PPARα基因多型性與代謝症候群之關聯

Association between PPARα Gene Polymorphism and Metabolic Syndrome

林斯晨(Sir-Chen Lin)

指導教授 ： 江福田

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02343

摘要

研究背景
本研究的目的，主要是關於代謝症候群（metabolic syndrome）之遺傳因子的探討。代謝症候群的特徵是：腹部肥胖、血脂異常、高血壓、胰島素抗性、葡萄糖耐受性不良、易產生血管粥狀硬化、易發生血栓、及易造成炎症反應。過氧化體增殖劑活化受體（peroxisome proliferator-activated receptors），簡稱PPARs，為賀爾蒙受器，它位於細胞核，在許多器官，包括肝臟、心臟、腎臟、及骨骼肌，掌管調控脂肪的輸送和新陳代謝，它也與血糖的恆定息息相關。甚至與降血脂肪或血糖的藥物機轉也有相關。其中過氧化體增殖劑活化受體α（PPARα）的功能主要在調控血脂異化代謝（lipid catabolism）的過程。本研究的目的在探討過氧化體增殖劑活化受體基因中的多形性（polymorphism）是否和代謝症候群及其組成有關聯，及進一步探討過氧化體增殖劑活化受體α基因多形性與代謝症候群相關的疾病（如高血壓、糖尿病）之間的關聯。我們主要是採取病例對照研究（case-control study）的模式，來進行研究。

研究方法與材料
第一部分：我們根據民國93年衛生署代謝症候群的之臨床診斷準則定義，選擇300位代謝症候群成年病患，並選擇300位無代謝症候群成年人為對照組，這些病人我們以聚合脢連鎖反應（polymerase chain reaction，PCR）及限制酵素片段長度多型性（restriction fragment length polymorphism，RFLP）等方法，判定PPARα基因第三插入子（intron 3）的三個單一核苷多型性（single nucleotide polymorphism，SNP），分別是：IMS-JST019819 C/A、IMS-JST019815 T/C、及rs4253730 A/G。
我們計算各個SNP的對偶基因頻率（allele frequency）。統計各個SNP基因型（genotypes）在下列情況的分佈：代謝症候群，高血壓、糖尿病、及冠狀動脈疾病。以卡方檢定（Chi-square）或費雪正確機率考驗（Fisher's exact test）計算其勝數比（odds ratio）。並且以邏輯迴歸分析（logistic regression）校正可能的混淆因素（confounding factors）。除了傳統上的單位址分析，我們也以配子基因型分析（haplotype analysis）等方法，來探討多位址基因效用。在連續變項方面，如身體質量指數，三酸甘油脂，總膽固醇，低密度脂蛋白膽固醇，高密度脂蛋白膽固醇，我們也統計在各個單一核苷多型性不同基因型間是否有差異。
第二部分：我們選擇第五表現子（exon 5）的單一核苷多型性Leu162Val （rs1800206）來進行研究。共選擇40位無代謝症候群成年人，我們以聚合脢連鎖反應及限制酵素片段長度多型性之方法，來判定基因型，計算台灣人此一多型性的對偶基因頻率（allele frequency）。
第三部分：我們選擇啟動子（promoter）區域來進行研究。我們選擇20位無代謝症候群成年人，以聚合脢連鎖反應及核苷酸定序之方法，來找出啟動子區域可能的單一核苷多型性。

