Substitution of Glu to Lys at Codon 332 on the GFAP Gene Alone Is Causative for Adult-onset Alexander Disease

Abstract

A 57-year-old man whose mother had been pathologically diagnosed with Alexander disease (ALXDRD), presented with cerebellar ataxia, pyramidal signs, and mild dysarthria. Brain magnetic resonance imaging revealed typical ALXDRD alterations, such as atrophy of the medulla oblongata (MO) and cervical spinal cord, a reduced sagittal diameter of the MO, and garland-like hyperintensity signals along the lateral ventricular walls. A genetic analysis of GFAP by Sanger sequencing revealed a single heterozygous mutation of Glu to Lys at codon 332 (c.994G＞A) in the GFAP gene. Our results newly confirmed that p.E332K alone is the pathogenic causative mutation for adult-onset ALXDRD.

Introduction

Alexander disease (ALXDRD) (MIM 203450) is an autosomal dominant, rare neurological disease (1) that is classified into three subtypes based on the age at the onset: infantile (before two years old), juvenile (until the middle teens), and adult (2,3). Adult-onset (AO) ALXDRD displays the most varied phenotype.

A common neuropathological feature of all forms of ALXDRD is the diffuse presence of Rosenthal fibers, or intracytoplasmic aggregates within astrocytes containing glial fibrillary acidic protein (GFAP; the principal intermediate filament of astrocytes). Various heterozygous missense mutations in the GFAP gene have been identified in patients with neuropathologically proven ALXDRD thus far (http://www.waisman.wisc.edu/alexander/index.html). These mutations have been found mostly in the 1A, 2A, and 2B segments of the conserved central rod domain of the GFAP gene and in the variable tail region (3,4). The absence of symptoms or pathology in GFAP-null mice indicates that the disease is caused by a dominant toxic gain of function of GFAP (4).

We herein report a case of ALXDRD harboring a p.E332K mutation in the GFAP gene in an adult patient.

Case Report

A 57-year-old man (Fig. 1A, II-2) experienced slow progressive gait impairment, frequent falls, generalized muscle weakness, intermittent position-independent vertigo, and general pain. The patient reported that his symptoms had emerged when he was 20 years old, causing a tendency to fall while riding a bicycle. Five years ago, at 52 old, he had developed mild dysarthria. Autonomic dysfunction was not observed. His mother (Fig. 1A, I-3) also showed gait instability and spasticity. She had died of lung cancer at 60 years old and been autopsied, where she was diagnosed with AO-ALXDRD postmortem by the presence of Rosenthal fibers in the white matter of her brain (Fig. 2).

Figure 1.

Family pedigree. Filled symbols represent patients; empty symbols represent healthy individuals; symbols with oblique slashes represent deceased patients. I-3: pathologically diagnosed. II-2: proband.

Figure 2.

Histopathologic and immunohistochemical features of the cerebellar dentate nucleus of the autopsied mother of the patient (case I-3). A: Numerous Rosenthal fibers (arrows) and prominent astrocytosis are observed in the brain. B: Glial fibrillary acidic protein (GFAP)-labeled Rosenthal fibers (arrowheads) and astrocytic processes are observed in the brain. C: α-B-crystallin-labeled Rosenthal fibers (arrowheads) are observed by immunohistochemistry of the brain. A: Hematoxylin and Eosin (H&E) staining. B, C: Immunostained and then counterstained with H&E staining. Bars=50 μm.

A clinical examination of the man revealed vertical gaze nystagmus, cerebellar ataxia, general muscle weakness, dysarthria, brisk tendon reflexes, and positive pyramidal signs. Brain magnetic resonance imaging (MRI) revealed atrophy of the medulla oblongata (MO) and cervical spinal cord (tadpole appearance), with a reduced sagittal diameter of the MO and abnormal signals of the pyramid of the MO. There were hyperintensities of the hilum of the dentate nucleus and midbrain periventricular rim and garland-like hyperintensity signals along the lateral ventricular walls (ventricular garland sign) (Fig. 3). These clinicoradiological findings are compatible with those of ALXDRD (5).

Figure 3.

The brain MRI of proband (case II-2). A: Sagittal T2-weighted imaging indicated marked atrophy and abnormal signals of the medulla oblongata (MO) (arrowhead) and cervical spinal cord. B: Midbrain ventricular rim (arrowheads) in axial fluid-attenuated inversion recovery is clearly observed. C: Hyperintense signals of the bilateral dentate nuclei (arrowheads) on axial T2-weighted images are observed. D: A garland-like low-intensity signal (arrowhead) along the lateral ventricular wall is present on T2-weighted imaging.

