本文選題：Nance-Horan綜合征(NHS) + 基因突變��； 參考：《浙江大學》2015年博士論文

【摘要】：研究目的 Nance-Horan綜合征(Nance-Horan syndrome, NHS)是一種罕見的x連鎖遺傳性疾病,其典型特征是先天性白內(nèi)障、牙齒及顱面部異常。其中30%的患者有智力發(fā)育障礙。NHS基因是該綜合征的致病基因。NHS基因包含10個外顯子,通過選擇性剪切編碼至少6種蛋白異構(gòu)體(亞型)。其中,NHS-A和NHS-1A是兩種主要的亞型,兩者均從NHS基因第一個外顯子轉(zhuǎn)錄,前者編碼一個長約1630個氨基酸的蛋白質(zhì),后者編碼一個長約1651個氨基酸的蛋白質(zhì)。在這兩個蛋白亞型的N端有一個WAVE蛋白同源結(jié)構(gòu)域(WHD),能夠與Abelson-interactor (Abi)蛋白家族,如Abi1和Abi2等相互作用。研究顯示NHS-1A參與調(diào)節(jié)肌動蛋白重塑和維持細胞形態(tài)。在本研究中,我們對一個四代的中國Nance-Horan綜合征家系的臨床特征和分子遺傳學機制進行分析,并在體外實驗研究NHS基因的相關(guān)細胞學功能,為闡明該疾病的分子遺傳學機制提供研究基礎(chǔ)。 研究方法 1.家系內(nèi)所有成員接受病史詢問,眼部及全身系統(tǒng)性檢查。使用Cyrlillic件繪制家系遺傳圖譜進行系譜分析。抽取家系成員全血提取基因組DNA。 2.對該家系的一個男性患者進行全外顯子組測序,結(jié)合此家系臨床表型及遺傳方式分析,選定x染色體上NHS基因上的一個無義突變c.322GT(E108X)為可疑致病突變。通過聚合酶鏈式反應(yīng)(polymerase chain reaction, PCR)和Sanger測序,對該家系內(nèi)其他成員進行NHS基因突變分析,同時對50名健康對照者的NHS基因的突變檢測結(jié)果進行對比。另外,使用CLC Sequence Viewer5.5(CLC bio A/S, http://www.clcbio.com)軟件對NHS蛋白序列在七個不同物種中進行多序列比對分析。 3.構(gòu)建表達NHS-1A蛋白的野生型質(zhì)粒pEGFP-C2-NHS(NHS-EGFP)及突變質(zhì)粒pEGFP-C2-NHS-c.322G T (NHSE108X-EGFP).將NHS-EGFP, NHSE108X-EGFP, pEGFP-C2質(zhì)粒瞬時轉(zhuǎn)染進HEK293T細胞和SRA01/04細胞中。使用倒置熒光顯微鏡觀察觀察三種質(zhì)粒在SRAO1/04細胞中的亞細胞定位。使用實時熒光定量PCR (Quantitative real-time polymerase chain reaction, QRT-PCR)分析轉(zhuǎn)染三種質(zhì)粒后HEK293T細胞中NHS mRNA的表達水平。化學合成針對NHS基因的小干擾RNA (small interfering RNA, siRNA),將其轉(zhuǎn)染進人晶體上皮細胞系SRAO1/04細胞中,通過Western blot分析Abi2蛋白的表達水平變化。通過Transwell小室遷移實驗觀察NHS表達受抑制后對SRA01/04細胞的遷移能力影響。 研究結(jié)果 1.該Nance-Horan綜合征家系一共四代,其中七名家庭成員參加了本研究,包括3名男性患者,2名女性攜帶者及2名男性正常對照。所有男性患者自幼因“先天性白內(nèi)障”接受雙眼白內(nèi)障手術(shù),眼部其他表型包括雙眼先天性小角膜,眼球震顫及斜視。除此之外,顱面部異常在三名患者中均有體現(xiàn),一名男性患者有典型的牙齒異常。所有女性攜帶者均有不同程度的晶體混濁及雙顳部縮窄。系譜分析該家系遺傳方式可能為X連鎖遺傳。 2.全外顯子組測序結(jié)合Sanger測序發(fā)現(xiàn)NHS基因第一個外顯子上的c.322GT(E108X)突變?yōu)橐鹪摷蚁蹬R床病變的突變位點；多物種NHS蛋白內(nèi)序列比對發(fā)現(xiàn)該突變位點第108位氨基酸殘基位于高度保守區(qū)。 3.倒置熒光顯微鏡顯示野生型質(zhì)粒定位于SRA01/04細胞的細胞質(zhì)中,而突變型質(zhì)粒定位于細胞核及細胞質(zhì)中。QRT-PCR顯示突變NHS基因的mRNA表達水平較野生型明顯降低(p0.05)。抑制SRA01/04細胞中NHS基因表達后,細胞內(nèi)Abi2蛋白表達水平降低(P0.05)；Transwell遷移實驗顯示siRNA組穿過小室細胞數(shù)較對照組明顯降低(P0.05)。 結(jié)論： 1.報道了國內(nèi)首例Nance-Horan綜合征家系。臨床表型分析顯示在家系中存在表型異質(zhì)性。 2.首次發(fā)現(xiàn)一個新的NHS基因無義突變c.322GT(E108X).多物種NHS蛋白內(nèi)序列比對發(fā)現(xiàn)該突變位點第108位氨基酸殘基位于高度保守區(qū)。 3.該突變導致NHS蛋白細胞學定位改變,并引發(fā)NHS基因mRNA降解。干擾NHS基因表達后,晶體細胞中Abi2蛋白表達水平下降,細胞遷移能力降低。
[Abstract]:research objective
Nance-Horan syndrome (Nance-Horan syndrome, NHS) is a rare X linked hereditary disease, characterized by congenital cataract, tooth and craniofacial abnormalities. 30% of the patients have the.NHS gene for mental retardation, which is the pathogenetic gene of the syndrome, the.NHS gene contains 10 exons, and at least 6 species are encoded by selective shear coding. Protein isomers (subtypes). Among them, NHS-A and NHS-1A are two major subtypes, both are transcribed from the first exon of the NHS gene, the former encodes a protein of about 1630 amino acids, and the latter encodes a protein with a length of about 1651 amino acids. There is a WAVE protein homologous domain (WHD) in the N terminal of the two protein subtypes. The interaction with the Abelson-interactor (Abi) protein family, such as Abi1 and Abi2, has shown that NHS-1A is involved in regulating actin remodeling and maintaining cell morphology. In this study, we analyzed the clinical and molecular genetic mechanisms of a four generation of Chinese Nance-Horan family, and studied the NHS gene in vitro. The related cytological functions provide a basis for elucidating the molecular genetic mechanism of the disease.
research method
All members of the 1. families received medical history inquiry, eye and systemic examination. Using Cyrlillic parts to draw family genetic map for pedigree analysis. Extraction of genomic DNA. from whole blood of family members.