研究結果
本研究在第一部分的單位址分析方面，發現IMS-JST019819 C/A多型性之基因型中，AA的人得到冠狀動脈疾病的機會比CC及CA高（勝數比＝1.531，p＝0.038）。但是我們若以多重邏輯迴歸分析（multiple logistic regression）校正性別、年齡、高血壓、糖尿病等4項變數，來分析與冠狀動脈疾病之關聯性，我們得到的p值仍然有顯著意義（勝數比＝1.777，p＝0.039）。若校正性別、年齡、高血壓、糖尿病、低密度脂蛋白膽固醇，高密度脂蛋白膽固醇6項變數，我們得到的p值反而無顯著意義（勝數比＝1.567，p＝0.224）。我們也發現IMS-JST019815 T/C多型性，帶有CC及TC基因型的人得到糖尿病的機會比TT基因型的人低（勝數比＝0.63，p＝0.014）。但是我們若校正性別、年齡兩項變數，來分析與糖尿病之關聯性。呈現出的p值仍然有顯著意義（勝數比＝0.596，p＝0.014）。而在rs4253730 A/G多型性方面，帶有GG或AG基因型的人得到冠狀動脈疾病的機會比AA基因型的人高（勝數比＝1.954，p＝0.0003）。但是我們若校正性別、年齡、高血壓、糖尿病等4項變數，來分析與冠狀動脈疾病之關聯性，我們得到的p值仍然有顯著意義（勝數比＝1.648，p＝0.027）。若校正性別、年齡、高血壓、糖尿病、低密度脂蛋白膽固醇，高密度脂蛋白膽固醇6項變數，我們得到的p值同樣具有顯著意義（勝數比＝2.388，p＝0.005）。同時經由變異數分析（ANOVA）分析，我們也發現帶有G allele越多的人，有越高的低密度脂蛋白膽固醇（p＝0.017）。
但是三種SNP與代謝症候群、高血壓的相關性都不顯著。三種SNP與身體質量指數、三酸甘油脂、總膽固醇、高密度脂蛋白膽固醇的相關性也都不顯著。
在多位址配子基因型頻率分析（multilocus haplotype analysis）方面，計算這三個SNP與代謝症候群、高血壓、糖尿病、冠狀動脈疾病四種情況之關聯性。整體而言，總合配子基因型分析在有病或對照組之間的差異都不顯著。而在個別配子基因型分析方面，也沒有一種組合顯示有統計學上的意義。
本研究在第二部分的結果顯示，全部40組皆為LL同型結合子（homozygote），因此估計台灣人PPARα基因第五表現子162V的對偶基因頻率可能小於1/80（即小於1.25％）。
本研究在第三部分的結果顯示，台灣人PPARα基因啟動子區域-1664至-712處可能沒有單一核苷多型性。

結論
本研究證實過PPARα基因intron 3的多形性與代謝症候群相關的疾病（糖尿病、冠狀動脈疾病）的發生有關聯性，也與低密度脂蛋白膽固醇的血中濃度有關聯性。

關鍵字

過氧化體增殖劑活化受體 ； 代謝症候群 ； 基因多型性

並列摘要

Introduction
Characteristics of the metabolic syndrome are abdominal obesity, atherogenic dyslipidemia, raised blood pressure, insulin resistance, prothrombotic and proinflammatory states. Patients with metabolic syndrome are at increased risk for developing diabetes mellitus (DM) and cardiovascular diseases. Peroxisome proliferator-activated receptors (PPARs) are nuclear proteins that belong to the superfamily of nuclear hormone receptors. PPARs are found in liver, heart, liver and skeletal muscle. PPARs are ligand-induced transcription factors that regulate the transcription of target genes in response to specific ligands, both synthetic and endogenous. PPARα regulates many genes involved in the uptake, transport, and oxidation of fatty acids. Therefore, PPARα is a potential candidate gene that may influence the risk of developing the metabolic syndrome. This study is aimed to find out the association between PPARα gene polymorphism and metabolic syndrome, its components or associated diseases.

