本文關(guān)鍵詞：BPES綜合征伴不孕癥家系FOXL2基因突變檢測(cè)及功能研究　出處：《山東大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 眼瞼下垂內(nèi)眥贅皮眼裂狹窄綜合征(BPES) FOXL2 基因突變 不孕癥

【摘要】：研究背景瞼裂狹小、倒轉(zhuǎn)型內(nèi)眥贅皮和上瞼下垂綜合征(blepharophimosis-ptosis-epicanthus inversus syndrome,BPES)的發(fā)病率為 1/5,000,作為一種罕見(jiàn)的常染色體顯性遺傳病,它的主要臨床表現(xiàn)是雙側(cè)上瞼下垂、瞼裂狹小、逆向內(nèi)眥贅皮、內(nèi)眥間距過(guò)寬等,次要表現(xiàn)是鼻梁扁平、低位耳等。BPES共分為兩型:Ⅰ型和Ⅱ型。Ⅰ型表現(xiàn)為眼瞼發(fā)育異常并且受累女性伴有不孕,Ⅱ型僅僅表現(xiàn)為眼瞼發(fā)育的異常,男女患者生育功能未受影響。兩型的主要區(qū)別主要是Ⅰ型受累女性同時(shí)伴有不孕癥,然而由于BPES Ⅰ型女性的基因型與表型并不完全一致,其所引起的女性不孕的發(fā)病機(jī)制尚不明確。迄今為止,臨床上尚未找到可靠有效的方法來(lái)治療BPES Ⅰ型患者的不孕癥。雖然可以通過(guò)重建手術(shù)來(lái)矯正BPES患者的上瞼下垂,以改善其面貌,但BPES Ⅰ型所伴發(fā)的不孕癥給女性患者造成身心上的巨大痛苦。此外,由于BPES患者尤其是Ⅰ型女性患者的臨床表型變異程度較大,使得臨床醫(yī)生較難對(duì)該病進(jìn)行準(zhǔn)確的診斷,從而使該類(lèi)患者無(wú)法得到及時(shí)有效的治療。更重要的是,該病具有較高的家族遺傳性,根據(jù)孟德?tīng)栠z傳定律計(jì)算,BPES患者子代的再發(fā)病風(fēng)險(xiǎn)約為50%,因此對(duì)BPES患者進(jìn)行常規(guī)的產(chǎn)前遺傳咨詢(xún),通過(guò)產(chǎn)前篩查、診斷等必要措施避免患病兒的出生就顯得尤為重要。Forkhead boxL2(FOXL2)基因是定位于染色體3q23(3號(hào)染色體2區(qū)3帶)的單一外顯子基因。FOXL2基因是一個(gè)在維持卵泡發(fā)育、卵巢正常功能中發(fā)揮重要作用的常染色體基因,主要在中小卵泡期的顆粒細(xì)胞中表達(dá),通過(guò)抑制下游靶基因CYP11A1、CYP1941、CCND2等啟動(dòng)子的轉(zhuǎn)錄活性,從而在卵泡顆粒細(xì)胞的增殖、分化、卵巢類(lèi)固醇激素的生成過(guò)程中發(fā)揮重要作用。FOXL2基因突變會(huì)導(dǎo)致卵巢早衰的發(fā)生。研究表明FOXL2基因是BPES最常見(jiàn)的、首位致病候選基因,在BPES Ⅰ型和Ⅱ型患者中均發(fā)現(xiàn)有FOXL2基因突變的存在。但FOXL2基因突變?nèi)绾螌?duì)下游基因啟動(dòng)子的轉(zhuǎn)錄功能產(chǎn)生影響、引起POF并導(dǎo)致BPES不孕癥發(fā)生的具體分子機(jī)制尚未完全明確。研究目的本研究中,我們主要關(guān)注FOXL2基因突變?cè)贐PES Ⅰ型患者不孕癥發(fā)生發(fā)展中的作用機(jī)制。我們對(duì)一個(gè)BPES不孕癥家系進(jìn)行家系遺傳學(xué)分析、對(duì)FOXL2基因突變進(jìn)行篩查并對(duì)新發(fā)突變進(jìn)行功能預(yù)測(cè)和體外實(shí)驗(yàn)功能驗(yàn)證,從而探討FOXL2基因突變與該家系患者不孕癥發(fā)病的相關(guān)性,以期給相關(guān)不孕癥病人的臨床診療及遺傳咨詢(xún)等提供更多的依據(jù)和幫助。研究方法1.以2015年就診于山東大學(xué)附屬生殖醫(yī)院的伴有BPES表現(xiàn)的不孕癥患者的家系為研究對(duì)象,對(duì)家系成員進(jìn)行臨床表型及遺傳規(guī)律分析。2.抽取家系成員的外周血,以在本院招募的223例卵巢功能正常且不伴有BPES的育齡期健康婦女外周血為正常對(duì)照,采用直接測(cè)序法對(duì)FOXL2基因的外顯子區(qū)進(jìn)行突變位點(diǎn)篩查檢測(cè)。3.應(yīng)用生物信息學(xué)軟件Polyphen-2、PROVEN和SIFT等對(duì)FOXL2基因突變對(duì)蛋白質(zhì)的影響進(jìn)行功能預(yù)測(cè)。4.構(gòu)建野生型和突變型FOXL2基因質(zhì)粒,轉(zhuǎn)染至HEK293細(xì)胞后,采用雙熒光素酶報(bào)告系統(tǒng)檢測(cè)基因轉(zhuǎn)錄活性、細(xì)胞免疫熒光技術(shù)檢測(cè)蛋白表達(dá)及細(xì)胞定位。研究結(jié)果1.家系中的兩例患者均符合BPES的臨床診斷,且受累的女性均患有不孕癥,符合BPES分型中的I型,遺傳方式符合單基因常染色體顯性遺傳方式。2.在該BPES不孕癥家系中發(fā)現(xiàn)了FOXL2基因編碼區(qū)第188位的胸腺嘧啶突變?yōu)橄汆堰?為新發(fā)突變c.188IA,第63位氨基酸由Ile變?yōu)锳sn(p.I63N),突變位點(diǎn)位于FKH結(jié)構(gòu)域,該突變位點(diǎn)(p.I63N)及其上下游序列在物種間高度保守。3.功能預(yù)測(cè)提示p.I63N突變對(duì)FOXL2蛋白功能有損害。這個(gè)新發(fā)突變位點(diǎn)為我們首次發(fā)現(xiàn)。4.體外轉(zhuǎn)染HEK293細(xì)胞的功能實(shí)驗(yàn)證實(shí),該錯(cuò)義突變p.I63N減弱了FOXL2對(duì)下游基因CYP19A1和CCND2啟動(dòng)子的轉(zhuǎn)錄抑制作用(P0.05),但對(duì)CYP11A1啟動(dòng)子的轉(zhuǎn)錄抑制無(wú)明顯影響(P0.05),而且對(duì)CYP11A1、CYP19A1和CCND2三個(gè)下游靶基因的啟動(dòng)子都沒(méi)有顯性負(fù)效應(yīng);亞細(xì)胞定位及蛋白表達(dá)定位同野生型相比未見(jiàn)明顯改變。研究結(jié)論1.對(duì)一個(gè)不孕癥家系進(jìn)行家系分析,符合BPES的臨床診斷,患者有典型的眼瞼異常并伴有不孕癥屬于Ⅰ型BPES患者。2.在患者FOXL2基因中檢測(cè)到了一個(gè)新發(fā)突變,位于第188位堿基的胸腺嘧啶突變?yōu)橄汆堰?c.188TA),導(dǎo)致第63位氨基酸的異亮氨酸變?yōu)樘於０?p.Ile63Asn,p.I63N),該突變位于FKH結(jié)構(gòu)域,拓展了BPES基因錯(cuò)義突變譜。3.蛋白功能預(yù)測(cè)軟件顯示,p.I63N突變對(duì)FOXL2蛋白功能的影響較大。4.突變p.I63N有可能通過(guò)單倍劑量不足的機(jī)制,影響FOXL2對(duì)下游調(diào)控基因CYP19A1和CCND2啟動(dòng)子的轉(zhuǎn)錄抑制,進(jìn)而導(dǎo)致顆粒細(xì)胞功能及類(lèi)固醇激素生成的異常,最終導(dǎo)致不孕癥的發(fā)生。