Blood tests and cerebrospinal fluid analysis results were normal. A magnetic motor-evoked potential study identified prolonged central motor conduction time on both sides. His central motor conduction times (Cortex-Spine) of the upper extremities were 9.8 ms on the right side and 9.0 ms on the left side (normal range: 6.1±0.7 ms). A nerve conduction study revealed no abnormalities in the peripheral nerves. The Wechsler Adult Intelligence Scale-III revealed a normal cognitive function [mean intelligence quotients (IQs) as follows: full scale IQ, 104; verbal IQ, 99; performance IQ, 110].

His DNA was extracted from peripheral blood according to standard extraction methods. Sanger sequencing of the entire exonic sequences revealed a heterozygous substitution of Glu to Lys at codon 332, p.E332K, of the GFAP gene (NM_002055.5, c.994G＞A) (Fig. 4). Unfortunately, a DNA sample from his mother was not available in stocked brain samples.

Figure 4.

Genetic analysis results. The sequence analysis of the GFAP gene of the patient identified a Glu-to-Lys mutation at codon 332.

Discussion

The mutation p.E332K of GFAP was previously reported as a coupled mutation in the same allele, p.[R330G; E332K] (6), and probably as a single mutation (7). Our review (Table) showed that clinical features of patients with these mutations are similar and include spasticity, bulbar symptoms, ataxia, no seizure, and severe MO atrophy with mild signal changes. Interestingly, the age at the onset varied widely, ranging from 20 to over 50 years old, and ataxia was not severe, but severe MO atrophy was observed. The mutations were located in a mutation-poor segment of the α-helical 2B domain within the central rod structure of three domains of GFAP (3), and p.[R330G; E332K] was predicted to break the α-helix stretch of GFAP (8).

Table.

Clinical Features of Alexander Disease with Mutations at Codon 332 Alone or 330 and 332.

	(7)	(6)	The present patient
Mutation on GFAP
Protein	p.E332K	p.[R330G; E332K]	p.E332K
cDNA	c.994G>A	c.988C>G and c.994G>A	c.994G>A
Sex	M	M, F, M	M
Form	AO	AO	AO
Age at onset (yo)	57	56, 53, 28	20
Duration at examination (y)	4	4, 13, 4	37
Clinical symptoms
Seizures	ND	ND, ND, ND	No
Spasticity	Mild	Yes, Yes, No	Yes
Nystagmus	Yes	ND, ND, ND	Yes
Ptosis	No	ND, ND, ND	No
Dysphagia	Yes	Yes, Yes, No	No
Dysarthria	Yes	Yes, Yes, No	Mild
Palatal myoclonus	No	Yes, No, No	No
Ataxia	Mild	Yes, Yes, No	Mild
Cognitive defet	ND	ND, ND, ND	No
MRI
Cerebral white matter abnormalities	No	No, No, No	No
Medulla oblongata atrophy	Severe	Severe, Severe, Slight	Severe
Medulla oblongata signal change	Severe	Yes, Yes, No	Mild
Dentate hilum signal changes	Yes	ND, ND, ND	Yes
Spinal cord atrophy	Yes	ND, ND, ND	Yes
Postcontrast enhancement	No	Yes, ND, No	No
Pathology
Rosental fibers	ND	ND, ND, ND	Yes (mother)

M: male, F: female, AO: adult onset, ND: not described

Bachetti et al. conducted in vitro intracellular overexpression experiments using human astrocytoma U251-MG cells; the cells were transfected using constructs carrying p.[R330G;E332K], p.R330G, or p.E332K green fluorescent protein-conjugated GFAP (8). Formation of aggregates was induced by both p.[R330G;E332K] and p.E332K constructs, whereas no significant effect was observed by p.R330G. Accordingly, p.E332K is speculated to play a major pathological role in the p.[R330G;E332K] allele and is described as ‘likely pathogenic' in the database of Alexander Disease Lab (https://alexander-disease.waisman.wisc.edu/diagram-mutations/). However, no single mutation had been described in detailed clinical and pathological cases (5,6). Therefore, the pathogenicity of each mutation has remained inconclusive.

The present study identified a patient with AO-ALXDRD harboring p.E332K, confirming the pathogenicity of p.E332K. The present p.E332K phenotypes were similar to previously reported p.[R330G; E332K] cases, which may suggest low pathogenicity of p.R330G, in agreement with the in vitro cellular analysis by Bachetti et al. (8)

In conclusion, we confirmed that p.E332K alone is a pathogenic mutation for AO-ALXDRD associated with mild clinical features.

Written informed consent for this study was obtained from the patient. The Ethics Committee of Kyoto Prefectural University of Medicine approved the genetic testing. All protocols were approved by the Ethics Committee of Tokyo Medical and Dental University and were thus performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

The authors state that they have no Conflict of Interest (COI).