Associations Of Genetic Variants With Metabolic Syndrome

Metabolic syndrome (MS) is a complex disorder defined by a cluster of interconnected factors that increase the risk of diabetes mellitus type 2 and cardiovascular atherosclerotic diseases. This disorder consists of central obesity, glucose intolerance, dyslipidemia, hypertension, and hyperinsulinemia, which is characterized by insulin resistance as its core. The prevalence of metabolic syndrome, which has become a global public health problem, has been increased dramatically in recent years. According to our investigations of several cities in China, the prevalence of metabolic syndrome is 18.1% in adult and 3.8% in children and adolescents. Our research explored the associations of genetic variations with metabolic syndrome. The thesis is organized into 2 parts as follows:1. Associations of genetic variants in inflammatory pathway related genes with metabolic syndromeThe subjects were selected by case-control strategy. And the genetic variants were genotyped using Illumina Human-OmniExpress-12v1.0 Chip. We performed the study to explore the association between genetic variations in inflammation related genes and metabolic syndrome, assess the genetic susceptibility and establish the assessment model for metabolic syndrome.Allele specific influence of the SNPs (FPRP<0.2) on transcription factors binding was predicted by online sofeware TFSEARCH and PATCH, and dual luciferase reporter gene assay (DLR) and enzyme linked immunosorbent assay (ELISA) were performed to verify the predicted influence.2. Associations of LIPC C-514T polymorphism with obesity and plasma lipids levels in Chinese children and adolescentsSubjects were recruited from a cross-sectional study on metabolic syndrome of children and adolescents and selected by case-control strategy. LIPC C-514T polymorphism was genotyped using the Sequenom MassARRAY iPLEX platform. We evaluated the association between the polymorphism with obesity and plasma lipid levels, and the potential interaction effect of gender on plasma lipid levels. Additionally we conducted a meta-analysis of all results from published studies as well as our own data.Part I:Associations of Genetic Variants in Inflammatory Pathway Related Genes with Metabolic SyndromeMetabolic syndrome is a cluster of interrelated metabolic disorders, which includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension, and hyperinsulinemia. The disorder may increase the risk of diabetes mellitus type 2 and cardiovascular atherosclerotic diseases. With insulin resistance as its central pathophysiologic mechanism, the detailed mechanism has not been fully elucidated. Metabolic syndrome per se is a state of low-grade chronic systemic inflammation, and inflammation has been found to take part in the occurrence and development of metabolic syndrome with a mount of inflammatory molecules involved in the process. According to the previous studies, there were mainly three signal pathways, NF-κB pathway, JNK pathway and PKR pathway, involved in the metabolic and inflammatory processes. The genetic variations in inflammatory pathway related gene may influence the risk of suffering metabolic syndrome and its related diseases by its biological functions.Purpose:To explore the association between genetic variations in inflammation related genes and metabolic syndrome, assess the genetic susceptibility and establish the assessment model for metabolic syndrome.Matetials and Methods:1. Screening for metabolic syndrome related SNPsThe CDS (Chinese Diabetes Society) criteria was used to confirm the diagnosis of metabolic syndrome. Subjects were recruited from a cross-sectional study on metabolic syndrome of adults without intervention. The case-control strategy was employed to selected the control group as health people without metabolic syndrome and related diseases including diabetes, hypertension, dyslipidemia, stroke and coronary heart disease, and has no blood relationship with the case group. We selected 914 SNP in 80 inflammation related genes from NF-κB pathway, JNK pathway and