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Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
Official Journal of the European Paediatric Neurology Society
Original article
Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder Daniella Nishri a,b,c, Simon Edvardson d, Dorit Lev a,e, Esther Leshinsky-Silver a,e,f, Liat Ben-Sira g, Marco Henneke h, Tally Lerman-Sagie a,c, Lubov Blumkin a,c,* a
Metabolic-Neurogenetic Clinic, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel b Child Development Center, Central District, Maccabi Health Services, Tel Aviv, Israel c Pediatric Neurology Unit, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Israel d Pediatric Neurology Unit, Hadassah Medical Center, Jerusalem, affiliated to Hebrew University, Jerusalem, Israel e Institute of Medical Genetics, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Israel f Molecular Genetics Laboratory, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel g Pediatric Radiology Unit, Tel Aviv Medical Center, Tel Aviv, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel h Department of Pediatrics and Adolescent Medicine, Division of Pediatric Neurology, University Medical Center Go¨ttingen, Germany
article info
abstract
Article history:
Introduction: There are many similarities, both clinical and radiological, between mito-
Received 22 September 2013
chondrial leukoencephalopathies and Alexander disease, an astrogliopathy. Clinically,
Received in revised form
both can manifest with a myriad of symptoms and signs, arising from the neonatal period
25 March 2014
to adulthood. Radiologically, both can demonstrate white matter changes, signal abnor-
Accepted 28 March 2014
malities of basal ganglia or thalami, brainstem abnormalities and contrast enhancement of white matter structures. Magnetic resonance spectroscopy may reveal elevation of lactate
Keywords:
in the abnormal white matter in Alexander disease making the distinction even more
Leukoencephalopathy
challenging.
Alexander disease
Patient and Methods: We present a child who was considered to have an infantile onset
Mitochondrial disorders
mitochondrial disorder due to a combination of neurological symptoms and signs (devel-
Lactic acid
opmental regression, failure to thrive, episodic deterioration, abnormal eye movements, pyramidal and cerebellar signs), urinary excretion of 3-methyl-glutaconic acid and imaging findings (extensive white matter changes and cerebellar atrophy) with a normal head circumference. Whole exome sequence analysis was performed.
* Corresponding author. Pediatric Neurology Unit, Wolfson Medical Center, Holon, Israel. Tel.: þ972 3 5028458. E-mail address: [email protected] (L. Blumkin). http://dx.doi.org/10.1016/j.ejpn.2014.03.009 1090-3798/ª 2014 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
2
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
Results: The child was found to harbor the R416W mutation, one of the most prevalent mutations in the glial fibrillary acidic protein (GFAP) gene that causes Alexander disease. Conclusions: Alexander disease should be considered in the differential diagnosis of infantile leukoencephalopathy, even when no macrocephaly is present. Next generation sequencing is a useful aid in unraveling the molecular etiology of leukoencephalopathies. ª 2014 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.
1.
Introduction
Alexander disease is an astrogliopathy caused by dominant mutations in the glial fibrillary acidic protein gene (GFAP), usually characterized on magnetic resonance imaging (MRI) by leukodystrophy with a frontal predominant pattern.1 Three forms of Alexander disease are recognized, based on age of onset and clinical features.2 Type I (cerebral) is characterized by infantile onset (birth to 2 years) and features macrocephaly, developmental delay, encephalopathy, seizures, failure to thrive, paroxysmal deterioration and typical MRI findings, mainly superior frontal white matter changes. Type II (bulbospinal) is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, palatal myoclonus, weakness, ataxia, bulbar or pseudo-bulbar symptoms, with preserved motor and cognitive functions and a milder progression than type I. The MRI features in this type are considered atypical and include signal abnormalities and atrophy in the medulla oblongata and upper cervical spinal cord. Type III is an intermediate form which has the characteristics of both.1,3 We present an atypical case that was suspected as having a mitochondrial disorder (Leigh-like syndrome) due to a fluctuating deterioration since infancy following infections, no megalencephaly, with subtle signal changes in both basal ganglia and brainstem on the initial MRI and elevated excretion of 3-methylglutaconic acid. Exome sequencing revealed a heterozygote mutation in the GFAP gene consistent with Alexander syndrome.
2.