2. of a male patient in the family was sequenced, combined with the clinical phenotype and genetic analysis of the family, a non sense mutation c.322GT (E108X) on the NHS gene of the X chromosome was selected as a suspected pathogenic mutation. The other adult in the family was made by polymerase chain reaction (polymerase chain reaction, PCR) and Sanger sequencing. The NHS gene mutation analysis was carried out and the NHS gene mutation detection results of 50 healthy controls were compared. In addition, the CLC Sequence Viewer5.5 (CLC bio A/S, http://www.clcbio.com) software was used to analyze the multiple sequence ratio of the NHS protein sequence in seven different species.
3. to construct the wild plasmids pEGFP-C2-NHS (NHS-EGFP) and mutant plasmid pEGFP-C2-NHS-c.322G T (NHSE108X-EGFP) expressing NHS-1A protein. NHS-EGFP, NHSE108X-EGFP, and pEGFP-C2 plasmids were transiently transfected into HEK293T cells and SRA01/04 cells. The subcellular localization of the three plasmids in the SRAO1/04 cells was observed by inverted fluorescence microscope. The expression level of NHS mRNA in HEK293T cells transfected with three plasmids was analyzed by real-time fluorescent quantitative PCR (Quantitative real-time polymerase chain reaction, QRT-PCR). Chemical synthesis of small interference RNA for NHS gene was transfected into the human lens epithelial cell line. The expression level of Abi2 protein was analyzed by blot. Transwell cell migration assay was used to observe the effect of NHS expression on the migration of SRA01/04 cells.
Research results
1. a total of four generations of the Nance-Horan family, of which seven family members participated in the study, including 3 male patients, 2 female carriers and 2 male normal controls. All male patients received double cataract surgery for "congenital cataract", and other ocular phenotypes included binocular congenital small cornea, nystagmus, and the other phenotypes of the eyes. In addition to this, craniofacial abnormalities are manifested in three patients, and a male patient has typical dental abnormalities. All female carriers have different degrees of crystal turbidity and double temporal constriction. Genealogy may be a X linkage inheritance.
2. the total exon sequencing combined with Sanger sequencing found that the c.322GT (E108X) mutation on the first exon of the NHS gene was the mutation site that caused the clinical pathological changes of the family, and the comparison of the internal sequence of the multiple species NHS protein was located in the highly conserved area of the 108th amino acid residues found at the mutation site.
3. inverted fluorescence microscopy showed that the wild plasmids were located in the cytoplasm of SRA01/04 cells, while the.QRT-PCR expression level of mutant NHS in the nucleus and cytoplasm was significantly lower than that of the wild type (P0.05). The level of Abi2 protein expression in the cells decreased (P0.05) in the SRA01/04 cells (P0.05). Transwell migration test showed that the number of cells passing through the compartments in siRNA group was significantly lower than that in the control group (P0.05).
Conclusion:
1. the first case of Nance-Horan syndrome family was reported in China. Phenotypic analysis showed phenotypic heterogeneity in the family.
2. a new NHS gene nonsense mutation c.322GT (E108X) was found for the first time. The comparison of the internal sequence of the multiple species NHS protein found that the 108th amino acid residues in the mutation site were located in the highly conserved region.
3. this mutation leads to the change of NHS protein cytology and the degradation of the NHS gene mRNA. After interfering with the expression of NHS gene, the expression of Abi2 protein in the crystal cells decreases and the cell migration ability is reduced.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R596

【共引文獻】

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本文編號：1999146