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A case of infantile Alexander disease with a milder phenotype and a novel GFAP mutation, L90P
Brain & Development 26 (2004) 206–208 www.elsevier.com/locate/braindev
Case report
A case of infantile Alexander disease with a milder phenotype and a novel GFAP mutation, L90P Yoshiko Suzukia,*, Naomi Kanazawab, Junko Takenakaa, Akihisa Okumuraa, Tamiko Negoroa, Seiichi Tsujinob a
Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan b Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8502, Japan Received 9 January 2003; received in revised form 1 July 2003; accepted 1 July 2003
Abstract Alexander disease is a leukoencephalopathy that usually presents during infancy with developmental delay, macrocephaly and seizures. Several sequencing analyses have identified mutations in the gene encoding glial fibrillary acidic protein (GFAP) of patients with Alexander disease. We described a girl who developed seizures in infancy with atypical CT findings and in whom a novel heterozygous mutation, L90P (283T ! C), was detected in exon 1 of the GFAP gene. The neurological deterioration was mild and appeared relatively late for infantile onset. q 2003 Elsevier B.V. All rights reserved. Keywords: Alexander disease; Mutation; GFAP; Infantile; Computed tomography
1. Introduction Alexander disease is an uncommon degenerative disorder that is associated with myelin loss in a rostrocaudal gradient. The pathologic hallmark of the disorder is the presence of Rosenthal fibers located within astrocytes. Although brain biopsy or postmortem examination has been the only confirmatory test for a diagnosis, glial fibrillary acidic protein (GFAP) mutations have now been found in several cases of Alexander disease [1 – 7]. These mutations found in child patients are considered to be de novo and dominant. We describe a girl who had white matter abnormalities with frontal preponderance and a novel mutation within the GFAP gene. The patient had developed seizures in infancy but developmental deterioration was mild and appeared relatively late for infantile onset.
2. Case report The patient was a 5-year-old girl born as the first child of Japanese non-consanguineous parents. Her mother had *
Corresponding author. Tel.: þ81-52-744-2294; fax: þ 81-52-744-2974. E-mail address: [email protected] (Y. Suzuki).
0387-7604/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00132-3
a history of systemic lupus erythematosus. The rest of the family were healthy. Her prenatal and perinatal history were unremarkable. At the age of 5 months, she developed a cluster of complex partial seizures, controlled by phenobarbitone. Macrocephaly was not present. Brain CT at 5 months of age revealed scattered high density areas in the basal ganglia (Fig. 1A), which remained the same on the latest CT scan at 4 years of age. Normal development continued until 7 months of age, motor development then slowed down, such that she could only walk with support at 12 months and without only at 21. She was referred to our hospital at 4 years 6 months of age. Her height was 90 cm (below 3rd percentile) and head circumference 50.8 cm (75th percentile). She walked clumsily and could speak simple sentences. Deep tendon reflexes and muscle tone were normal. MR imaging showed predominantly frontal involvement of the white matter with low signal intensity on T1-weighted images and high signal intensity on T2-weighted. The subcortical U-fibers were relatively spared (Fig. 1B). Lesions were also observed in the putamen, globus pallidus, caudate nucleus, medulla oblongata and dorsal pons. Proton MR spectroscopy of the frontal white matter showed reduced N-acetylaspartic acid/ creatine, increased choline/creatine ratios and accumulation of lactate, though the accumulation of lactate was not
Y. Suzuki et al. / Brain & Development 26 (2004) 206–208
207
Fig. 1. (A) Axial CT image shows scattered high-density areas in the bilateral basal ganglia at 5 months of age. Abnormal low density is not evident in the frontal deep white matter. (B) Axial T2-weighted MR image shows high signal intensity in the bilateral frontal white matter, putamen, globus pallidus and caudate nucleus.