Materials and methods
Case study: The patient is a product of a spontaneous twin pregnancy. She was born at term by caesarian section due to breech presentation; birth weight and Apgar scores were normal. The family history is noted for hypertrophic cardiomyopathy and several cases of auto-immune thyroid disorders on the maternal side. The patient’s older brother had hypotonia and gross motor delay and is now clumsy; her non-identical gender-matched twin is healthy. Early development was normal until the age of 10 months; then she underwent a febrile illness followed by protracted vomiting. She became lethargic, and abnormal eye movements were noted (alternating esotropia and nystagmus). Gradually, she lost all acquired developmental milestones and
failed to thrive. A metabolic workup (ammonia, blood gases, lactate, pyruvic acid, carnitine, acyl carnitine profile, amino acids, very-long-chain fatty acids) was normal except for urinary organic acids that showed mild excretion of ketones, dicarboxylic acids, Krebs cycle intermediates and 3methylglutaconic acid (suggestive of a mitochondrial disorder). A brain MRI showed hyper-intense T2 signal in the periaqueductal area and mild signal changes in the caudate nucleus bilaterally (Fig. 1). She received cyproheptadine (for its appetizing affect) and developmental therapies, and gradually regained weight and developmental milestones. When examined for the first time in our metabolicneurogenetic clinic at the age of 2 years 5 months, she had alternating strabismus, pyramidal signs (brisk reflexes, clonus and extensor plantar responses) and a gait abnormality. She showed gross motor and expressive speech delay; formal developmental assessment using the Griffiths developmental scales performed at the age of 20 months found a developmental quotient of 77. An additional MRI performed at the age of 2 years and 7 months demonstrated mild hyper-intense signal changes in the caudate head, diffuse white matter signal changes in the frontal area bilaterally (Fig. 2 top panel) and mild atrophy of the superior vermis (Fig. 3 top panel). She continued follow up at our clinic and in the next 1.5 years seemed to have a static clinical condition with residual pyramidal and cerebellar signs. Head circumference growth was consistently on the 25th percentile. At the age of 4 years, she suffered from bacterial pneumonia and a few weeks later, a nonspecific viral illness. During these two febrile illnesses, she had frequent emesis, claimed to be weak and refused to walk. When made to stand, she appeared unstable. When examined at our clinic about a month after the onset of this episode, her neurological examination demonstrated pyramidal signs (that were present before), marked ataxia, tremor and dysmetria, oral dystonia and mild ptosis. A mitochondrial “cocktail” consisting of carnitine, antioxidants and coenzyme Q10 was recommended. She began to recuperate about a week after her visit; she regained walking and the ptosis disappeared. MRI performed at the age of 4 years 2 months (1 month after the onset) demonstrated extensive signal changes in the frontal white matter with a discrete lesion in the left anterior periventricular region, showing a restrictive pattern on diffusion-weight imaging (DWI) and enhancement with gadolinium. Also noted were diffuse, symmetric, bilateral signal changes in the caudate heads, brainstem and white matter including the peri-aqueductal region and in the cerebellar dentate nuclei, with worsening cerebellar atrophy.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
3
against muscle biopsy. Two months later, she underwent another febrile illness during which she refused to walk. Neurological examination demonstrated horizontal and vertical nystagmus, mild ptosis, dysmetria and worsening ataxia. The antioxidant doses were elevated and again she recuperated clinically. MRI performed at the age of 4 years and 9 months demonstrated more pronounced similar bilateral signal changes in the frontal white matter and the caudate heads, with progressive supra- and infra-tentorial atrophy and signal changes in the brainstem and cerebellar peduncles areas (Fig. 4). MR spectroscopy of the left anterior periventricular region showed an increased choline peak and suspected lactate peak (Fig. 5). Currently her main symptoms are frequent falls and occasionally tremor. Neurological examination at the age of 5 years 9 months demonstrated mild cerebellar signs (horizontal and vertical nystagmus, dysmetria, kinetic tremor and ataxia) and pyramidal signs. Developmental assessment using the Gesell developmental schedule found a developmental quotient of 80. She had significant gross motor delay but the other sub-scales scores were in the low-normal values. Her cognitive development was considered normal. She never had seizures. MRI performed at the age of 5 years 9 months was basically without change from the one performed a year before (Fig. 2 middle and bottom panels and Fig. 3 bottom panel).
3.
Methods
Whole exome sequencing was performed on DNA from the patient using SureSelect Human All Exon v.2 Kit (Agilent Technologies, Santa Clara, CA) on HiSeq2000 (Illumina, San Diego, CA).4
4.
