Novel GFAP mutation in patient with adult‐onset Alexander disease presenting with spastic ataxia

Adult-onset Alexander disease is rare and clinically characterized by slowly progressive signs of brainstem and spinal cord involvement. Missense mutations in the gene encoding the glial fibrillary acidic protein (GFAP) have been identified as a genetic basis for Alexander disease. We here report a Japanese patient with adult-onset Alexander disease with a novel GFAP mutation. A 36-year-old man of Japanese descent, a child of nonconsanguineous parents, with a 10-year history of slowly progressive gait disturbance, was referred to us. His early motor and intellectual development were normal. Neurological examination revealed rhythmic ocular nystagmoid movement, dysarthria, truncal and limb ataxia, increased muscle stretch reflex with bilateral Babinski sign, and spasticity in his lower extremities. Palatal myoclonus was not noted. He was ambulatory, but his gait was unsteady owing to ataxia and spasticity in the lower extremities. Brain MRI demonstrated a marked atrophy of the medulla oblongata and cervical spinal cord, and a mild atrophy of the cerebellar hemisphere (Fig. 1A). Fluid attenuation inversion recovery (FLAIR) images revealed abnormal hyperintensities in cerebellar dentate nucleus (Fig. 1B) and the periventricular white matter (Fig. 1C). Molecular genetic analysis of GFAP was performed using the patient’s genomic DNA after obtaining written informed consent. Sequence analysis revealed a heterozygous 302T > C substitution in exon 1 of GFAP, leading to an L101P substitution. The L101P substitution is located in the C-terminal end of the 1A rod domain of GFAP occurring in a highly conserved amino acid residue across species (Fig. 1D). The sequence change was confirmed by restriction fragment length polymorphism (RFLP) using enzyme digestion by BcgI in the patient and 100 normal control subjects (Fig. 1E). To obtain biochemical evidence of pathogenecity of the novel GFAP L101P mutant, we transfected wild-type and mutant GFAP, and examined the solubility of the GFAP protein. Samples were sequentially extracted with different stringent buffers and subjected to western blot analysis (see Supplementary methods). The well-characterized mutant R416W GFAP was largely recovered from the detergent-resistant S2 fraction because of the decreased solubility of mutant GFAP (Fig. 1F, lane 8) as previously reported. In this assay, wild-type GFAP was predominantly detected in the soluble S1 fraction (Fig. 1F, lane 2). In contrast, the mutant L101P GFAP was largely observed in the detergent-resistant S2 fraction (Fig. 1F, lane 7). Transfected cells were further analyzed for GFAP assembly by confocal microscopy (see Supplementary methods). Whereas wild-type GFAP displayed cytoplasmic distribution with a filamentous network, the L101P mutant yielded an irregular dot-like structure largely lacking the filamentous structure (Supp. Info. Fig.). In this study, we identified a novel GFAP mutation in a Japanese patient with adult-onset Alexander disease presenting with slowly progressive spastic ataxia. The parents of the patient are unaffected; hence, the mutation seems to arise de novo in the patient as in most cases of Alexander disease. Indeed, the mother did not carry the mutation. Unfortunately, DNA sample was unavailable from the father, who has recently died of heart disease. GFAP is a member of the intermediate filament family with a conserved central helical rod domain flanked by the head and tail domains (Fig. 1D). The L101P mutation detected in the patient is located in the coil 1A rod domain, which is considered to play an essential role in filament formation. Mutations of the a-helix regions in the rod domain are considered to alter the charge and hydrophobic interactions within coiled coils. Thus, mutations in the domain may affect the solubility of GFAP, which is supported by our biochemical experiments using cells expressing mutant GFAP. To date, more than 10 GFAP missense mutations associated with adult-onset Alexander disease have been found, with nearly all occurring in the rod domain. The genotype–phenotype correlation in Alexander disease has been poorly understood particularly in adult-onset cases, probably owing to the very small number of patients. Alexander disease in our patient is clinically characterized by slowly progressive spastic ataxia with bulbar signs without palatal myoclonus. In patients with adult-onset Alexander disease, bulbar symptoms, gait ataxia, and spasticity are common clinical features, whereas ocular motor abnormalities, autonomic dysfunctions, and palatal myoclonus have been reported with varying frequency. Our patient exhibited atrophy of the medulla and spinal cord, and abnormal hyperintensities of the periventricular white matter and cerebellar dentate nucleus on FLAIR images. The characteristic atrophy of the medulla and spinal cord is invariably present in adult-onset Alexander disease. In contrast, leukoencephalopathy and abnormal signal intensities of the cerebellum are not always observed in adultonset cases. The question of why missense mutations in the same critical domain of GFAP result in such different clinical phenotypes and MRI findings in Alexander disease is intriguing and deserves further attention and elucidation.