyses. Second, we also could not rule out the effect of asymptomatic diseases. ird, an association between the PLTP rs4810479 SNP and serum lipid concentrations was observed in this study, but many unmeasured factors should be considered including genetic and environmental risk factors. Finally, the sample size in our study populations is a bit small. erefore, it is necessary to further expand the sample size, especially the gene-gene, gene-environment, and environment-environment interactions on serum lipid parameters to confirm our findings.

Conclusions
ere was a significant difference in the genotype and allele distribution of the PLTP rs4810479 SNP between the Maonan and Han populations. e association between the PLTP rs4810479 SNP and serum lipid parameters was also different between the two nationalities and between males and females.
ere may be a racial/ethnic-and/or sexspecific association between the PLTP rs4810479 SNP and serum lipid concentrations in our study populations.

Data Availability
e datasets generated during the present study are not publicly available because detailed genetic information of each participant was included in these materials.

Disclosure
ere was no role of the funding body in the design of the study and collection, analysis, and interpretation of the data and in writing the manuscript.

Conflicts of Interest
e authors declare no conflicts of interest.