Results
The child was found to harbor a heterozygous mutation (R416W), in the glial fibrillary acidic protein (GFAP) gene that causes Alexander disease.
5. Fig. 1 e Brain MRI at the age of 11 months: Top: axial image showing subtle hyper-intense T2 signal in the periaqueductal area (arrow); Bottom: axial imaging demonstrating minimal T2 signal abnormalities in the caudate head bilaterally (arrows) (almost normal).
Laboratory tests demonstrated mildly elevated inflammation markers and normal liver functions, ammonia, blood gases, carnitine levels and urine organic acid profile. Lactate levels were marginally elevated on one occurrence (2.56 mmol/l, N < 2.4), and normal on two others; autoimmune markers (antinuclear antigen, anticardiolipin, double-stranded DNA, anti B2 glycoprotein, complement and immunoglobulins) levels were normal. The mitochondrial marker FGF21 was tested and found to be within normal limits. The parents opt
Discussion
There are many similarities, both clinical and radiological, between mitochondrial leukoencephalopathies and Alexander disease. The misdiagnosis of a primary mitochondrial disorder in a patient with Alexander disease has previously been reported in two patients.5,6 All the patients, including ours had elevated blood lactic acid, Krebs cycle intermediates in urinary organic acids, brainstem and white matter changes on brain MRI and a normal head circumference. In addition, one of the patients showed pathologic findings on muscle biopsy characteristic of a mitochondrial disorder.6 The diagnosis of Alexander disease was only made by neuropathology in the patient reported by Gingold et al.5 A new rare mutation was discovered in the second case.6 Clinically, both disorders can manifest with a myriad of neurological symptoms and signs arising from the neonatal period to adulthood and can demonstrate paroxysmal
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
Fig. 2 e Top: MRI at the age of 2 years 7 months: axial imaging showing bilateral and symmetric T2 signal changes in the frontal white matter (arrows); middle: MRI at the age of 5 years 9 months showing progression of frontal white matter T2 signal changes; bottom: MRI at the age of 5 years 9 months: axial fluid attenuated inversion recovery (FLAIR) image at the same level accentuating the signal changes in the frontal white matter and caudate heads (arrows).
deterioration. Radiologically, central nervous system abnormalities in mitochondrial disorders include focal or diffuse white matter lesions, focal or diffuse atrophy (cerebral and/or cerebellar), stroke-like lesions, basal ganglia necrosis, calcifications, cysts, elevated lactate and various vascular changes.7 Contrast enhancement can also be a feature of mitochondrial leukoencephalopathies.8 Some lesions may remain unchanged for years, some may show contiguous spread and progression, and some may disappear spontaneously or in response to medication.7 Van der Knaap et al.9 proposed specific MRI criteria for the diagnosis of Alexander disease (4 of 5 are needed): (1) extensive cerebral white matter changes with frontal predominance; (2) a periventricular rim of low signal on T2, high signal on T1; (3) signal abnormalities of basal ganglia or thalami; (4) brainstem abnormalities; and (5) contrast enhancement of
one or more of the gray and white matter structures. Patients having less than 4 criteria are defined as having an atypical presentation.9 The association of signal abnormalities in basal ganglia and brainstem with a leukoencephalopathy is characteristic of both disorders. We considered the child to have an infantile onset mitochondrial disorder due to multifocal central nervous system involvement, urinary excretion of 3-methyl-glutaconic acid and Krebs cycle intermediates, and the association of white matter, cerebellar, and basal ganglia lesions with contrast enhancement and an elevated lactate peak on brain spectroscopy. Alexander disease was not considered since the patient presented in infancy and did not demonstrate macrocephaly or seizures that are classic features of type I. She developed ptosis, which is considered an uncommon feature of Alexander disease.10 Furthermore the elevated urinary 3-
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
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few years. This lack of frontal white matter abnormalities in young infants with Alexander disease has been reported and the signal abnormality in the peri-aqueductal region was included by van der Knaap et al. as an early MRI finding in
Fig. 3 e Vermis atrophy with time as demonstrated on Sagittal T1 weighted images. Top: MRI at the age of 2 years 7 months (arrow); Bottom: MRI at the age of 5 years 9 months. Also note slight atrophy of brainstem (lower arrow).
methylglutaconic acid is typical of a mitochondrial disorder.11 The progressive course combined with episodes of clinical and neuroradiological worsening after infections coexisting with temporary improvement following treatment with a mitochondrial cocktail further suggested a mitochondrial disorder. The signal abnormalities on MRI were initially noted only in the peri-aqueductal region; the frontal white matter changes typical of Alexander disease appeared only after a
Fig. 4 e MRI at the age of 4 years 9 months: Top: axial FLAIR image showing symmetric hyper intense signal in the brainstem (arrow); bottom: axial FLAIR image showing signal changes in the cerebellar peduncles (top arrow) and dentate nucleus (bottom arrow).
