Association of Polymorphisms in Stress-Related TNFα and NPY Genes with the Metabolic Syndrome in Han and Hui Ethnic Groups

Metabolic syndrome (MS) is widely defined as a complex of interrelated risk factors, including dysglycemia, raised triglyceride (TC) levels, raised lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels, obesity and hypertension (Grundy et al., 2004; Ferrannini et al., 2006; McGrowder et al., 2012; Saukkonen et al., 2012; Song et al., 2012). With rapid changes in society and increasing demands of intense competition, modern life presents increasing exposure to different kinds of stress. Increasing stress is believed to play a critical role in the development of MS (Maier et al., 2000; Black et al., 2003; Almadi et al., 2013; Li et al., 2013) however, the relationship between stress and MS still remains uncertain, especially for different ethnic groups. The tumor necrosis factor alpha (TNFα) gene is


Introduction
Metabolic syndrome (MS) is widely defined as a complex of interrelated risk factors, including dysglycemia, raised triglyceride (TC) levels, raised lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels, obesity and hypertension (Grundy et al., 2004;Ferrannini et al., 2006;McGrowder et al., 2012;Saukkonen et al., 2012;Song et al., 2012).With rapid changes in society and increasing demands of intense competition, modern life presents increasing exposure to different kinds of stress.Increasing stress is believed to play a critical role in the development of MS (Maier et al., 2000;Black et al., 2003;Almadi et al., 2013;Li et al., 2013) however, the relationship between stress and MS still remains uncertain, especially for different ethnic groups.
The tumor necrosis factor alpha (TNFα) gene is

Association of Polymorphisms in Stress-Related TNFα and NPY Genes with the Metabolic Syndrome in Han and Hui Ethnic Groups
De-Yun Bu, Wen-Wu Ji, Dan Bai, Jian Zhou, Hai-Xia Li, Hui-Fang Yang* located in the 6p21.1-21.3MHC-III chromosomal region, which is also the location of heredity susceptibility for schizophrenia (Peltonen et al., 1995;Teran-Garcia et al., 2007).An increasing number of researches consider TNFα to be a candidate gene for the development of MS (Vendrell et al., 2003;Sookoian et al., 2005;Nobili et al., 2012).A G/A substitution at position 308 upstream of the TNFα gene promoter increases the release of TNF-α protein , augmenting insulin resistance and playing an important role in the pathogenesis of diabetes (Vendrell et al., 2003;Sookoian et al., 2005;Li et al., 2012).However, a study suggested there was no specific evidence that variation in TNFα influences susceptibility to type 2 diabetes (Zeggini et al., 2005).In addition, another study found no association between TNFα gene polymorphism with characteristics of MS, including type 2 diabetes, hypertension, dyslipidemia and obesity in Hong Kong Chinese (Lee et al., 2000).
Neuropeptide Y (NPY) is a 36 amino acid polypeptide (Gehlert, 1999) released from sympathetic nerves by various stressors, particularly chronic stress (Zukowska-Grojec, 1995;Morales-Medina et al., 2010;Gülsün et al., 2012).Increased NPY activity has been found to lead to vasoconstriction and results in high blood pressure (Haefliger et al., 1999;McDermott et al., 2007).NPY also affects the tendency for obesity, which is linked to the development of MS (Kuo et al., 2008;Ruohonen et al., 2009).Kalra et al suggested that NPY and its interacting proteins may be the basis for a new treatment modality to curb the pandemic of obesity and MS resulting from the suppression of NPY that occurs in age-related dietary obesity (Kalra et al., 2004).As the rs16147 single nucleotide polymorphism (SNP) of NPY, occurring in the promoter, has been shown to effect the expression of the gene (Itokawa et al., 2003;Zhou et al., 2008), our study was focused on the relationship between NPYrs16147 and MS.
Inter-individual variability in the regulation of the human stress system accounts, in part, for differences in the onset of stress-related diseases.These differences are also influenced by environmental and genetic factors.This study aimed to investigate the relationship between psychological stress and MS with respect to genetic susceptibility.

