BPES综合征伴不孕症家系FOXL2基因突变检测及功能研究

发布时间：2018-01-03 21:22

本文关键词：BPES综合征伴不孕症家系FOXL2基因突变检测及功能研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景睑裂狭小、倒转型内眦赘皮和上睑下垂综合征(blepharophimosis-ptosis-epicanthus inversus syndrome,BPES)的发病率为 1/5,000,作为一种罕见的常染色体显性遗传病,它的主要临床表现是双侧上睑下垂、睑裂狭小、逆向内眦赘皮、内眦间距过宽等,次要表现是鼻梁扁平、低位耳等。BPES共分为两型:Ⅰ型和Ⅱ型。Ⅰ型表现为眼睑发育异常并且受累女性伴有不孕,Ⅱ型仅仅表现为眼睑发育的异常,男女患者生育功能未受影响。两型的主要区别主要是Ⅰ型受累女性同时伴有不孕症,然而由于BPES Ⅰ型女性的基因型与表型并不完全一致,其所引起的女性不孕的发病机制尚不明确。迄今为止,临床上尚未找到可靠有效的方法来治疗BPES Ⅰ型患者的不孕症。虽然可以通过重建手术来矫正BPES患者的上睑下垂,以改善其面貌,但BPES Ⅰ型所伴发的不孕症给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型女性患者的临床表型变异程度较大,使得临床医生较难对该病进行准确的诊断,从而使该类患者无法得到及时有效的治疗。更重要的是,该病具有较高的家族遗传性,根据孟德尔遗传定律计算,BPES患者子代的再发病风险约为50%,因此对BPES患者进行常规的产前遗传咨询,通过产前筛查、诊断等必要措施避免患病儿的出生就显得尤为重要。Forkhead boxL2(FOXL2)基因是定位于染色体3q23(3号染色体2区3带)的单一外显子基因。FOXL2基因是一个在维持卵泡发育、卵巢正常功能中发挥重要作用的常染色体基因,主要在中小卵泡期的颗粒细胞中表达,通过抑制下游靶基因CYP11A1、CYP1941、CCND2等启动子的转录活性,从而在卵泡颗粒细胞的增殖、分化、卵巢类固醇激素的生成过程中发挥重要作用。FOXL2基因突变会导致卵巢早衰的发生。研究表明FOXL2基因是BPES最常见的、首位致病候选基因,在BPES Ⅰ型和Ⅱ型患者中均发现有FOXL2基因突变的存在。但FOXL2基因突变如何对下游基因启动子的转录功能产生影响、引起POF并导致BPES不孕症发生的具体分子机制尚未完全明确。研究目的本研究中,我们主要关注FOXL2基因突变在BPES Ⅰ型患者不孕症发生发展中的作用机制。我们对一个BPES不孕症家系进行家系遗传学分析、对FOXL2基因突变进行筛查并对新发突变进行功能预测和体外实验功能验证,从而探讨FOXL2基因突变与该家系患者不孕症发病的相关性,以期给相关不孕症病人的临床诊疗及遗传咨询等提供更多的依据和帮助。研究方法1.以2015年就诊于山东大学附属生殖医院的伴有BPES表现的不孕症患者的家系为研究对象,对家系成员进行临床表型及遗传规律分析。2.抽取家系成员的外周血,以在本院招募的223例卵巢功能正常且不伴有BPES的育龄期健康妇女外周血为正常对照,采用直接测序法对FOXL2基因的外显子区进行突变位点筛查检测。3.应用生物信息学软件Polyphen-2、PROVEN和SIFT等对FOXL2基因突变对蛋白质的影响进行功能预测。4.构建野生型和突变型FOXL2基因质粒,转染至HEK293细胞后,采用双荧光素酶报告系统检测基因转录活性、细胞免疫荧光技术检测蛋白表达及细胞定位。研究结果1.家系中的两例患者均符合BPES的临床诊断,且受累的女性均患有不孕症,符合BPES分型中的I型,遗传方式符合单基因常染色体显性遗传方式。2.在该BPES不孕症家系中发现了FOXL2基因编码区第188位的胸腺嘧啶突变为腺嘌呤,为新发突变c.188IA,第63位氨基酸由Ile变为Asn(p.I63N),突变位点位于FKH结构域,该突变位点(p.I63N)及其上下游序列在物种间高度保守。3.功能预测提示p.I63N突变对FOXL2蛋白功能有损害。这个新发突变位点为我们首次发现。4.体外转染HEK293细胞的功能实验证实,该错义突变p.I63N减弱了FOXL2对下游基因CYP19A1和CCND2启动子的转录抑制作用(P0.05),但对CYP11A1启动子的转录抑制无明显影响(P0.05),而且对CYP11A1、CYP19A1和CCND2三个下游靶基因的启动子都没有显性负效应;亚细胞定位及蛋白表达定位同野生型相比未见明显改变。研究结论1.对一个不孕症家系进行家系分析,符合BPES的临床诊断,患者有典型的眼睑异常并伴有不孕症属于Ⅰ型BPES患者。2.在患者FOXL2基因中检测到了一个新发突变,位于第188位碱基的胸腺嘧啶突变为腺嘌呤(c.188TA),导致第63位氨基酸的异亮氨酸变为天冬酰胺(p.Ile63Asn,p.I63N),该突变位于FKH结构域,拓展了BPES基因错义突变谱。3.蛋白功能预测软件显示,p.I63N突变对FOXL2蛋白功能的影响较大。4.突变p.I63N有可能通过单倍剂量不足的机制,影响FOXL2对下游调控基因CYP19A1和CCND2启动子的转录抑制,进而导致颗粒细胞功能及类固醇激素生成的异常,最终导致不孕症的发生。该研究揭示了该BPES家系女性患者不孕症发生的可能机制,为BPES的进一步研究提供了实验依据。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.6;R596

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