在臺灣婦女族群中探討基因多型性與子宮內膜異位症之間的相關性研究

Association study between gene polymorphisms and endometriosis in Taiwan women

張憲汛(Hsien-Hsun Chang)

指導教授 ： 楊維中

臺北醫學大學/口腔醫學院/生醫材料暨工程研究所/碩士(2011年)

摘要

子宮內膜異位症(endometriosis)是一種常見的婦科疾病，傳統上認為當內膜細胞(endometrium)隨經血逆流(retrograde menstruation)至腹腔(abdominal cavity)，並在腹膜(peritoneum)或其他器官上存活增生，對其他組織器官造成壓迫、沾黏及破壞，稱為子宮內膜異位症。造成子宮內膜異位症的原因不明，已知包含基因(genetic)、內分泌(endocrine)、免疫(immune)及環境(environmental factor)等因素都與子宮內膜異位症有關。我們實驗室先前的實驗數據顯示患有子宮內膜異位症婦女之子宮內膜組織與未患有子宮內膜異位症婦女之子宮內膜組織比較，其基質金屬蛋白酶-14(matrix metalloproteinase-14, MMP-14)、組織蛋白酶L1 (cathepsin L1, CTSL1)和富含半胱胺酸的酸性蛋白(secreted protein acidic and rich in cysteine, SPARC)的蛋白質表現量有增加的趨勢。然而，以上分子其基因的因素中單一核苷酸多型性位點(single nucleotide polymorphism; SNPs)與子宮內膜異位症是否有關，文獻報導較少，同時，屬於本土的子宮內膜異位症基因體研究資料亦較少。所以，本研究以基因學的角度探索子宮內膜異位症與蛋白質水解作用(proteolysis)相關的基因-基質金屬蛋白酶-14(MMP-14)；與免疫反應(immune response)相關的基因-組織蛋白酶L1(CTSL1)；與細胞外基質(extracellular matrix;ECM)調控相關的基因-富含半胱胺酸的酸性蛋白(SPARC)的關聯性。
本研究實驗檢體包含85位子宮內膜異位症的婦女和99位未患有子宮內膜異位症婦女之DNA(deoxyribonucleic acid)檢體，總共184例，利用限制酶片段長度多型性的技術(Restriction Fragment Length Polymorphism, RFLP)得到基因定型的結果(SNP genotyping data)，最後用卡方檢定及風險的分析來評估選定基因上SNP位點的基因型與子宮內膜異位症的相關程度。在SNP位點的選擇上，我們在HapMap Project (www.hapmap.org)資料中選取標誌性SNPs (tagging SNPs)，以達到更強的統計效果及更佳的基因覆蓋率，且在連鎖不平衡檢定(linkage disequilibrium test)(r2)必須 ≧0.8，來挑選出高度與致病基因連鎖的SNP位點；另外也在中國人族群中次要的對偶基因頻率(minor allele frequency)必須 ≧10%，當作選擇SNP位點之標準。
結果顯示在基質金屬蛋白酶-14(MMP-14)基因的SNP位點
-rs743257(在轉錄序列區域十;exon 10)上，其次要的對偶基因T
(minor allele T)，與基因型CT相比，基因型CC存在患有子宮內
膜異位症的患者與健康控制組作比較，有統計上之顯著差異(43.4%
vs 30.6%，P值=0.048，odds ratio: 1.89)；單就具優勢的基因
組合來看，SNP位點-rs743257之基因型CC+TT，與基因型CT相比，
存在患有子宮內膜異位症的患者與健康控制組作比較，有統計上之
顯著差異(56.6% vs 41.8%，P值 =0.047，odds ratio: 1.82)，在
富含半胱胺酸的酸性蛋白(SPARC)基因的SNP位點-rs2304052(在轉
錄序列區域三;exon 3)上，其次要的對偶基因T (minor allele C)，
與基因型TT相比，基因型CT存在患有子宮內膜異位症的患者與健
康控制組作比較，有統計上之顯著差異(19.3% vs 8.1%，P值=0.028，odds ratio: 2.69)。
基質金屬蛋白酶-14(MMP-14)基因的SNP位點-rs743257之基因型CC和在富含半胱胺酸的酸性蛋白(SPARC)基因的SNP位點-rs2304052之基因型CT，可能會提高患病風險，是否與調節其蛋白質轉錄或轉譯作用有關，尚待釐清，未來將收集更多的檢體驗證此兩個位點與子宮內膜異位症的相關性以及比較與西方族群的差異性。