recognized at 5 years 9 months of age. Except for mildly elevated CSF levels of lactate and pyruvate (lactate 26 mg/dl, pyruvate 1.2 mg/dl), laboratory values were normal including plasma amino acids, urine organic acids, serum levels of lactate, pyruvate, very long-chain fatty acids, and phytanic acid, and the activities of arylsulfatase A, beta-galactosidase, beta-hexosaminidase A and galactocerebrosidase. Ophthalmologic findings were normal. EEG showed high voltage slow waves in frontal areas. Brainstem auditory responses demonstrated a prolongation in I –V interval and delay in peak latencies of waves III, IV and V. After informed consent was obtained from the patient’s parents, genomic DNA was isolated from peripheral leukocytes, and amplified by PCR as previously described [4]. The PCR products were electrophoresed through 1.5% agarose gels, purified by GeneElute Agarose Spin Column (Sigma, St. Louis, MO) or QIAquick PCR Purification kit (Qiagen, Hilden, Germany), and directly sequenced using a BigDye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems, Foster City, CA), the same primers as used for PCR or occasionally inner primers, and ABI Prism 310 Genetic Analyzer (Applied Biosystems), according to manufacturer’s instructions. Direct sequencing of all PCR fragments revealed a T-to-C transition at nucleotide 283 (283T ! C) in exon 1 (Fig. 2A), resulting in the substitution of proline for leucine at codon 90 (L90P). A PCR fragment in exon 1 was amplified using primers (50 -AGCTCAATGACCGCTTTGCC-30 and 50 GTAGACGTCTGCCAGCTTGG-30 ), and was digested with the restriction endonuclease Msp I (New England Biolabs, Inc., Beverly, MA). Restriction digests were analyzed in 3% NuSieve agarose (BMA, Rockland, ME). In the presence of the mutation, the PCR product (125 bp) was cleaved into two segments (79 and 46 bp) by Msp I, whereas the wild-type PCR product was not cleaved (Fig. 2B). This PCR-restriction fragment length polymorphism (RFLP) confirmed the nucleotide change, and showed that the patient was heterozygous for 283T ! C and that
Fig. 2. (A) Sequencing data around the site of the mutation, 283T ! C. (B) Schematic representation of the mutant and wild-type PCR fragments digested with Msp I. Numbers indicate length (in bp) of the segments cleaved by Msp I. (C) Pedigree of the patient and an electrophoresis on 3% NuSieve agarose of PCR products digested with Msp I.
neither parent had this mutation (Fig. 2C). Furthermore, this mutation was not detected in 51 control DNAs (102 alleles) by PCR-RFLP. At age 5 years, she had prolonged febrile convulsions with subsequent mild regression of motor function. The latest examination at 5 years 10 months of age revealed truncal hypotonia and hyperreflexia of the lower limbs. She walked with a wide-based, stooped posture. 3. Discussion Recently, mutations in the GFAP gene have been reported in patients with Alexander disease, and they are becoming accepted as a reliable molecular marker. Together with typical MRI findings [8] and normal metabolic laboratory examination, the identification of the de novo L90P mutation, which was not observed in 102 normal control alleles, strongly supported the diagnosis of Alexander disease. All intermediate filament proteins, including GFAP, consist of a central alpha-helical rod domain flanked by randomly coiled head and tail regions. The central rod is further subdivided into four segments, 1A, 1B, 2A and 2B. The L90P mutation is located in rod domain 1A. The leucine at position 90 is conserved in rat, mouse, bovine and human
208
Y. Suzuki et al. / Brain & Development 26 (2004) 206–208
