Molecular analysis of a new type of spinocerebellar degeneration with GFAP mutations

胶质纤维酸性蛋白(GFAP)是星形胶质细胞表达的一种中间丝蛋白。最近，在不同形式的Alexander病(ALX)患者中已经报道了GFAP杂合点突变。ALX按发病年龄分为婴儿型、青少年型和成年型。婴儿型的特征是进行性精神运动恶化、癫痫、脑积水，并在婴儿期死亡。少年型的特点是精神运动恶化、延髓体征、四肢无力和痉挛，但智力低下和进展没有婴儿型严重。成人型以共济失调、腭肌阵挛和痉挛性瘫痪为特征，但从严重的神经功能缺陷到无症状状态的症状差异极大。我们报告了伴有V87G GFAP突变的腭肌阵挛和脊髓萎缩的家族性病例。我们还报告了一例新的腭肌阵挛、锥体束征、小脑征…。更多的是S，延髓和脊髓明显萎缩，杂合子R416W突变，在婴幼儿和青少年中也存在。为了阐明临床表型与GFAP突变的相关性，我们分析了4个日本家系的9名患者，并回顾了65名报道的GFAP突变点患者。此外，我们还构建了突变型GFAP表达载体，并对突变型GFAP在培养细胞中的表达模式进行了研究。74例患者中，婴儿型42例，幼年型13例，成年型19例。在19例成人型患者中，2例无主观症状，7个家系中有6个有家族史。幼体和成体共存于同一家庭成员中。腭肌阵挛只有四名成人患者被识别。在MRI表现方面，额部优势白质损害在婴儿期被发现，但脊髓萎缩在成人型中显著。GFAP突变主要定位于外显子1、4和8。R416W突变可检测到各种类型的ALX，其临床表现与突变性质无明显相关性。在1例幼年型患者中，发现线粒体异常、红色纤维、mtDNA A8291G替换和9BP缺失。我们通过定点突变产生了四种不同的GFAP突变(T235C、T274G、C276T、C1260T)。将这些突变体导入克隆的人脑胶质瘤细胞。用抗GFAP抗体进行免疫组织化学染色，研究GFAP蛋白的表达特性。与野生型GFAP相比，GFAP基因突变的ALX细胞中GFAP的表达增强。每种ALX的表型差异不仅是由于GFAP突变的不同部位和性质所致，还与其他修饰因子(S)有关，如其他中间丝和线粒体功能。比较每种突变在转基因小鼠和培养系统中的表达模式，可以阐明ALX与GFAP突变的表型和遗传相关性。较少

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocyte. Recently heterozygous point mutations in the GFAP have been reported in patients with various forms of Alexander disease (ALX). ALX is classified into infantile, juvenile and adult forms by age of disease onset. Infantile form is characterized by progressive psychomotor deterioration, seizure, hydrocephalus, and dies in infancy. Juvenile form is characterized by psychomotor deterioration, bulbar signs, and weakness and spasticity of lower limbs, but mental retardation and progression are less severe than those of the infantile form. Adult form is characterized by ataxia, palatal myoclonus and spastic paraparesis, but the symptoms are extremely variable from severe neurological defects to asymptomatic state.We reported familial cases of palatal myoclonus and spinal cord atrophy with V87G GFAP mutation. We also reported a new case of palatal myoclonus, pyramidal tract signs, cerebellar sign … More s, and marked atrophy of the medulla oblongata and spinal cord, and heterozygous R416W mutation which was also found in both of infantile and juvenile forms. To clarify the correlation of the clinical phenotypes and GFAP mutations, we analyzed nine patients in four Japanese families and reviewed 65 reported patients on the point of GFAP mutations. In addition, we produced the mutant GFAP expression vectors and investigated the expression pattern of the mutant GFAP in the culture cells.In the 74 patients, 42, 13 and 19 patients were infantile, juvenile and adult forms, respectively. In the 19 patients with adult form, two of them had no subjective symptoms and six of seven families had family history of the disease. The juvenile and adult forms coexisted in the same family members. Palatal myoclonus was recognized only four patients with the adult form. Regarding MRI findings, frontal dominant white matter lesion was detected in the infantile form, but spinal cord atrophy was prominent in the adult form. The sites of GFAP mutation mainly localized at exon 1, 4 and 8. R416W mutation was detected all forms of ALX and no clear correlation between the clinical forms and nature of the mutations. Mitochondrial abnormalities, ragged-red fibers, mtDNA A8291G substitution and 9bp deletion, were detected in one patient with the juvenile form. No nestin mutation was detected in the adult form.We produced four different GFAP mutations (T235C, T274G, C276T, C1260T) by site specific mutagenesis. These mutants were transfected into cloned human glioma cells. We investigated the expressions properties of the GFAP protein through immunohistochemical staining using anti-GFAP antibody. Over expression of GFAP was found in the cells with GFAP mutations in comparison with those of wild type GFAP.In conclusion, we confirmed the phenotypic and genetic features of adult form ALX with GFAP mutations. The phenotypic difference between each form of ALX is due not only to the different site and nature of mutations in GFAP, but also to other modifying factor(s) like other intermediate filaments and mitochondrial functions. Comparison of the expression patterns of each mutation in transgenic mice and culture system may clarify the phenotypic and genetic correlation in ALX with GFAP mutations. Less