Subjects
Ningxia Province has the largest Hui population in China.This matched (1:1) case-control study was carried out in the city of Wu Zhong in Ningxia Province, China.All participants had been living in Wu Zhong for at least 5 years and were between 35 and 74 years of age.Patients were diagnosed according to the definition and criteria published by the International Diabetes Federation (IDF 2005), and participants were considered to have MS if they had central obesity (i.e., waist circumference, WC, ≥90 cm for Chinese men, ≥80 cm for Chinese women) and at least two of the following criteria: (1) TC ≥1.7 mmol/L; (2) reduced HDL-C levels <1.03 mmol/L for men and <1.29 mmol/l for women; (3) blood pressure of ≥130/85 mm Hg or previously diagnosed hypertension; and (4) raised fasting blood glucose (FBG) level of ≥5.6 mmol/L or previously diagnosed type 2 diabetes mellitus.The case group was comprised of 200 patients with MS (Hui 102, Han 98) and the control group of 200 healthy individuals (Hui 102, Han 98).Case-control pairs were individuals that were matched 1:1 with respect to gender, age and nationality.People with mental disease, history of drug abuse, metabolic diseases (such as diabetes mellitus), and other serious diseases were excluded from this study.The study was approved by The Medical Ethics Review Committee of Ningxia Medical University, and all patients agreed to participate and signed a consent form before the study was conducted.

Symptom Checklist-90
The Symptom Checklist-90 (SCL-90), designed by Derogatis in 1975, is one of the most widely used measures of psychological distress in clinical practice and research.The SCL-90 includes 90 items, which are evaluated on 5-point scale: 1, not at all; 2, a little bit; 3, moderately; 4, quite a bit; and 5, extremely.The SCL-90 measures nine primary distress dimensions (somatization, obsessive compulsive behavior, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation and psychoticism).Four global indices of distress (global severity index, global severity index on equal, positive symptom total, factor score) were obtained from SCL-90-R scores.The subjects completed survey questions by entering the appropriate number which best describe their true feelings in the past one week, including the day of the survey.

Anthropometric measurements
Anthropometric measurements, including height, weight, blood pressure, WC and hip circumference (HC), were recorded by trained physicians for all subjects.Body mass index (BMI) was calculated as kg/m 2 .Standing height was measured once using a portable ruler (made in China), and body weight was measured on a digital scale while subjects were wearing light clothes and no shoes.Arterial blood pressure was measured three times in a sitting position.

Laboratory measurements
E l b o w v e i n b l o o d w a s c o l l e c t e d i n t w o tubes (3-5 ml each) containing the anticoagulant ethylenediaminetetraacetic acid in the morning before participants had eaten.One blood sample was used to measure FBG, total cholesterol (TC), TG, LDL and HDL.Among them, FBG measurement should be finished in less than1 h, the others in less than 3 h.Serum was separated from the second tube and stored at -20°C.

Statistical analysis
All survey data were entered with and checked by EPIDATA 3.01.Statistical analysis was carried out using the Statistical Package for the Social Science (SPSS ® ) version 17.0 (IBM, IL, USA).Quantitative variables were analyzed by adopting t-test and one-way analysis of variance and presenting them as the mean ± standard deviation (SD).All statistical tests were two-tailed, and p<0.05 was chosen as the level of significance.The chi-square test was employed to analyze the association between genetic polymorphism and MS.Genotype frequencies of control groups were found to be in Hardy-Weinberg equilibrium.

General subject characteristics
The anthropometric and biochemical characteristics of the case and control groups are shown Table 1.Compared to the control group, the case group exhibited higher values for height, weight, BMI, WC, HC, systolic blood pressure, diastolic blood pressure, TC, TG, HDL and FBG (p<0.05), and these differences were statistically significant.

Analysis of psychological status
Table 2 shows the SCL-90 scores (mean ± SD) for nine factors.Significant differences (p<0.05) were observed for somatization, compulsion, interpersonal sensitivity, depression, anxiety, hostility, fear, paranoia and psychoticism between case and control groups.These results indicate that psychological stress was associated with MS.

Analysis of TNFα-308 polymorphism
Distribution of TNFα-308 genotypes (c 2 = 13.39,p=0.001) and alleles (c 2 = 14.32, p=0.001) are presented in Table 3. Differences were statistically significant (p<0.05).The homozygous mutant genotype (AA) and the mutant allele (A) were less frequent in the control group compared to the case group.Table 3 presents the distribution of TNFα-308 genotypes and genotypic frequencies for case and control subjects of Han and Hui ethnicity.These differences were also statistically significant (p<0.05) for the Han and Hui ethnic groups.This result indicates that TNFα-308 is associated with MS.