GFAP, and in other human intermediate filaments, vimentin, desmin and keratin 5 [9]. MRI and CT scans are highly suggestive of the disease, with the CT scans showing low density in the frontal white matter that gradually extends posteriorly into the parietal region [10]. Our patient showed scattered high density areas in the basal ganglia on CT images at a very early stage of the disease, even when an abnormal low density was not evident in the frontal white matter. Basal ganglia calcification was reported in an adult case of Alexander disease on CT scanning and at necropsy. The authors speculated that the calcification might be related to the prolonged duration of the illness [11]. But this is not true of the present case. Early MR findings include signal abnormality of the basal ganglia, probably due to an extreme density of Rosenthal fibers [8]. It is possible that numerous Rosenthal fibers are responsible for signal abnormality of the basal ganglia on CT images. The clinical phenotype of Alexander disease has been divided into three types based on the age of onset: infantile, juvenile and adult onset. Typical features of infantile onset include developmental delay, macrocephaly and seizures. Juvenile onset Alexander disease has been described with normal head circumference, predominant bulbar involvement and a slowly progressive course [10]. These subgroups, however, are not strictly age-dependent and the clinical picture is highly variable among patients [12]. This patient developed seizures in infancy, but with some similar features to juvenile form, that is, no apparent macrocephaly and a slowly progressive clinical course. A recent review of the Alexander disease-associated mutations in the GFAP gene revealed that the same mutation could cause both infantile and juvenile phenotypes [6]. Rodriguez et al., however, reported that the phenotype of the R79 mutations appeared much less severe than that of the R239 mutation [3]. Our patient’s novel mutation, L90P, is located close to the R88C mutation, which was identified in juvenile and asymptomatic cases and the V87G mutation, identified in adult cases [6]. Therefore, it is possible that mutations in this region may contribute to the mild phenotype of this disease.
Acknowledgements We thank Dr Yasuyuki Suzuki (Gifu University School of Medicine) for measuring very long-chain fatty acids and phytanic acid and Dr Koji Inui (Osaka University Graduate School of Medicine) for galactocerebrosidase.
References [1] Aoki Y, Haginoya K, Munakata M, Yokoyama H, Nishio T, Togashi N, et al. A novel mutation in glial fibrillary acidic protein gene in a patient with Alexander disease. Neurosci Lett 2001;312:71 –4. [2] Brenner M, Johnson AB, Boespflug-Tanguy O, Rodriguez D, Goldman JE, Messing A. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat Genet 2001;27:117 –20. [3] Rodriguez D, Gauthier F, Bertini E, Bugiani M, Brenner M, N’guyen S, et al. Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation. Am J Hum Genet 2001;69:1134 –40. [4] Shiroma N, Kanazawa N, Izumi M, Sugai K, Fukumizu M, Sasaki M, et al. Diagnosis of Alexander disease in a Japanese patient by molecular genetic analysis. J Hum Genet 2001;46:579–82. [5] Gorospe JR, Naidu S, Johnson AB, Puri V, Raymond GV, Jenkins SD, et al. Molecular findings in symptomatic and pre-symptomatic Alexander disease patients. Neurology 2002;58:1494– 500. [6] Li R, Messing A, Goldman JE, Brenner M. GFAP mutations in Alexander disease. Int J Dev Neurosci 2002;20:259–68. [7] Shiroma N, Kanazawa N, Kato Z, Shimozawa N, Imamura A, Ito M, et al. Molecular genetic study in Japanese patients with Alexander disease: a novel mutation, R79L. Brain Dev 2003;25:116 –21. [8] van der Knaap MS, Naidu S, Breiter SN, Blaser S, Stroink H, Springer S, et al. Alexander disease: diagnosis with MR imaging. Am J Neuroradiol 2001;22:541–52. [9] Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem 1994;63:345–82. [10] Kaye EM. Disorders primarily affecting white matter. In: Swaiman KF, Ashwal S, editors. Pediatric neurology principles and practice. Missouri: Mosby; 1999. p. 855–6. [11] Walls TJ, Jones RA, Cartlidge NEF, Saunders M. Alexander’s disease with Rosenthal fibre formation in an adult. J Neurol Neurosurg Psychiatry 1984;47:399–403. [12] Springer S, Erlewein R, Naegele T, Becker I, Auer D, Grodd W, et al. Alexander disease—classification revisited and isolation of a neonatal form. Neuropediatrics 2000;31:86–92.
