Nance-Horan综合征家系致病基因突变定位及功能研究

发布时间：2018-06-09 05:26

本文选题：Nance-Horan综合征(NHS) + 基因突变　；参考：《浙江大学》2015年博士论文

【摘要】：研究目的 Nance-Horan综合征(Nance-Horan syndrome, NHS)是一种罕见的x连锁遗传性疾病,其典型特征是先天性白内障、牙齿及颅面部异常。其中30%的患者有智力发育障碍。NHS基因是该综合征的致病基因。NHS基因包含10个外显子,通过选择性剪切编码至少6种蛋白异构体(亚型)。其中,NHS-A和NHS-1A是两种主要的亚型,两者均从NHS基因第一个外显子转录,前者编码一个长约1630个氨基酸的蛋白质,后者编码一个长约1651个氨基酸的蛋白质。在这两个蛋白亚型的N端有一个WAVE蛋白同源结构域(WHD),能够与Abelson-interactor (Abi)蛋白家族,如Abi1和Abi2等相互作用。研究显示NHS-1A参与调节肌动蛋白重塑和维持细胞形态。在本研究中,我们对一个四代的中国Nance-Horan综合征家系的临床特征和分子遗传学机制进行分析,并在体外实验研究NHS基因的相关细胞学功能,为阐明该疾病的分子遗传学机制提供研究基础。研究方法 1.家系内所有成员接受病史询问,眼部及全身系统性检查。使用Cyrlillic件绘制家系遗传图谱进行系谱分析。抽取家系成员全血提取基因组DNA。 2.对该家系的一个男性患者进行全外显子组测序,结合此家系临床表型及遗传方式分析,选定x染色体上NHS基因上的一个无义突变c.322GT(E108X)为可疑致病突变。通过聚合酶链式反应(polymerase chain reaction, PCR)和Sanger测序,对该家系内其他成员进行NHS基因突变分析,同时对50名健康对照者的NHS基因的突变检测结果进行对比。另外,使用CLC Sequence Viewer5.5(CLC bio A/S, http://www.clcbio.com)软件对NHS蛋白序列在七个不同物种中进行多序列比对分析。 3.构建表达NHS-1A蛋白的野生型质粒pEGFP-C2-NHS(NHS-EGFP)及突变质粒pEGFP-C2-NHS-c.322G T (NHSE108X-EGFP).将NHS-EGFP, NHSE108X-EGFP, pEGFP-C2质粒瞬时转染进HEK293T细胞和SRA01/04细胞中。使用倒置荧光显微镜观察观察三种质粒在SRAO1/04细胞中的亚细胞定位。使用实时荧光定量PCR (Quantitative real-time polymerase chain reaction, QRT-PCR)分析转染三种质粒后HEK293T细胞中NHS mRNA的表达水平。化学合成针对NHS基因的小干扰RNA (small interfering RNA, siRNA),将其转染进人晶体上皮细胞系SRAO1/04细胞中,通过Western blot分析Abi2蛋白的表达水平变化。通过Transwell小室迁移实验观察NHS表达受抑制后对SRA01/04细胞的迁移能力影响。研究结果 1.该Nance-Horan综合征家系一共四代,其中七名家庭成员参加了本研究,包括3名男性患者,2名女性携带者及2名男性正常对照。所有男性患者自幼因“先天性白内障”接受双眼白内障手术,眼部其他表型包括双眼先天性小角膜,眼球震颤及斜视。除此之外,颅面部异常在三名患者中均有体现,一名男性患者有典型的牙齿异常。所有女性携带者均有不同程度的晶体混浊及双颞部缩窄。系谱分析该家系遗传方式可能为X连锁遗传。 2.全外显子组测序结合Sanger测序发现NHS基因第一个外显子上的c.322GT(E108X)突变为引起该家系临床病变的突变位点；多物种NHS蛋白内序列比对发现该突变位点第108位氨基酸残基位于高度保守区。 3.倒置荧光显微镜显示野生型质粒定位于SRA01/04细胞的细胞质中,而突变型质粒定位于细胞核及细胞质中。QRT-PCR显示突变NHS基因的mRNA表达水平较野生型明显降低(p0.05)。抑制SRA01/04细胞中NHS基因表达后,细胞内Abi2蛋白表达水平降低(P0.05)；Transwell迁移实验显示siRNA组穿过小室细胞数较对照组明显降低(P0.05)。结论： 1.报道了国内首例Nance-Horan综合征家系。临床表型分析显示在家系中存在表型异质性。 2.首次发现一个新的NHS基因无义突变c.322GT(E108X).多物种NHS蛋白内序列比对发现该突变位点第108位氨基酸残基位于高度保守区。 3.该突变导致NHS蛋白细胞学定位改变,并引发NHS基因mRNA降解。干扰NHS基因表达后,晶体细胞中Abi2蛋白表达水平下降,细胞迁移能力降低。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R596

【共引文献】