Discussion
Differences in culture, lifestyle and heredity exist between the Hui and Han ethnic groups.We found that psychological stress was associated with MS, and results from our previous study that suggested differences in the level of psychological stress exist between Hui and Han ethnic groups.Further, we found the stress-related TNFα gene was associated with MS in the Hui and Han ethnic In contrast, the NPY gene might possibly contribute to MS in the Han ethnic group.Metabolic syndrome has become a major public health problem within our society, several studies suggested that some components of the metabolic syndrome have been associated with the risk of breast cancer and prostate cancer (Ronco et al., 2012;Ozbek et al., 2014).Therefore, it is no time to delay to focus on the risk factors of MS Jermendy et al performed an interesting twin study that explains differences between genetic and environmental factors in the pathogenesis of type 2 diabetes and cardiovascular diseases (Jermendy et al., 2011).A study investigating the prevalence of MS and its factors in a Dutch multi-ethnic cohort of obese and overweight children found that Turkish children had significantly higher prevalence of cardiometabolic risk factors relative to their peers of Moroccan descent (Van Vliet et al., 2009).Given racial differences in genetic susceptibility and aggravating stress levels, differential methods must be developed to treat and prevent MS for different groups.
Exposure to chronic psychological stress increased vulnerability to MS.Two major components mediate stress responses: the autonomic nervous system, encompassing the sympathetic and parasympathetic nervous systems, and the hypothalamus-pituitary-adrenocortical (HPA) axis (Bose et al., 2009).HPA has been widely recognized as a potential mechanism for coping with stress (McEwen, 1998).Dallman et al. suggest psychological stress leads to weight gain through behavioral pathways, such as increased food assumption and sedentary behavior, as well as through prolonged exposure to biological stress mediators such as cortisol (Dallman et al., 2010).In contrast, another study suggested that hypocortisolism caused by chronic stress might have beneficial effects for the individual (Fries et al., 2005).Analysis of psychological status in our study suggests that there is association between psychological stress and MS.Exploring the association of polymorphisms in the stressrelated TNFα and NPY genes with metabolic syndrome in Han and Hui ethnic groups is very important.
Recent studies indicate that TNFα is related to insulin resistance, hypertension, leptin levels and hypercholesterolemia, which lead to MS ( Moller 2000;Trayhurn et al., 2004;Lo et al., 2007).The G-to-A variant of TNFα-308 increased the excretion of cytokine TNF, which plays a critical role in the onset of MS (Dalziel et al., 2002;Gupta et al., 2012) However, TNFα exerts complex regulatory actions on the development of MS.Curti et al, by assessing the association of TNFα-308G/A with changes induced by lifestyle intervention, found that the TNFα-308 G/A SNP may predispose a better glucose metabolism response (Curti et al., 2012).However, several other studies suggest the TNFα G-308A polymorphism is unlikely to play an important role in the development of insulin resistance and obesity related to MS (Meirhaeghe et al., 2005).In our study, the mutant A allele was less frequent in the control group, and a significant association of the TNFα-308 G/A polymorphism with MS was observed.
Kuo et al found that exposure to stress lead to the release of NPY from sympathetic nerves, which increased white adipose tissue and mediated stress-induced obesity and MS (Kuo et al., 2007).In our study, NPY associated with MS only in ethnically Han individuals.Differences in these conclusions could be explained by differences in ethnicity as well as by the difference between the test organisms used (human and non-human animals).Differences in transcription factors regulating NPY expression has been observed for different races (Gallagher et al., 2010).For instance, feeding wild-type mice a high-fat diet for 2 weeks does not affect mRNA expression levels of NPY (Heijboer et al., 2005).However, a recent study of Spanish children confirmed the association between NPYrs16147 and obesity and demonstrated for the first time an association of NPYrs16131 with childhood obesity (Olza et al., 2013).Extreme stress combined with a high fat/sugar diet increases the endogenous release of NPY into visceral fat, causing the formation of new adipocytes and accelerating abdominal obesity and the development of MS (Rasmusson et al., 2010).
In conclusion, our findings suggest that psychological stress contributes to the development of MS.In addition, variants of the stress-related TNFα gene could constitute a useful predictive marker.We found that the NPY gene was associated with MS only in the Han ethnic group.To find potential and specific strategies to prevent and control the development of MS, a cohort study with subjects of different ethnicities that experience different levels of psychological stress is necessary to confirm and evaluate this study.

Table 3 . Distribution of TNFα Genotypes in Case and Control Groups
Data given as Mean ± SD; *Significant value (p< 0.05) 5898De-Yun Bu et al