Case report
A case of infantile Alexander disease with a milder phenotype and a novel GFAP mutation, L90P Yoshiko Suzukia,*, Naomi Kanazawab, Junko Takenakaa, Akihisa Okumuraa, Tamiko Negoroa, Seiichi Tsujinob a
Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan b Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, 4-1-1 Ogawahigashi-Cho, Kodaira, Tokyo 187-8502, Japan Received 9 January 2003; received in revised form 1 July 2003; accepted 1 July 2003
Abstract Alexander disease is a leukoencephalopathy that usually presents during infancy with developmental delay, macrocephaly and seizures. Several sequencing analyses have identified mutations in the gene encoding glial fibrillary acidic protein (GFAP) of patients with Alexander disease. We described a girl who developed seizures in infancy with atypical CT findings and in whom a novel heterozygous mutation, L90P (283T ! C), was detected in exon 1 of the GFAP gene. The neurological deterioration was mild and appeared relatively late for infantile onset. q 2003 Elsevier B.V. All rights reserved. Keywords: Alexander disease; Mutation; GFAP; Infantile; Computed tomography
1. Introduction Alexander disease is an uncommon degenerative disorder that is associated with myelin loss in a rostrocaudal gradient. The pathologic hallmark of the disorder is the presence of Rosenthal fibers located within astrocytes. Although brain biopsy or postmortem examination has been the only confirmatory test for a diagnosis, glial fibrillary acidic protein (GFAP) mutations have now been found in several cases of Alexander disease [1 – 7]. These mutations found in child patients are considered to be de novo and dominant. We describe a girl who had white matter abnormalities with frontal preponderance and a novel mutation within the GFAP gene. The patient had developed seizures in infancy but developmental deterioration was mild and appeared relatively late for infantile onset.
2. Case report The patient was a 5-year-old girl born as the first child of Japanese non-consanguineous parents. Her mother had *
Corresponding author. Tel.: þ81-52-744-2294; fax: þ 81-52-744-2974. E-mail address: [email protected] (Y. Suzuki).
0387-7604/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0387-7604(03)00132-3
a history of systemic lupus erythematosus. The rest of the family were healthy. Her prenatal and perinatal history were unremarkable. At the age of 5 months, she developed a cluster of complex partial seizures, controlled by phenobarbitone. Macrocephaly was not present. Brain CT at 5 months of age revealed scattered high density areas in the basal ganglia (Fig. 1A), which remained the same on the latest CT scan at 4 years of age. Normal development continued until 7 months of age, motor development then slowed down, such that she could only walk with support at 12 months and without only at 21. She was referred to our hospital at 4 years 6 months of age. Her height was 90 cm (below 3rd percentile) and head circumference 50.8 cm (75th percentile). She walked clumsily and could speak simple sentences. Deep tendon reflexes and muscle tone were normal. MR imaging showed predominantly frontal involvement of the white matter with low signal intensity on T1-weighted images and high signal intensity on T2-weighted. The subcortical U-fibers were relatively spared (Fig. 1B). Lesions were also observed in the putamen, globus pallidus, caudate nucleus, medulla oblongata and dorsal pons. Proton MR spectroscopy of the frontal white matter showed reduced N-acetylaspartic acid/ creatine, increased choline/creatine ratios and accumulation of lactate, though the accumulation of lactate was not
Y. Suzuki et al. / Brain & Development 26 (2004) 206–208
207
Fig. 1. (A) Axial CT image shows scattered high-density areas in the bilateral basal ganglia at 5 months of age. Abnormal low density is not evident in the frontal deep white matter. (B) Axial T2-weighted MR image shows high signal intensity in the bilateral frontal white matter, putamen, globus pallidus and caudate nucleus.
