脆性X综合征的筛查、诊断和社会适应能力及Fmr1基因敲除小鼠的神经机制研究

发布时间：2018-04-27 12:59

本文选题：脆性X综合征 + 筛查　；参考：《浙江大学》2012年博士论文

【摘要】：脆性X综合征(Fragile X mental retardation Syndrome, FXS)是一种常见的遗传性精神发育迟滞性疾病,其发病率高达1/4000。绝大多数的FXS是由于X染色体FMR1(fragile X mental retardation-1)基因(CGG)n重复序列过度扩展导致基因甲基化而引起的。男性患者临床表现较女性患者严重,主要表现为中、重度的精神发育迟滞,伴特殊面容,如长脸、大或招风耳,青春期以后出现大睾丸,许多患者还表现为多动、冲动、焦虑、社交退缩、刻板语言等孤独症样行为。早期发现、早期治疗和干预,可以显著改善患儿预后,并为家系成员提供遗传咨询,避免家族中再次出现同样患者,具有不可估量的社会效益和经济效益。本研究应用双重实时荧光定量PCR技术,即普通实时荧光定量PCR联合甲基化敏感性限制性内切酶定量PCR (methylation-sensitive restriction enzymes-based quantitative PCR, MSRE-QPCR)技术,进行FXS患者FMR1基因(CGG)n序列和CpG岛甲基化程度的定量检测,结果发现,该技术对男性患者的诊断效率极高,既可以诊断全突变男性,也可以辨别男性全突变/前突变嵌合体,与Southern blot相当；对女性全突变患者的甲基化分辨率也较高,可以作出诊断,但对于女性全突变和全突变/前突变嵌合体的分辨率欠佳。该技术自动化程度高,重复性好,快速经济,需要DNA量少,可以进行高通量的大样本检测,因此,我们建议可以将其推广用于脆性X综合征(尤其是男性患者)的分子生物学诊断和人群筛查。目前,关于我国脆性X综合征儿童临床特征和社会适应能力的研究尚属空白。本研究通过临床检查表和社会适应能力量表对FXS男童进行评估,结果发现,六项简易临床检查表≥5分的切值更适合我国FXS男童的早期筛查,该切值既可以早期发现可能患FXS的男童,对其进行分子生物学诊断,提高了FXS的检出率,又可以使61.6%的可疑患儿避免进行脆性X综合征的实验室诊断,大大减少了诊断成本,而不会漏诊任何患儿。对确诊儿童的评估发现,中国FXS男童的注意缺陷/多动障碍(attention deficiency hyperactivity disorder, ADHD)的发生率明显高于其他国家的报道,可能与我们的患儿未积极进行药物治疗有关。同时,研究还发现,我国脆性X综合征男童的社会适应能力较差,明显落后于同年龄的正常儿童,与同智龄正常儿童和同智龄同年龄的唐氏综合征(Down syndrome, DS)儿童相仿,但是,各个维度发展不均衡,作业操作、参加集体活动和自我管理能力甚至落后于DS儿童。因此,要改善我国FXS儿童的临床症状和生活质量,需要加强早期诊断和积极治疗,以及针对患儿临床特征的特殊训练和教育。脆性X综合征动物模型的应用,为神经病理学机制的研究提供了条件,进而促进了针对神经机制的靶向药物治疗的发展。本研究通过实时荧光定量RT-PCR和免疫组化、Western blot实验方法,对Fmr1基因敲除(knockout, KO)小鼠三个主要脑区(大脑皮层、海马和小脑)突触后致密物蛋白95(postsynaptic density protein95,PSD-95)mRNA及其蛋白质时空表达的改变进行了探索。结果发现,在这三个主要脑区中,PSD-95mRNA及其蛋白质的表达在生后第7天(P7)最低,14天明显上升,在青春期或成年期达到高峰。与野生型对照组小鼠相比,Fmr1KO小鼠不管在生长发育期(P7、P14、P21、P28)还是成年期(P90),海马部位PSD-95mRNA与蛋白质的表达水平均显著降低。因此,我们认为PSD-95在海马部位的表达受FMRP的调节,海马区PSD-95的受损与脆性X综合征患者海马依赖的学习功能障碍有关。该结果为进一步针对PSD-95途径的靶向治疗研究提供了依据。
[Abstract]:Fragile X syndrome (Fragile X mental retardation Syndrome, FXS) is a common hereditary mental retardation disease. The incidence of the disease is as high as that of the vast majority of 1/4000., which is caused by the excessive expansion of the X chromosome FMR1 (fragile) repeat sequence leading to gene methylation. The performance of the bed is more severe than that of the female patients, mainly manifested in moderate, severe mental retardation with a special face, such as long face, large or wind ear, large testicles after puberty, and many patients with hyperactivity, impulsiveness, anxiety, social withdrawal, stereotyped language and other autism like behavior. Early discovery, early treatment and intervention can significantly improve the patient's suffering. To provide genetic counseling for family members and avoid reoccurrence of the same family members, it has immeasurable social and economic benefits.
In this study, the dual real-time fluorescence quantitative PCR technique, namely, the quantitative detection of FMR1 gene (methylation-sensitive restriction enzymes-based quantitative PCR, MSRE-QPCR), was used to detect the FMR1 gene (CGG) sequence and the degree of methylation of the island of FXS patients. The results of the quantitative detection of the methylation-sensitive restriction enzymes-based quantitative PCR, and the quantitative detection of the degree of methylation in FXS patients were carried out. It is found that the technique is very efficient for the diagnosis of male patients, not only for the diagnosis of all mutant men, but also for the male total mutation / pre mutation chimeras, which is equivalent to the Southern blot; the methylation resolution of the women with full mutation is also higher, but the diagnosis can be made, but the resolution of full mutation and total mutation / pre mutation chimerism in women is distinguished. The rate is poor. The technology has a high degree of automation, reproducibility, rapid economy, and less DNA. It can be used for large sample detection of high flux. Therefore, we suggest that it can be applied to the molecular biology diagnosis and population screening of fragile X syndrome (especially for male patients).