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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Fig. 5 e MR Spectroscopy with short TE of 35 in the periventricular left white matter at the age of 4 years 9 months: suspected lactate double peak at 1.3 ppm (star) and elevated choline (cho) peak.
patients with Alexander disease.9 Additionally, there have been several cases showing elevated lactic acid in both blood2,5,6,12 and the abnormal white matter demonstrated by MR spectroscopy.13 In retrospect, our patient complies with MRI features of Alexander disease. Prust et al. correlated the genotype-phenotype of 215 Alexander disease patients.1 The authors defined a 2-class model based on age of onset. Patients with type I Alexander disease presented with macrocephaly, failure to thrive, encephalopathy, seizures, motor and cognitive delay, and paroxysmal deterioration within the first 4 years of life. Patients with type II presented with bulbar symptoms, autonomic dysfunction, ataxia, dysarthria, dysphonia, ocular movement abnormalities, and palatal myoclonus across the lifespan.1 The radiologic features in type I patients were the typical MRI features defined by van der Knaap et al.9 Type II patients had atypical MRI features characterized most commonly by a predominance of posterior fossa white matter abnormalities, brainstem, cerebellar, and spinal cord atrophy.1 Despite the high quality fit, this model did not allow for type classification in all the cases: the authors confirmed genotype-phenotype correlation for the R79 and R239 mutations but failed to find typical clinical features associated with R88 or R416 mutations.1 Our patient was found to harbor the R416W, a c-terminus mutation in the GFAP gene. This mutation, the 4th most common, was found to be causative in 5.6% of cases.1 The R416W mutation is located in the tail domain of GFAP and may be a hot spot mutation that presents in multiple forms of Alexander disease.3 Der Perng et al.14 demonstrated that the R416W mutation significantly perturbs in vitro filament assembly. Consistent with the heterozygosity of the mutation, this effect was found to be dominant over wild-type GFAP in coassembly experiments. The R416W mutation changes the assembly process in a way that encourages aberrant filamentefilament interactions that then lead to
protein aggregation and chaperone sequestration.14 Yoshida et al.3 found that most of the astrocytes with R416W gene mutation were capable of cell division. The authors speculated that the alteration of R416W GFAP function in astrocytes may depend on other elements interacting with GFAP. This may support the notion that the heterogeneous phenotypes caused by GFAP mutations in the tail domain are influenced by other genetic or environmental factors. The relationship between GFAP mutations and aberrant mitochondrial function seems too frequent to be incidental. The mitochondria are known to take a pivotal role in the pathophysiology of various neuro-degenerative disorders, either directly, via proteins participating in known mitochondrial biochemical pathways and structure, or indirectly, via proteins that are not necessarily targeted to mitochondria, but affect their function secondarily. Impaired sub-cellular mitochondrial trafficking is one the mechanisms accounting for neurodegenerative disorders. The mitochondria are transported on cytoskeletal elements (microtubules and actin cables) often in association with intermediate filaments.15 Hence, disruption of the intermediate filaments’ frame may cause mitochondrial mal-distribution and disruption of the normal energetic function of the cell, causing accumulation of lactic acid. The increased urinary 3-methylglutaconic acid and Krebs cycle intermediates as well as elevated blood lactic acid found in our patient, may reflect a secondary mitochondrial dysfunction or be a marker of another, unknown mutation in a mitochondrial-related gene, modifying the overall phenotype. Most infants with Alexander disease develop a rapidly increasing head circumference; however, this feature is not found in all cases.2,6 The explanation for a normal head circumference in patients with Alexander disease and prominent mitochondrial features is not clear. It has been suggested that the acquired macrocephaly, seen in Alexander disease patients, results from a proliferation of atypical astrocytes.6 It has been reported that the astrocytes in Alexander disease may demonstrate oxidative stress and ultrastructural mitochondrial abnormalities.16 It is possible that disruption of the normal energetic equilibrium of astrocytes in certain cases causes a decrease in their proliferation and early death thereby preventing rapid acceleration of head growth. Why certain patients with Alexander disease manifest overt mitochondrial dysfunction is not clear and requires further research.