recognized at 5 years 9 months of age. Except for mildly elevated CSF levels of lactate and pyruvate (lactate 26 mg/dl, pyruvate 1.2 mg/dl), laboratory values were normal including plasma amino acids, urine organic acids, serum levels of lactate, pyruvate, very long-chain fatty acids, and phytanic acid, and the activities of arylsulfatase A, beta-galactosidase, beta-hexosaminidase A and galactocerebrosidase. Ophthalmologic findings were normal. EEG showed high voltage slow waves in frontal areas. Brainstem auditory responses demonstrated a prolongation in I –V interval and delay in peak latencies of waves III, IV and V. After informed consent was obtained from the patient’s parents, genomic DNA was isolated from peripheral leukocytes, and amplified by PCR as previously described [4]. The PCR products were electrophoresed through 1.5% agarose gels, purified by GeneElute Agarose Spin Column (Sigma, St. Louis, MO) or QIAquick PCR Purification kit (Qiagen, Hilden, Germany), and directly sequenced using a BigDye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems, Foster City, CA), the same primers as used for PCR or occasionally inner primers, and ABI Prism 310 Genetic Analyzer (Applied Biosystems), according to manufacturer’s instructions. Direct sequencing of all PCR fragments revealed a T-to-C transition at nucleotide 283 (283T ! C) in exon 1 (Fig. 2A), resulting in the substitution of proline for leucine at codon 90 (L90P). A PCR fragment in exon 1 was amplified using primers (50 -AGCTCAATGACCGCTTTGCC-30 and 50 GTAGACGTCTGCCAGCTTGG-30 ), and was digested with the restriction endonuclease Msp I (New England Biolabs, Inc., Beverly, MA). Restriction digests were analyzed in 3% NuSieve agarose (BMA, Rockland, ME). In the presence of the mutation, the PCR product (125 bp) was cleaved into two segments (79 and 46 bp) by Msp I, whereas the wild-type PCR product was not cleaved (Fig. 2B). This PCR-restriction fragment length polymorphism (RFLP) confirmed the nucleotide change, and showed that the patient was heterozygous for 283T ! C and that
Fig. 2. (A) Sequencing data around the site of the mutation, 283T ! C. (B) Schematic representation of the mutant and wild-type PCR fragments digested with Msp I. Numbers indicate length (in bp) of the segments cleaved by Msp I. (C) Pedigree of the patient and an electrophoresis on 3% NuSieve agarose of PCR products digested with Msp I.
neither parent had this mutation (Fig. 2C). Furthermore, this mutation was not detected in 51 control DNAs (102 alleles) by PCR-RFLP. At age 5 years, she had prolonged febrile convulsions with subsequent mild regression of motor function. The latest examination at 5 years 10 months of age revealed truncal hypotonia and hyperreflexia of the lower limbs. She walked with a wide-based, stooped posture. 3. Discussion Recently, mutations in the GFAP gene have been reported in patients with Alexander disease, and they are becoming accepted as a reliable molecular marker. Together with typical MRI findings [8] and normal metabolic laboratory examination, the identification of the de novo L90P mutation, which was not observed in 102 normal control alleles, strongly supported the diagnosis of Alexander disease. All intermediate filament proteins, including GFAP, consist of a central alpha-helical rod domain flanked by randomly coiled head and tail regions. The central rod is further subdivided into four segments, 1A, 1B, 2A and 2B. The L90P mutation is located in rod domain 1A. The leucine at position 90 is conserved in rat, mouse, bovine and human
208
Y. Suzuki et al. / Brain & Development 26 (2004) 206–208