At present, the study on the clinical characteristics and social adaptability of fragile X syndrome in China is still a blank. This study evaluated the FXS boys by the clinical checklist and the social adaptability scale. The results showed that the six simple clinical checklists more than 5 points were more suitable for the early screening of FXS boys in our country, and the cut value could be early in the early stage. It was found that the boys who were likely to suffer from FXS had a molecular biology diagnosis, increased the detection rate of FXS, and could make 61.6% of the suspected children avoid the laboratory diagnosis of fragile X syndrome, greatly reducing the cost of diagnosis and not missing any children. The assessment of the diagnosed children found that the attention deficit / hyperactivity disorder (at) of Chinese FXS boys (at The incidence of tention Deficiency Hyperactivity Disorder, ADHD) is obviously higher than that in other countries, and it may be related to the lack of active drug treatment in our children. Meanwhile, the study also found that the social adaptation ability of the boys with fragile X syndrome is poor, obviously behind the normal children of the same age, with the same age normal children and the children with the same age. Down syndrome (DS) children are similar to the age of the same age. However, the development of all dimensions is uneven, operation, participation in collective activities and self management are even behind DS children. Therefore, to improve the clinical symptoms and quality of life of FXS children in China, it is necessary to strengthen early diagnosis and active treatment, as well as for children. Special training and education of clinical features.
The application of fragile X syndrome animal model provides conditions for the study of neuropathological mechanism, and further promotes the development of targeted drug therapy for neural mechanisms. This study was carried out by real-time quantitative RT-PCR and immunohistochemistry and Western blot experimental method for Fmr1 gene knockout (knockout, KO) mice (knockout, KO) mice (brain cortex). The changes in spatio-temporal expression of postsynaptic density protein 95 (postsynaptic density protein95, PSD-95) mRNA and their protein in the layer, hippocampus and cerebellum were explored. The results showed that in these three major brain regions, the expression of PSD-95mRNA and its protein was lowest at seventh days after birth (P7), and 14 days rose obviously, and reached its peak during adolescence or adulthood. Compared with the wild type control group, the expression level of PSD-95mRNA and protein in the hippocampus of Fmr1KO mice decreased significantly in the growth and development period (P7, P14, P21, P28) or adult stage (P90). Therefore, we believe that the expression of PSD-95 in the hippocampus is regulated by FMRP, the impairment of PSD-95 in the hippocampus and the hippocampus dependence on fragile X syndrome. The results provide a basis for further targeted therapy of PSD-95 pathway.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.94

【参考文献】