6.
Conclusions
Alexander disease should be considered in the differential diagnosis of an infant presenting in the first year of life with a clinical picture suggesting a mitochondrial disorder, even without macrocephaly and when typical white matter changes are not yet present. Screening for the four most common mutations in the GFAP gene is warranted in these cases. When they are negative, next generation sequencing is a useful tool in unraveling the molecular etiology of these rare genetic neurodegenerative disorders.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
Acknowledgments This work has been supported by a trilateral grant from the German Research Foundation (Ga354/9-1).
references
1. Prust M, Wang J, Morizono H, et al. GFAP mutations, age at onset, and clinical subtypes in Alexander disease. Neurology 2011;77:1287e94. 2. Gordon N. Alexander disease. Eur J Paediatr Neurol 2003;7:395e9. 3. Yoshida T, Nakagawa M. Clinical aspects and pathology of Alexander disease, and morphological and functional alteration of astrocytes induced by GFAP mutation. Neuropathology 2012;32:440e6. 4. Edvardson S, Cinnamon Y, Jalas C, et al. Hereditary sensory autonomic neuropathy caused by a mutation in dystonin. Ann Neurol 2012;71:569e72. 5. Gingold MK, Bodensteiner JB, Schochet SS, Jaynes M. Alexander’s disease: unique presentation. J Child Neurol 1999;14:325e9. 6. Ca´ceres-Marzal C, Vaquerizo J, Gala´n E, Ferna´ndez S. Early mitochondrial dysfunction in an infant with Alexander disease. Pediatr Neurol 2006;35:293e6. 7. Finsterer J. Central nervous system imaging in mitochondrial disorders. Can J Neurol Sci 2009;36:143e53.
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8. Schiffmann R, van der Knaap MS. Invited article: an MRIbased approach to the diagnosis of white matter disorders. Neurology 2009;72:750e9. 9. van der Knaap MS, Naidu S, Breiter SN, et al. Alexander disease: diagnosis with MR imaging. Am J Neuroradiol 2001;22:541e52. 10. Pfeffer G, Abegg M, Vertinsky AT, et al. The ocular motor features of adult-onset alexander disease: a case and review of the literature. J Neuroophthalmol 2011;31:155e9. 11. Wortmann SB, Duran M, Anikster Y, et al. Inborn errors of metabolism with 3-methylglutaconic as discriminative feature: proper classification and nomenclature. J Inherit Metab Dis 2013;36:923e8. 12. Kang PB, Hunter JV, Kaye EM. Lactic acid elevation in extramitochondrial childhood neurodegenerative diseases. J Child Neurol 2001;16:657e60. 13. van der Voorn JP, Pouwels PJ, Salomons GS, et al. Unraveling pathology in juvenile Alexander disease: serial quantitative MR imaging and spectroscopy of white matter. Neuroradiology 2009;51:669e75. 14. Der Perng M, Su M, Wen SF, et al. The Alexander diseasecausing glial fibrillary acidic protein mutant, R416W, accumulates into Rosenthal fibers by a pathway that involves filament aggregation and the association of alpha B-crystallin and HSP27. Am J Hum Genet 2006;79:197e213. 15. Schon EA, Przedborski S. Mitochondria: the next (neurode) generation. Neuron 2011;70:1033e53. 16. Johnson AB, Brenner M. Alexander’s disease: clinical, pathologic and genetic features. J Child Neurol 2003;18:625e32.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
Official Journal of the European Paediatric Neurology Society
Original article
Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder Daniella Nishri a,b,c, Simon Edvardson d, Dorit Lev a,e, Esther Leshinsky-Silver a,e,f, Liat Ben-Sira g, Marco Henneke h, Tally Lerman-Sagie a,c, Lubov Blumkin a,c,* a
Metabolic-Neurogenetic Clinic, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel b Child Development Center, Central District, Maccabi Health Services, Tel Aviv, Israel c Pediatric Neurology Unit, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Israel d Pediatric Neurology Unit, Hadassah Medical Center, Jerusalem, affiliated to Hebrew University, Jerusalem, Israel e Institute of Medical Genetics, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Israel f Molecular Genetics Laboratory, Wolfson Medical Center, Holon, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel g Pediatric Radiology Unit, Tel Aviv Medical Center, Tel Aviv, affiliated to Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel h Department of Pediatrics and Adolescent Medicine, Division of Pediatric Neurology, University Medical Center Go¨ttingen, Germany
article info
abstract
Article history:
Introduction: There are many similarities, both clinical and radiological, between mito-
Received 22 September 2013
chondrial leukoencephalopathies and Alexander disease, an astrogliopathy. Clinically,
Received in revised form
both can manifest with a myriad of symptoms and signs, arising from the neonatal period
25 March 2014
to adulthood. Radiologically, both can demonstrate white matter changes, signal abnor-
Accepted 28 March 2014
malities of basal ganglia or thalami, brainstem abnormalities and contrast enhancement of white matter structures. Magnetic resonance spectroscopy may reveal elevation of lactate
Keywords:
in the abnormal white matter in Alexander disease making the distinction even more
Leukoencephalopathy
challenging.
