本文選題：脆性X綜合征 + 篩查��； 參考：《浙江大學》2012年博士論文

【摘要】：脆性X綜合征(Fragile X mental retardation Syndrome, FXS)是一種常見的遺傳性精神發(fā)育遲滯性疾病,其發(fā)病率高達1/4000。絕大多數(shù)的FXS是由于X染色體FMR1(fragile X mental retardation-1)基因(CGG)n重復序列過度擴展導致基因甲基化而引起的。男性患者臨床表現(xiàn)較女性患者嚴重,主要表現(xiàn)為中、重度的精神發(fā)育遲滯,伴特殊面容,如長臉、大或招風耳,青春期以后出現(xiàn)大睪丸,許多患者還表現(xiàn)為多動、沖動、焦慮、社交退縮、刻板語言等孤獨癥樣行為。早期發(fā)現(xiàn)、早期治療和干預,可以顯著改善患兒預后,并為家系成員提供遺傳咨詢,避免家族中再次出現(xiàn)同樣患者,具有不可估量的社會效益和經(jīng)濟效益。 本研究應用雙重實時熒光定量PCR技術,即普通實時熒光定量PCR聯(lián)合甲基化敏感性限制性內(nèi)切酶定量PCR (methylation-sensitive restriction enzymes-based quantitative PCR, MSRE-QPCR)技術,進行FXS患者FMR1基因(CGG)n序列和CpG島甲基化程度的定量檢測,結果發(fā)現(xiàn),該技術對男性患者的診斷效率極高,既可以診斷全突變男性,也可以辨別男性全突變/前突變嵌合體,與Southern blot相當；對女性全突變患者的甲基化分辨率也較高,可以作出診斷,但對于女性全突變和全突變/前突變嵌合體的分辨率欠佳。該技術自動化程度高,重復性好,快速經(jīng)濟,需要DNA量少,可以進行高通量的大樣本檢測,因此,我們建議可以將其推廣用于脆性X綜合征(尤其是男性患者)的分子生物學診斷和人群篩查。 目前,關于我國脆性X綜合征兒童臨床特征和社會適應能力的研究尚屬空白。本研究通過臨床檢查表和社會適應能力量表對FXS男童進行評估,結果發(fā)現(xiàn),六項簡易臨床檢查表≥5分的切值更適合我國FXS男童的早期篩查,該切值既可以早期發(fā)現(xiàn)可能患FXS的男童,對其進行分子生物學診斷,提高了FXS的檢出率,又可以使61.6%的可疑患兒避免進行脆性X綜合征的實驗室診斷,大大減少了診斷成本,而不會漏診任何患兒。對確診兒童的評估發(fā)現(xiàn),中國FXS男童的注意缺陷/多動障礙(attention deficiency hyperactivity disorder, ADHD)的發(fā)生率明顯高于其他國家的報道,可能與我們的患兒未積極進行藥物治療有關。同時,研究還發(fā)現(xiàn),我國脆性X綜合征男童的社會適應能力較差,明顯落后于同年齡的正常兒童,與同智齡正常兒童和同智齡同年齡的唐氏綜合征(Down syndrome, DS)兒童相仿,但是,各個維度發(fā)展不均衡,作業(yè)操作、參加集體活動和自我管理能力甚至落后于DS兒童。因此,要改善我國FXS兒童的臨床癥狀和生活質(zhì)量,需要加強早期診斷和積極治療,以及針對患兒臨床特征的特殊訓練和教育。 脆性X綜合征動物模型的應用,為神經(jīng)病理學機制的研究提供了條件,進而促進了針對神經(jīng)機制的靶向藥物治療的發(fā)展。本研究通過實時熒光定量RT-PCR和免疫組化、Western blot實驗方法,對Fmr1基因敲除(knockout, KO)小鼠三個主要腦區(qū)(大腦皮層、海馬和小腦)突觸后致密物蛋白95(postsynaptic density protein95,PSD-95)mRNA及其蛋白質(zhì)時空表達的改變進行了探索。結果發(fā)現(xiàn),在這三個主要腦區(qū)中,PSD-95mRNA及其蛋白質(zhì)的表達在生后第7天(P7)最低,14天明顯上升,在青春期或成年期達到高峰。與野生型對照組小鼠相比,Fmr1KO小鼠不管在生長發(fā)育期(P7、P14、P21、P28)還是成年期(P90),海馬部位PSD-95mRNA與蛋白質(zhì)的表達水平均顯著降低。因此,我們認為PSD-95在海馬部位的表達受FMRP的調(diào)節(jié),海馬區(qū)PSD-95的受損與脆性X綜合征患者海馬依賴的學習功能障礙有關。該結果為進一步針對PSD-95途徑的靶向治療研究提供了依據(jù)。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R749.94

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本文編號：1810827