關鍵字

子宮內膜異位症 ； 基質金屬蛋白酶-14 ； 組織蛋白酶L1 ； 富含半胱胺酸的酸性蛋白 ； 單一核苷酸多型性位點 ； 蛋白質水解作用 ； 細胞外基質 ； 限制酶片段長度多型性 ； 連鎖不平衡檢定

並列摘要

Endometriosis is a common gynecologic disorder. Endometriosis formation is generally believed to involve
retrograde menstruation, attachment of endometrial fragments
to the epithelium of the peritoneum, invasion of the
epithelium, establishment of a blood supply. The cause of endometriosis is unclear. Genetic, endocrine, immune, and environmental factors have been suggested in the pathogenesis. Our previous experimental data showed that matrix metalloproteinase-14(MMP-14), cathepsin L1(CTSL1), secreted protein acidic and rich in cysteine (SPARC) protein expression levels in endometriotic lesions of the patients with endometriosis were significantly higher than those in normal eutopic endometrium. Whether their expressions at gene level are associated with endometriosis remain unclear. A case-control study to investigate the association between endometriosis and the proteolysis-related genes, MMP-14, the immune response-related genes, CTSL1, the extracellular matrix (ECM) regulation-related genes, SPARC.
Eighty five cases with pathologically proved endometriosis and ninety nine disease-free women as control subjects were participated in this study. Venous blood was withdrawn from each subject, and DNA (Deoxyribonucleic acid) was extracted from the peripheral blood lymphocytes using the standard method.Genotyping was carried out by using the RFLP (Restriction Fragment Length Polymorphism) technology. Genotypes at each SNP were tested for genetic effect by using the X2 test. Genotypic effects were correlated with endometriosis by calculating the odds ratio. All statistical analyses were performed with SPSS statistical software. All tagging SNPs (tSNPs) from each candidate gene were selected from the HapMap Project (www.hapmap.org). The tSNPs were chosen according to the following criteria: r2 ≧0.8 and minor allele frequency ≧10% in the Han Chinese population.
For most of the selected SNPs, no significant differences were identified between endometriosis patients and controls (P value >0.05), except the SNP-rs743257 (exon 10; minor allele T) at MMP-14 gene was significantly associated to the disease. The CC genotype was significantly more frequent in the endometriosis patients than in the healthy controls (43.4% vs 30.6%, P value =0.048). The presence of the rs743257 genotype-CC is associated with an increased risk of developing endometriosis (odds ratio: 1.89). We also found that the SNP- rs2304052 (in exon 3, minor allele C) at SPARC gene was significantly associated to the disease. The CT genotype was significantly more frequent in the endometriosis patients than in the healthy controls (19.3% vs 8.1%, P value =0.028). The presence of the rs2304052 genotype-CT is associated with an increased risk of developing endometriosis (odds ratio: 2.69). The selected two SNPs, rs2274611 and rs3118869 of cathepsin L1 (CTSL1) gene, however, were not associated with endometriosis.
In conclusion, the present study suggests that the MMP-14 and SPARC gene may play a role to confer a risk for women with endometriosis in Taiwan. Whether the identified 2 SNPs in MMP-14 and SPARC associated with endometriosis requires more case number for statistic analysis. In addition, whether the 2 SNPs are genetic or environmental factor associated requires further studies to elucidate.

並列關鍵字

endometriosis ； MMP-14 ； CTSL1 ； SPARC ； single nucleotide polymorphism ； proteolysis ； extracellular matrix ； RFLP ； linkage disequilibrium test

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