該研究揭示了該BPES家系女性患者不孕癥發(fā)生的可能機(jī)制,為BPES的進(jìn)一步研究提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:On the background of blepharophimosis, epicanthus inversus and ptosis syndrome (blepharophimosis-ptosis-epicanthus inversus, syndrome, BPES) the incidence rate of 1/5000, is a rare autosomal dominant genetic disease, it is the main clinical manifestation of bilateral ptosis, blepharophimosis, reverse epicanthus the angular distance is too wide, the main performance is the bridge of the nose flat, low ear.BPES is divided into two types: type I and type II. Type I showed abnormal development and involvement of eyelid women with infertility, type II only showed abnormal eyelid development, patients with reproductive function were not affected. The main difference between men and women with type two is the main type of female involvement associated with infertility, however due to genotype BPES type female phenotype is not completely consistent, the pathogenesis of the cause of female infertility is not clear. So far, the bed has not been found To a reliable and effective method for treatment of BPES type patients with infertility. Although the reconstruction surgery to correct BPES patients with ptosis, to improve its appearance, but the BPES type associated with infertility caused great pain of the heart to female patients. In addition, because the BPES patients especially the clinical phenotype variation of female patients with larger, more difficult to make clinicians for accurate diagnosis of the disease, so that the patients can not get timely and effective treatment. More importantly, the hereditary disease is high, according to the calculation of Mendel's law of inheritance, with BPES offspring then the risk is about 50%, so routine prenatal genetic counseling of BPES patients, through prenatal screening, diagnosis and other necessary measures to avoid the risk of children born is particularly important.Forkhead boxL2 (FOXL2) gene is located on chromosome 3q23 (No. 3 3) the area of chromosome 2 with single exon of.FOXL2 gene is a development in the maintenance of follicle, autosomal genes play an important role in normal ovarian function, mainly expressed in granulosa cells and follicular phase, through inhibition of downstream target genes CYP11A1, CYP1941, transcriptional activity of CCND2 promoter, resulting in granulosa cell proliferation, differentiation, the important role of.FOXL2 gene mutations can lead to premature ovarian failure play generation process of ovarian steroid hormones. The study showed that the FOXL2 gene is the most common BPES, the first candidate gene BPES in type I and type II patients were found to have mutations in the FOXL2 gene. But FOXL2 gene how the function of promoter mutant transcription of downstream genes caused by impact, POF and lead to the molecular mechanism of BPES infertility is not completely clear. The purpose of the study in this study, our Lord To pay attention to the FOXL2 gene mutation mechanism in the development of infertility BPES type patients. We analyzed the family genetics to a family of BPES infertility, FOXL2 gene mutation screening and functional prediction and in vitro experiments to verify the function of the new mutation, so as to explore the relationship between FOXL2 gene mutation and the families of patients with infertility the incidence of infertility patients in