斑状角膜营养不良CHST6基因突变及组织病理学研究

发布时间：2018-05-20 04:04

本文选题：斑状角膜营养不良 + CHST6基因　；参考：《大连医科大学》2011年硕士论文

【摘要】：目的:斑状角膜营养不良(Macular Corneal Dystrophy,MCD)是一种少见的角膜基质层营养不良,属于常染色体隐性遗传病。Groenouw于1980年首次阐述,故又称Groenouw II型角膜营养不良。临床表现为进行性视力丧失、畏光和眼表不适感等,裂隙灯检查可见双眼进行性角膜基质弥漫雾状混浊,间(或)有局灶性斑片状白色混浊,由中央向周边及深层发展,最终需要进行角膜移植手术。2000年Akama等[1]首次发现斑状角膜营养不良的致病基因为一种新的碳水化合物磺基转移酶基因即CHST6基因,并将其定位于染色体16q22上。CHST6基因编码角膜N-乙酰葡萄糖胺-6-磺基转移酶(corneal N-acetylglucosamine-6-sulphotransferase,GlcNAc6ST),这种酶把硫酸根离子传递给N-乙酰葡萄糖胺,该物质参与角膜中硫酸角质素蛋白多糖的生物合成。CHST6基因突变导致这种酶活性的降低或消失,使角膜中硫酸角质素(keratan sulphate,KS)等粘多糖合成异常,异常物质沉积于角膜,破坏角膜细胞及纤维结构,最终导致角膜混浊[2]。自Akama后众多学者相继在其所在国家的MCD患者CHST6基因中发现了突变,我国对斑状角膜营养不良也有较多研究,但是多数仅限于病例报告及组织病理学研究,较少有关于基因突变研究的报告。本实验对我国东北地区一斑状角膜营养不良家系三个阳性体征患者进行研究:抽取外周静脉血提取DNA并进行CHST6基因序列正反双向测序;对穿透性角膜移植术后的患者角膜进行组织病理学检查,进一步探讨斑状角膜营养不良与CHST6基因突变的关系及研究其组织病理学改变。方法:选取我国东北地区一斑状角膜营养不良家系中已发现的同代三名患者,其中男性2人,女性1人。分别抽取外周静脉血2ml,提取基因组DNA。CHST6基因共有四个外显子,编码区位于第三个外显子上,应用聚合酶链反应(PCR)对CHST6基因的编码区进行基因扩增,然后进行基因测序。分别取两名患者角膜移植术后的1/2片病变角膜行4%甲醛溶液固定,石蜡包埋、切片,进行阿辛蓝(AB)和阿辛蓝-过碘酸雪氏(AB-PAS)染色,于光学显微镜下进行观察;对另1/2角膜片行2.5%戊二醛溶液固定,环氧树脂包埋,染色后进行透射电镜观察并摄像。结果:对该家系三名患者的CHST6基因编码区进行基因正反双向测序,发现的共同突变点为:第三外显子第62个碱基插入碱基G,原第62个碱基T→A,造成开放阅读框的改变;AB染色阳性:可见角膜基质中细胞质及弹性纤维蓝色着染;AB-PAS染色中可见角膜上皮变薄,上皮下大片的红蓝着染的斑块状物质;透射电镜观察到角膜上皮下可见大量圆形物质。结论:本研究证实了CHST6编码区的基因突变c.61_62insG是导致这一斑状角膜营养不良家系角膜混浊的直接原因,迄今为止该突变尚未被发现。
[Abstract]:Objective: macular Corneal dystrophy (Macular Corneal dystrophy) is a rare corneal stromal dystrophy. It is an autosomal recessive hereditary disease. Groenouw was first described in 1980, so it is also called Groenouw II corneal dystrophy. The clinical manifestations were progressive loss of vision, photophobia and ocular surface discomfort. Slit-lamp examination showed diffuse haze opacity of the corneal stroma in both eyes, focal flaky white opacity in the middle and / or local areas, which developed from the center to the peripheral and deep layers. In 2000, Akama et al first found a novel carbohydrate sulfotransferase gene (CHST6 gene), which is a novel carbohydrate sulfotransferase gene. The CHST6 gene was located on the chromosome 16q22 and encoded the keratoacetal N-acetylglucosamine-6-sulphotransferase (GlcNAc6STN), which transfered sulfate ion to N-acetylglucosamine-6-sulphotransferase (N-acetylglucosamine-6-sulphotransferase). This substance involved in the biosynthesis of keratin sulfate proteoglycan. CHST6 gene mutation resulted in the decrease or disappearance of the enzyme activity, the abnormal synthesis of mucopolysaccharide such as keratan sulphate KSin in the cornea, and the deposition of abnormal substances in the cornea. Destruction of corneal cells and fiber structure, eventually leading to corneal opacity [2]. Since Akama, many scholars have found mutations in the CHST6 gene of MCD patients in their country. In China, there are many studies on corneal speckle dystrophy, but most of them are limited to case reports and histopathological studies. There are few reports on gene mutation. In this study, we studied three patients with positive signs in a pedigree with corneal dystrophy in northeast China: DNA was extracted from peripheral venous blood and CHST6 gene sequence was sequenced in both positive and negative directions. Histopathological examination was performed on cornea after penetrating keratoplasty to investigate the relationship between speckle corneal dystrophy and mutation of CHST6 gene and its histopathological changes. Methods: three patients of the same generation were selected from a pedigree with corneal dystrophy in Northeast China, including 2 males and 1 female. There were four exons of genomic DNA.CHST6 gene extracted from peripheral venous blood. The coding region was located on the third exon. The coding region of CHST6 gene was amplified by polymerase chain reaction (PCR) and then sequenced. After keratoplasty, 1 / 2 of the lesions were fixed with 4% formaldehyde solution, embedded in paraffin wax, sectioned, stained with ABA and AB-PASs, and observed under optical microscope. Another 1 / 2 cornea was immobilized in 2.5% glutaraldehyde solution, embedded with epoxy resin, then stained with transmission electron microscope and photographed. Results: the CHST6 gene coding region of three patients from this family was sequenced in both directions. The common mutation sites were found as follows: the 62nd base of exon 3 was inserted into base Gand the original 62 base of T _ (2) A, which caused the change of open reading frame to be positive for AB staining. The results showed that the cytoplasm and elastic fibers of corneal stroma were blue stained with AB-PAS. In the staining, the corneal epithelium became thinner, A large area of red and blue stained plaques; a large number of circular substances were observed under transmission electron microscope. Conclusion: this study confirmed that c.61_62insG, a gene mutation in the CHST6 coding region, was the direct cause of corneal opacity in this pedigree with corneal dystrophy. So far, this mutation has not been found.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R772.2

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