Methods
A total of 300 patients with diagnosis of metabolic syndrome were enrolled in this study. The diagnosis of every patient is based on the criteria of Department of Health, Executive Yuan (1994). 300 controls were recruited who do not have metabolic syndrome. DNA was extracted from 10cc venous blood. Three single nucleotide polymorphisms (SNP) in intron 3: C/A (IMS-JST019819), T/C (IMS-JST019815), A/G (rs4253730) were selected from the JSNP and NCBI database. PCR-RFLP was performed to identify genotypes.
We calculated the genotypes distribution and allele frequency among control groups. Chi-square was done and odds ratio was calculated concerning the association between gene polymorphism and metabolic syndrome, associated diseases such as hypertension or DM or coronary artery disease. In different genotypes, ANOVA was used to test the difference of the continuous variables, such as triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and body mass index (BMI). Statistical significance is confirmed if p value<0.05. In addition, multilocus haplotype analysis was also done.
We also select another polymorphism Leu162Val (rs1800206) over exon 5 for the second part of study. A total of 40 subjects without metabolic syndrome were genotyped by the means of PCR-RFLP. Allele frequency was calculated, too.
Furthermore, in the third part of study, we focused on promoter region of this gene. There were totally 20 samples from subjects without metabolic syndrome were collected. In order to find possible SNPs in promoter region, PCR and sequencing was performed. Sequences were compared with each other to observe nucleotide variants.

Results
Single locus analysis revealed significant association between these three SNP and metabolic syndrome-associated diseases. SNP C/A (IMS-JST019819): For AA genotype, there was higher risk to have coronary artery disease than CC+CA group (odds ratio=1.531, p=0.038). P value was still significant after adjusting for sex, age, hypertension and DM. However, p value was not significant if we adjust sex, age, hypertension, DM, LDL and HDL; SNP T/C (IMS-JST019815): Subjects with C allele (CC+TC group) had lower probability value of DM than TT group (odds ratio=0.63, p=0.014). P value was still significant after adjusting for sex and age; SNP A/G (rs4253730): Subjects with G allele (GG+AG group) were at higher risk of developing coronary artery disease than AA group (odds ratio=1.954, p=0.0003). P value was still significant even after adjustment for sex, age, hypertension, DM, LDL and HDL. Subjects having more G alleles were also prone to have higher LDL (p=0.017); nonetheless, there was no strong correlation between these three polymorphisms and following items: metabolic syndrome, hypertension, triglyceride, total cholesterol, HDL or BMI.
In multilocus haplotype analysis, concerning following 4 situations including metabolic syndrome, hypertension, DM and coronary artery disease, all the p values of omnibus likelihood ratio test were not significant, which indicated the overall haplotype frequency profile difference between cases and controls were not significant. Besides, using individual haplotype analysis, we were not able to identify any haplotype with significant p value either.
In the second part of study, all the 40 samples were Leu162 homozygote. So we could predict that the actual allele frequency of 162Val is less than 1.25% among Taiwanese.
In the third part of study, totally 20 sets of DNA samples were successfully sequenced. There was no identified SNP in the promoter region between nucleotide -1664 and -712 among Taiwanese.

Conclusion
This study demonstrated that polymorphisms of PPARα gene intron 3 might be associated with coronary artery disease, DM and LDL level.

並列關鍵字

PPARα ； metabolic syndrome ； gene polymorphism

參考文獻

Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. 1998; 15:539-53.

Balkau B, Charles MA. Comment on the provisional report from the WHO consultation. European Group for the Study of Insulin Resistance (EGIR) Diabet Med. 1999;16:442-3.

Barish GD, Narkar VA, Evans RM. PPAR delta: a dagger in the heart of the metabolic syndrome. J Clin Invest. 2006;116:590-7.

Bossé Y, Pascot A, Dumont M, Brochu M, Prud'homme D, Bergeron J, Després JP, Vohl MC. Influences of the PPAR alpha-L162V polymorphism on plasma HDL(2)-cholesterol response of abdominally obese men treated with gemfibrozil. Genet Med. 2002;4:311-5.

Bossé Y, Després JP, Bouchard C, Pérusse L, Vohl MC. The peroxisome proliferator-activated receptor alpha L162V mutation is associated with reduced adiposity. Obes Res. 2003;11:809-16.