p53基因調節區之SNPs與婦科疾病相關性之探討

Abstract

背景與目的：針對PROGINS、p53基因轉譯區及p53基因調節區進行基因多型性分析，探討其與常見婦科疾病患者與健康個體的相關性。透過基因的研究可以瞭解疾病發生機轉或疾病發生的可能性，最典型的例子為p53基因之研究，已知55％以上的癌症與p53基因突變有關，然而目前研究主要著力於 p53 基因的譯碼區序列突變檢測，對於p53基因調節區的研究則相當少。許多婦科疾病發生機率高但都屬不易診斷之疾病，往往發現時都已很嚴重，我們希望透過p53基因調節區序列的多型性的分析，開發能預測這些婦科疾病發生之基因標誌。 材料與方法：本研究收集樣品數共有196人，其分別為子宮肌瘤43人、子宮肌腺瘤15人、子宮內膜異位症23人、卵巢癌22人、多囊性卵巢症15人、泌乳素過高症35人以及健康個體43人。本研究首先採收病患血樣，萃取其白血球總DNA以進行後續研究。PROGINS基因型分析策略採取直接進行PCR再以agarose膠體電泳分析；p53基因codon 11與codon 248多型性分析策略採取進行PCR後再以RFLPs分析；p53基因調節區分析策略採取定序分析p53基因調節區-433至+29間序列，所得序列以Clustal W程式進行multiple sequence alignment（MSA）分析，病患與健康族群間單一核苷酸多型性（SNPs）關連性以卡方檢定的獨立性檢定及勝數比分析。 研究結果：PROGINS基因、p53基因codon 11和codon 248多型性與婦科疾病關聯性分析中，共有154人樣品接受檢測，其中包括健康個體41人。在PROGINS基因分析中，結果顯示有T1（159 bp）與T2（480 bp）兩種基因型被檢測出，無論是疾病或正常族群中T1基因型頻度為98.4%，T2基因型頻度僅為1.6%，因此PROGINS基因型與本研究所探討的各種婦科疾病間並無相關。在p53基因codon 11與codon 248多型性分析中，所有受測的個體皆只有單一基因型，因此也無與各種婦科疾病間相關的p53基因codon 11與codon 248基因型被確認。在p53基因-433至 +29調節區序列的SNPs分析中，共有140人樣品被完成，其中包括健康個體43人。我們共發現98個突變點其中包括15種SNPs。本研究並無發現任何單一SNP與特定疾病有相關性（p>0.05）。-51 T/A位於NF-□B結合位及基因毒性逆境活化區域中，此SNP發生於子宮肌瘤的機率高達10%，可見子宮肌瘤的發生也許與這些結合位有相關性。-33 A/G位於c-Myc/Max heterodimer的結合位上，此SNP只發生於子宮肌瘤病患，因此其與該疾病的相關性值得探討。 結論：本實驗為p53調節區序列SNPs研究之濫觴。我們已分析出多種SNPs，將來研究者根據我們的成果得以進行大規模p53調節區多型性與疾病間關連性的研究，其中以我們已證實的特定婦科疾病研究與p53基因SNPs間相關，此相關可預期能在短期間獲得重要成果。

Objective: To investigate the genotypes of progesterone receptor gene（PROGINS ）, p53 coding region, and upstream of p53 gene from women with gynaecological diseases such as endometriosis, uterine leimyoma, adenomyosis, ovarian cancer, polycystic ovary syndrome (PCOD), and hyperprolactinemia. Background: Through the study of gene, scientists could understand the mechanism and probability of diseases, especial the studies of p53. About 55% of human cancers suffer mutations in both alleles of p53 gene. The high association between p53 mutations and many forms of cancers suggests that loss of p53 function can be used as a marker for high susceptibility to carcinogenesis. So far, genetic polymorphism of p53 gene regulatory region associated with carcinogenesis is unclear. Therefore, to find out the polymorphism of p53 regulatory region which related with gynaecological diseases remains to be explored. Materials and Methods: A total of 196 blood samples, including 43 uterine leiomyoma, 15 adenomyosis, 23 endometriosis, 22 ovarian cancer, 15 PCOD, 23 hyperprolactinemia, and 43 healthy individuals were collected and genomic DNA were prepared. Determination of PROGINS was performed by PCR and gel electrophoresis. Determination of p53 coding region codon 11 and 248 were performed by PCR and restriction fragment length polymorphism（RFLP）. The p53 regulatory region (-433 ~ +29) was amplified by PCR and then the sequence was idnetified. To find out the single nucleotide polymorphisms (SNPs) in the p53 regulatory region, the multiple sequences were aligned with program “Clustal W”. The SNPs between patients and healthy individuals were evaluated by chi-square test of independence and odds ratio. Results: A total of 154 samples, including 41 healthy individuals, of PROGINS and p53 coding region codon 11 and 248 were studied. Frequencies of T1 (159 bp) allele was 98.4% and T2 (480 bp) allele was 1.6% in the both populations of patients and healthy individuals. Morever, no mutant types of p53 codon 11 and 248 were discovered in the both populations. Therefore, there were no statistically significant association between PROGINS, p53 codon 11 and 248 from women with gynaecological diseases to the normal. In the analysis of p53 regulatory region, 140 sequences, including 43 healthy individuals have been sequenced. 98 mutations and 15 SNPs have been identified in the p53 regulatory region. There was no statistically significant association among the SNPs from the gynaecological diseases compared with the normal. However, the SNP of -51 T/A, on the binding site of NF-□B and in the response region of genotoxic stress, was happened in the patients of unterine leiomyomas and healthy individuals that the prevalence rate was up to 10% in the unterine leiomyomas. The SNP -33 A/G, on the c-Myc/Max heterodimer binding site, was only happened in the unterine leiomyomas. The result hints that though the p value is greater than 0.05, the relationship between the SNPs and gynaecological diseases need to be more discussions. Conclusion: This study is a pioneer to report the SNPs in the regulatory region of p53 gene. The results in the present study provide the capacity to study the relationship between gene polymorphism in the regulatory region and diseases in a large scale. We wonder to expect the critical results concerning with gynaecological diseases associated with the p53 SNPs will be published in the future.