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

胶质细胞酸性蛋白（GFAP）是星形胶质细胞表达的一种中间纤维蛋白。最近，在各种形式的亚历山大病（ALX）患者中报告了GFAP的杂合子点突变。根据发病年龄，ALX分为婴儿型、青少年型和成人型。婴儿型的特征是进行性精神衰退、癫痫发作、脑积水，并在婴儿期死亡。幼年型的特征是精神退化、延髓征、下肢无力和痉挛，但智力迟钝和进展不如婴儿型严重。成人型以共济失调、腭肌阵挛和痉挛性轻瘫为特征，但症状从严重的神经功能缺损到无症状的状态变化很大。我们报道了V87 G 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 ...更多信息 s，延髓和脊髓明显萎缩，杂合子R416W突变也见于婴儿型和少年型。为了阐明临床表型和GFAP突变的相关性，我们分析了4个日本家庭的9例患者，并回顾了65例报告的GFAP突变患者。此外，我们还制备了突变型GFAP表达载体，并研究了突变型GFAP在培养细胞中的表达模式，在74例患者中，婴儿型42例，青少年型13例，成人型19例。成人型19例中2例无自觉症状，7个家系中6个有家族史。幼年型和成年型共存于同一家系成员中。腭肌阵挛是公认的只有4例成人形式。关于MRI所见，在婴儿型中检测到额叶显性白色病变，但在成人型中脊髓萎缩突出。GFAP突变位点主要位于外显子1、4和8。所有形式的ALX均检测到R416W突变，且突变的临床形式和性质之间无明显相关性。在1例幼年型患者中检测到线粒体异常，红色纤维破碎，线粒体DNA A8291G取代和9bp缺失。通过定点突变技术，我们在成体中发现了四种不同的GFAP突变（T235C，T274G，C276T，C1260T）。将这些突变体转染到克隆的人胶质瘤细胞中。通过免疫组织化学方法检测胶质纤维酸性蛋白（GFAP）的表达特性。与野生型相比，突变型ALX细胞中GFAP的表达量明显增加，证实了突变型ALX的表型和遗传特征。每种形式的ALX之间的表型差异不仅是由于GFAP中突变的不同位点和性质，而且还由于其他修饰因子如其他中间丝和线粒体功能。比较各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。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