order to provide relevant clinical and genetic counseling more basis and help. Methods 1. to 2015 in the reproductive Hospital Affiliated to Shandong University with BPES manifestations of infertility patients in the family as the research object, peripheral blood clinical phenotype and genetic analysis of selected.2. family the members of the family members, with women in 223 cases of ovarian function in our hospital recruited with normal peripheral blood BPES as normal control, using direct Sequencing of the FOXL2 gene exon mutation detection of.3. using bioinformatics software Polyphen-2, PROVEN and SIFT etc. the effects of mutations on protein of FOXL2 gene was predicted to construct.4. and mutant FOXL2 gene plasmid was transfected into wild-type HEK293 cells, detected by dual luciferase reporter gene transcription. System, cell immunofluorescence technique to detect protein expression and cellular localization. Results two cases in 1. families of patients were consistent with the clinical diagnosis of BPES, and the involvement of women who were infertile, with BPES type I type, genetic manner consistent with autosomal dominant inheritance mode of.2. in the BPES family of infertility found in the FOXL2 gene encoding region 188th mutation adenine thymine, new mutation c.188IA, sixty-third amino acid from Ile to Asn (p.I63N), a point mutation in FKH Domain, the mutation site (p.I63N) and its downstream sequence is highly conserved among species.3. function prediction showed that the p.I63N mutation damage to the function of the FOXL2 protein. The new mutation was confirmed for the first time we function in experimental.4. in vitro transfection of HEK293 cells, the missense mutations p.I63N decreased transcription of FOXL2 downstream genes of CYP19A1 and the inhibitory effect of CCND2 promoter (P0.05), but had no obvious effect on inhibiting the transcription of CYP11A1 promoter (P0.05), and the CYP11A1, CYP19A1 and CCND2 three downstream target gene promoters have a dominant negative effect; the expression and subcellular localization of protein compared with wild type had no obvious change. The conclusion of the study on the 1. a family pedigree analysis of infertility, consistent with the clinical diagnosis of BPES, patients with typical eyelid disorders accompanied by infertility patients with.2. belongs to the type I BPES in patients with FOXL2 gene detected A new mutation in 188th base mutations for adenine thymine (c.188TA), resulting in sixty-third amino acid isoleucine to asparagine (p.Ile63Asn, p.I63N), the mutations in the FKH domain, BPES gene missense mutation spectrum of.3. protein function prediction software that extended p.I63N mutation mechanism of FOXL2 protein the function of larger.4. mutant p.I63N likely by haploinsufficiency, effects of FOXL2 on the transcription of downstream gene CYP19A1 and CCND2 promoter inhibited, leading to abnormal granulosa cell function and steroidogenesis, eventually leading to the occurrence of infertility. The study reveals the possible mechanism of female patients with infertility of the BPES family, provide the experimental basis for the further study of BPES.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R711.6;R596

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本文編號(hào)：1375637