Alexander disease
Patient and Methods: We present a child who was considered to have an infantile onset
Mitochondrial disorders
mitochondrial disorder due to a combination of neurological symptoms and signs (devel-
Lactic acid
opmental regression, failure to thrive, episodic deterioration, abnormal eye movements, pyramidal and cerebellar signs), urinary excretion of 3-methyl-glutaconic acid and imaging findings (extensive white matter changes and cerebellar atrophy) with a normal head circumference. Whole exome sequence analysis was performed.
* Corresponding author. Pediatric Neurology Unit, Wolfson Medical Center, Holon, Israel. Tel.: þ972 3 5028458. E-mail address: [email protected] (L. Blumkin). http://dx.doi.org/10.1016/j.ejpn.2014.03.009 1090-3798/ª 2014 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
2
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 4 ) 1 e7
Results: The child was found to harbor the R416W mutation, one of the most prevalent mutations in the glial fibrillary acidic protein (GFAP) gene that causes Alexander disease. Conclusions: Alexander disease should be considered in the differential diagnosis of infantile leukoencephalopathy, even when no macrocephaly is present. Next generation sequencing is a useful aid in unraveling the molecular etiology of leukoencephalopathies. ª 2014 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.
1.
Introduction
Alexander disease is an astrogliopathy caused by dominant mutations in the glial fibrillary acidic protein gene (GFAP), usually characterized on magnetic resonance imaging (MRI) by leukodystrophy with a frontal predominant pattern.1 Three forms of Alexander disease are recognized, based on age of onset and clinical features.2 Type I (cerebral) is characterized by infantile onset (birth to 2 years) and features macrocephaly, developmental delay, encephalopathy, seizures, failure to thrive, paroxysmal deterioration and typical MRI findings, mainly superior frontal white matter changes. Type II (bulbospinal) is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, palatal myoclonus, weakness, ataxia, bulbar or pseudo-bulbar symptoms, with preserved motor and cognitive functions and a milder progression than type I. The MRI features in this type are considered atypical and include signal abnormalities and atrophy in the medulla oblongata and upper cervical spinal cord. Type III is an intermediate form which has the characteristics of both.1,3 We present an atypical case that was suspected as having a mitochondrial disorder (Leigh-like syndrome) due to a fluctuating deterioration since infancy following infections, no megalencephaly, with subtle signal changes in both basal ganglia and brainstem on the initial MRI and elevated excretion of 3-methylglutaconic acid. Exome sequencing revealed a heterozygote mutation in the GFAP gene consistent with Alexander syndrome.
2.