PKR pathway. The genetic variants were genotyped using Illumina Human-OmniExpress-12v1.0 Chip. Statistical methods including t-test, chi-square test, logistic regression, and linear regression were applied to evaluate the association between SNPs with metabolic syndrome and its compositions. The minimum P value under 3 genetic models (additive, dominant and recessive model) made the min-P. False positive report probability (FPRP) was applied to assess the statistical results to reduce the probability of false positive results. Genetic risk score (GRS) calculated by variants (FPRP<0.2) was employed to evaluate the genetic susceptibility to metabolic syndrome.2. Preliminary study on biological function of metabolic syndrome related SNPsAllele specific influence of the SNPs (FPRP<0.2) on transcription factors binding was predicted by online sofeware TFSEARCH and PATCH, and dual luciferase reporter gene assay (DLR) and enzyme linked immunosorbent assay (ELISA) were performed to verify the predicted influence.Results:1. Screening for metabolic syndrome related SNPsThere were 32 SNPs which showed statistical significant in the association analysis between inflammation related genetic variants and metabolic syndrome, of which 4 SNPs’ FPRP<0.2, and PRKCE (rs2595192 and rs935653), TRAF2(rs4880073) might reduce the risk of suffering metabolic syndrome (OR=0.80,95% CI=0.70-0.91; OR=0.75,95% CI=0.62-0.90; OR=0.80,95% CI=0.69-0.92, respectively), while LEP (rs12706832) might increase the risk (OR=1.27,95% CI= 1.09-1.48).The associations analysis between metabolic syndrome and genotypes showed 82 SNPs’ min-P<0.05 of which 5 SNPs’ FPRP<0.2.All together there are 6 SNPs’ FPRP<0.2, of which PRKCE (rs3911797, rs2595192) showed statistically significant both in 3 genetic models and trend analysis (all P<0.05), and the mutant allele reduced the risk of MS; PRKCE (rs935653), ABCC4 (rs1729788), LEP (rs12706832) showed statistical significant in additive and dominant model, and trend analysis (all P<0.05), and the mutant allele of rs935653 and rs1729788 reduced the risk of MS, while rs 12706832 increased the risk; TRAF2 (rs4880073) showed statistically significant in additive and recessive model, and trend analysis (all P<0.05), and the mutant allele reduce the risk of metabolic syndrome. These results suggested the genetic variations in inflammatory pathway related genes may contribute to the genetic susceptibility to metabolic syndrome.The aforementioned 6 SNPs with FPRP<0.2 were used to calculate the GRS. Compared with the reference group (scored≤5), the risk of MS increased with the GRS score, the risk increased to 1.52 times (95% CI= 1.14-2.03) in the group scored 6-7, to 2.08 times (95% CI= 1.55-2.78) in the group scored 8-9, to 2.84 times (95% CI= 1.76-4.58) in the group scored 10-11. The result indicated the risk of suffering metabolic syndrome increase with the growing counting of risk allele in a dose-dependent way (Ptrend<0.0001).In the associations analysis between metabolic syndrome compositions (BMI, TC, TG, HDL-C, LDL-C, SBP, DBP, fasting blood glucose and uric acid)and the 6 SNPs, BMI was significantly related to rs3911797, rs935653, rs12706832, and rs1729788; TG was significantly related to rs2595192 and rs4880073; SBP was significantly related to rs3911797, rs2595192, rs935653, rsl729788 and rs4880073; DBP was significantly related to rs3911797, rs2595192 and rs1729788; fasting blood glucose was significantly related to rs4880073; uric acid was significantly related to rs3911797 and rs1729788.2. Preliminary study on biological function of metabolic syndrome related SNPsAllele specific influence of the SNPs (FPRP<0.2) on transcription factors binding was found in rs4880073, rs3911797, rs935653 and rs2167270 (r2 value of linkage equilibrium between rs12706832 and rs2167270 is 0.77). And DLR assay proven the mutant allele of rs2167270 could increase the expression of luciferase compared with the wild allele (luciferase relative expression (mean±SEM):GG:20.30±2.11; AA: 30.13±3.84, P=0.034). ELISA of serum in male showed the mutant allele homozygote