GFAP, and in other human intermediate filaments, vimentin, desmin and keratin 5 [9]. MRI and CT scans are highly suggestive of the disease, with the CT scans showing low density in the frontal white matter that gradually extends posteriorly into the parietal region [10]. Our patient showed scattered high density areas in the basal ganglia on CT images at a very early stage of the disease, even when an abnormal low density was not evident in the frontal white matter. Basal ganglia calcification was reported in an adult case of Alexander disease on CT scanning and at necropsy. The authors speculated that the calcification might be related to the prolonged duration of the illness [11]. But this is not true of the present case. Early MR findings include signal abnormality of the basal ganglia, probably due to an extreme density of Rosenthal fibers [8]. It is possible that numerous Rosenthal fibers are responsible for signal abnormality of the basal ganglia on CT images. The clinical phenotype of Alexander disease has been divided into three types based on the age of onset: infantile, juvenile and adult onset. Typical features of infantile onset include developmental delay, macrocephaly and seizures. Juvenile onset Alexander disease has been described with normal head circumference, predominant bulbar involvement and a slowly progressive course [10]. These subgroups, however, are not strictly age-dependent and the clinical picture is highly variable among patients [12]. This patient developed seizures in infancy, but with some similar features to juvenile form, that is, no apparent macrocephaly and a slowly progressive clinical course. A recent review of the Alexander disease-associated mutations in the GFAP gene revealed that the same mutation could cause both infantile and juvenile phenotypes [6]. Rodriguez et al., however, reported that the phenotype of the R79 mutations appeared much less severe than that of the R239 mutation [3]. Our patient’s novel mutation, L90P, is located close to the R88C mutation, which was identified in juvenile and asymptomatic cases and the V87G mutation, identified in adult cases [6]. Therefore, it is possible that mutations in this region may contribute to the mild phenotype of this disease.
Acknowledgements We thank Dr Yasuyuki Suzuki (Gifu University School of Medicine) for measuring very long-chain fatty acids and phytanic acid and Dr Koji Inui (Osaka University Graduate School of Medicine) for galactocerebrosidase.
References [1] Aoki Y, Haginoya K, Munakata M, Yokoyama H, Nishio T, Togashi N, et al. A novel mutation in glial fibrillary acidic protein gene in a patient with Alexander disease. Neurosci Lett 2001;312:71 –4. [2] Brenner M, Johnson AB, Boespflug-Tanguy O, Rodriguez D, Goldman JE, Messing A. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat Genet 2001;27:117 –20. [3] Rodriguez D, Gauthier F, Bertini E, Bugiani M, Brenner M, N’guyen S, et al. Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation. Am J Hum Genet 2001;69:1134 –40. [4] Shiroma N, Kanazawa N, Izumi M, Sugai K, Fukumizu M, Sasaki M, et al. Diagnosis of Alexander disease in a Japanese patient by molecular genetic analysis. J Hum Genet 2001;46:579–82. [5] Gorospe JR, Naidu S, Johnson AB, Puri V, Raymond GV, Jenkins SD, et al. Molecular findings in symptomatic and pre-symptomatic Alexander disease patients. Neurology 2002;58:1494– 500. [6] Li R, Messing A, Goldman JE, Brenner M. GFAP mutations in Alexander disease. Int J Dev Neurosci 2002;20:259–68. [7] Shiroma N, Kanazawa N, Kato Z, Shimozawa N, Imamura A, Ito M, et al. Molecular genetic study in Japanese patients with Alexander disease: a novel mutation, R79L. Brain Dev 2003;25:116 –21. [8] van der Knaap MS, Naidu S, Breiter SN, Blaser S, Stroink H, Springer S, et al. Alexander disease: diagnosis with MR imaging. Am J Neuroradiol 2001;22:541–52. [9] Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem 1994;63:345–82. [10] Kaye EM. Disorders primarily affecting white matter. In: Swaiman KF, Ashwal S, editors. Pediatric neurology principles and practice. Missouri: Mosby; 1999. p. 855–6. [11] Walls TJ, Jones RA, Cartlidge NEF, Saunders M. Alexander’s disease with Rosenthal fibre formation in an adult. J Neurol Neurosurg Psychiatry 1984;47:399–403. [12] Springer S, Erlewein R, Naegele T, Becker I, Auer D, Grodd W, et al. Alexander disease—classification revisited and isolation of a neonatal form. Neuropediatrics 2000;31:86–92.