Materials and methods
Case study: The patient is a product of a spontaneous twin pregnancy. She was born at term by caesarian section due to breech presentation; birth weight and Apgar scores were normal. The family history is noted for hypertrophic cardiomyopathy and several cases of auto-immune thyroid disorders on the maternal side. The patient’s older brother had hypotonia and gross motor delay and is now clumsy; her non-identical gender-matched twin is healthy. Early development was normal until the age of 10 months; then she underwent a febrile illness followed by protracted vomiting. She became lethargic, and abnormal eye movements were noted (alternating esotropia and nystagmus). Gradually, she lost all acquired developmental milestones and
failed to thrive. A metabolic workup (ammonia, blood gases, lactate, pyruvic acid, carnitine, acyl carnitine profile, amino acids, very-long-chain fatty acids) was normal except for urinary organic acids that showed mild excretion of ketones, dicarboxylic acids, Krebs cycle intermediates and 3methylglutaconic acid (suggestive of a mitochondrial disorder). A brain MRI showed hyper-intense T2 signal in the periaqueductal area and mild signal changes in the caudate nucleus bilaterally (Fig. 1). She received cyproheptadine (for its appetizing affect) and developmental therapies, and gradually regained weight and developmental milestones. When examined for the first time in our metabolicneurogenetic clinic at the age of 2 years 5 months, she had alternating strabismus, pyramidal signs (brisk reflexes, clonus and extensor plantar responses) and a gait abnormality. She showed gross motor and expressive speech delay; formal developmental assessment using the Griffiths developmental scales performed at the age of 20 months found a developmental quotient of 77. An additional MRI performed at the age of 2 years and 7 months demonstrated mild hyper-intense signal changes in the caudate head, diffuse white matter signal changes in the frontal area bilaterally (Fig. 2 top panel) and mild atrophy of the superior vermis (Fig. 3 top panel). She continued follow up at our clinic and in the next 1.5 years seemed to have a static clinical condition with residual pyramidal and cerebellar signs. Head circumference growth was consistently on the 25th percentile. At the age of 4 years, she suffered from bacterial pneumonia and a few weeks later, a nonspecific viral illness. During these two febrile illnesses, she had frequent emesis, claimed to be weak and refused to walk. When made to stand, she appeared unstable. When examined at our clinic about a month after the onset of this episode, her neurological examination demonstrated pyramidal signs (that were present before), marked ataxia, tremor and dysmetria, oral dystonia and mild ptosis. A mitochondrial “cocktail” consisting of carnitine, antioxidants and coenzyme Q10 was recommended. She began to recuperate about a week after her visit; she regained walking and the ptosis disappeared. MRI performed at the age of 4 years 2 months (1 month after the onset) demonstrated extensive signal changes in the frontal white matter with a discrete lesion in the left anterior periventricular region, showing a restrictive pattern on diffusion-weight imaging (DWI) and enhancement with gadolinium. Also noted were diffuse, symmetric, bilateral signal changes in the caudate heads, brainstem and white matter including the peri-aqueductal region and in the cerebellar dentate nuclei, with worsening cerebellar atrophy.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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against muscle biopsy. Two months later, she underwent another febrile illness during which she refused to walk. Neurological examination demonstrated horizontal and vertical nystagmus, mild ptosis, dysmetria and worsening ataxia. The antioxidant doses were elevated and again she recuperated clinically. MRI performed at the age of 4 years and 9 months demonstrated more pronounced similar bilateral signal changes in the frontal white matter and the caudate heads, with progressive supra- and infra-tentorial atrophy and signal changes in the brainstem and cerebellar peduncles areas (Fig. 4). MR spectroscopy of the left anterior periventricular region showed an increased choline peak and suspected lactate peak (Fig. 5). Currently her main symptoms are frequent falls and occasionally tremor. Neurological examination at the age of 5 years 9 months demonstrated mild cerebellar signs (horizontal and vertical nystagmus, dysmetria, kinetic tremor and ataxia) and pyramidal signs. Developmental assessment using the Gesell developmental schedule found a developmental quotient of 80. She had significant gross motor delay but the other sub-scales scores were in the low-normal values. Her cognitive development was considered normal. She never had seizures. MRI performed at the age of 5 years 9 months was basically without change from the one performed a year before (Fig. 2 middle and bottom panels and Fig. 3 bottom panel).
3.
Methods
Whole exome sequencing was performed on DNA from the patient using SureSelect Human All Exon v.2 Kit (Agilent Technologies, Santa Clara, CA) on HiSeq2000 (Illumina, San Diego, CA).4
4.
Results
The child was found to harbor a heterozygous mutation (R416W), in the glial fibrillary acidic protein (GFAP) gene that causes Alexander disease.
5. Fig. 1 e Brain MRI at the age of 11 months: Top: axial image showing subtle hyper-intense T2 signal in the periaqueductal area (arrow); Bottom: axial imaging demonstrating minimal T2 signal abnormalities in the caudate head bilaterally (arrows) (almost normal).