carriers had higher level of serum Leptin compared with the carriers with wild allele (GG:4.13±0.62, GA:4.79±0.69, AA:8.30±1.20, P<0.05), while no significant result was found in female (P>0.05).Conclusions:1. Inflammation related genetic variants are associated with genetic susceptibility for MS. The mutant alleles of rs3911797, rs2595192, rs935653, rs1929788 and rs4880073 may decrease the risk of suffering MS, while rs12706832 may increase the risk for the carriers. And the GRS built by the 6 SNPs could predict the risk of MS.2. The mutant allele of LEP rs2167270 may specifically influence on transcription factors binding to upregulate the expression of LEP to impact on the biological metabolism and metabolic syndrome.Part Ⅱ:Associations of LIPC C-514T Polymorphism with Obesity and Plasma Lipids Levels in Chinese Children and AdolescentsHepatic lipase (LIPC) is a key rate-limiting enzyme in lipoprotein catabolism pathways, which involved in the hydrolysis of triglycerides (TG), HDL metabolism, LDL particle deformation, phospholipids of all major classes lipoproteins and reverse cholesterol transport (RCT). The-514 C to T polymorphism (rs1800588) in the LIPC gene promoter region is associated with decreased LIPC activity and increased HDL-C level. Hepatic lipase also plays a role in energy homeostasis. LIPC was identified as an obesity candidate gene in mouse model, LIPC deficient mouse can protect against diet-induced obesity. Human hepatic lipase activity increased with increasing visceral adiposity, whereas loss of intraabdominal fat was associated with reduced hepatic lipase activity. LIPC C-514T polymorphism accounted for 30-40% of the variation in hepatic lipase activity. And numerous studies focusing on the relationship between the polymorphism and various disorders had been carried out in adult, but less attention was paid on children and adolescents with inconsistency results.Purpose:In the present study, we investigate the associations of LIPC C-514T polymorphism with obesity and plasma lipids in Chinese children and adolescents.Matetials and Methods:Subjects were recruited from a cross-sectional study on metabolic syndrome of children and adolescents and selected by case-control strategy. The China obesity working group (WGOC) criteria was used to confirm the diagnosis of obesity. A total of 2969 school children and adolescents (aged 7-17 yrs) were enrolled from 6 cities in China, including 850 obese cases and 2119 controls. LIPC C-514T polymorphism was genotyped using the Sequenom MassARRAY iPLEX platform. Statictical methods including chi-square test, t-test, logistic regression, trend test and linear regression were applied to evaluate the association between the polymorphism with obesity and plasma lipid levels, and the potential interaction effect of gender on plasma lipid levels. Additionally we conducted a meta-analysis of all results from published studies as well as our own data.Results:The polymorphism was associated with obesity in boys (P=0.042), but not in girls. And significant associations were observed with TC and HDL-C independent of obesity in boys. The T allele carriers had higher levels of LDL-C in obese boys, and TG, TC and LDL-C in non-obese girls (all P<0.05).In the meta-analysis, under dominant model the T allele increased BMI, HDL-C, TG compared with the CC homozygote in boys (WMD= 0.35,95%CI= 0.10～0.60, P= 0.007; WMD= 0.07,95%CI= 0.04～0.09, P< 0.001; WMD= 0.05,95%CI= 0.02～0.09, P= 0.002, respectively). The T allele oppositely effect on BMI in girls which decreased BMI level (WMD=-0.27,95%CI=-0.52～-0.02, P= 0.034), and no significant relationships were found with TG, LDL-C and HDL-C. And carriers with T allele had higher level of TC compared with the CC homozygote independent of gender (boys: WMD= 0.13,95%CI= 0.07～0.18, P< 0.001; girls:WMD= 0.09,95%CI= 0.01～0.16, P= 0.002; total:WMD= 0.11,95%CI= 0.07～0.16, P< 0.001).Conclusions:1. The T allele might carry an increased risk of obesity in Chinese boys.2. The polymorphism is associated with the levels of plasma lipids, which may be modulated by gender.