Laboratory tests demonstrated mildly elevated inflammation markers and normal liver functions, ammonia, blood gases, carnitine levels and urine organic acid profile. Lactate levels were marginally elevated on one occurrence (2.56 mmol/l, N < 2.4), and normal on two others; autoimmune markers (antinuclear antigen, anticardiolipin, double-stranded DNA, anti B2 glycoprotein, complement and immunoglobulins) levels were normal. The mitochondrial marker FGF21 was tested and found to be within normal limits. The parents opt
Discussion
There are many similarities, both clinical and radiological, between mitochondrial leukoencephalopathies and Alexander disease. The misdiagnosis of a primary mitochondrial disorder in a patient with Alexander disease has previously been reported in two patients.5,6 All the patients, including ours had elevated blood lactic acid, Krebs cycle intermediates in urinary organic acids, brainstem and white matter changes on brain MRI and a normal head circumference. In addition, one of the patients showed pathologic findings on muscle biopsy characteristic of a mitochondrial disorder.6 The diagnosis of Alexander disease was only made by neuropathology in the patient reported by Gingold et al.5 A new rare mutation was discovered in the second case.6 Clinically, both disorders can manifest with a myriad of neurological symptoms and signs arising from the neonatal period to adulthood and can demonstrate paroxysmal
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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Fig. 2 e Top: MRI at the age of 2 years 7 months: axial imaging showing bilateral and symmetric T2 signal changes in the frontal white matter (arrows); middle: MRI at the age of 5 years 9 months showing progression of frontal white matter T2 signal changes; bottom: MRI at the age of 5 years 9 months: axial fluid attenuated inversion recovery (FLAIR) image at the same level accentuating the signal changes in the frontal white matter and caudate heads (arrows).
deterioration. Radiologically, central nervous system abnormalities in mitochondrial disorders include focal or diffuse white matter lesions, focal or diffuse atrophy (cerebral and/or cerebellar), stroke-like lesions, basal ganglia necrosis, calcifications, cysts, elevated lactate and various vascular changes.7 Contrast enhancement can also be a feature of mitochondrial leukoencephalopathies.8 Some lesions may remain unchanged for years, some may show contiguous spread and progression, and some may disappear spontaneously or in response to medication.7 Van der Knaap et al.9 proposed specific MRI criteria for the diagnosis of Alexander disease (4 of 5 are needed): (1) extensive cerebral white matter changes with frontal predominance; (2) a periventricular rim of low signal on T2, high signal on T1; (3) signal abnormalities of basal ganglia or thalami; (4) brainstem abnormalities; and (5) contrast enhancement of
one or more of the gray and white matter structures. Patients having less than 4 criteria are defined as having an atypical presentation.9 The association of signal abnormalities in basal ganglia and brainstem with a leukoencephalopathy is characteristic of both disorders. We considered the child to have an infantile onset mitochondrial disorder due to multifocal central nervous system involvement, urinary excretion of 3-methyl-glutaconic acid and Krebs cycle intermediates, and the association of white matter, cerebellar, and basal ganglia lesions with contrast enhancement and an elevated lactate peak on brain spectroscopy. Alexander disease was not considered since the patient presented in infancy and did not demonstrate macrocephaly or seizures that are classic features of type I. She developed ptosis, which is considered an uncommon feature of Alexander disease.10 Furthermore the elevated urinary 3-
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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few years. This lack of frontal white matter abnormalities in young infants with Alexander disease has been reported and the signal abnormality in the peri-aqueductal region was included by van der Knaap et al. as an early MRI finding in
Fig. 3 e Vermis atrophy with time as demonstrated on Sagittal T1 weighted images. Top: MRI at the age of 2 years 7 months (arrow); Bottom: MRI at the age of 5 years 9 months. Also note slight atrophy of brainstem (lower arrow).
methylglutaconic acid is typical of a mitochondrial disorder.11 The progressive course combined with episodes of clinical and neuroradiological worsening after infections coexisting with temporary improvement following treatment with a mitochondrial cocktail further suggested a mitochondrial disorder. The signal abnormalities on MRI were initially noted only in the peri-aqueductal region; the frontal white matter changes typical of Alexander disease appeared only after a
Fig. 4 e MRI at the age of 4 years 9 months: Top: axial FLAIR image showing symmetric hyper intense signal in the brainstem (arrow); bottom: axial FLAIR image showing signal changes in the cerebellar peduncles (top arrow) and dentate nucleus (bottom arrow).
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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Fig. 5 e MR Spectroscopy with short TE of 35 in the periventricular left white matter at the age of 4 years 9 months: suspected lactate double peak at 1.3 ppm (star) and elevated choline (cho) peak.
patients with Alexander disease.9 Additionally, there have been several cases showing elevated lactic acid in both blood2,5,6,12 and the abnormal white matter demonstrated by MR spectroscopy.13 In retrospect, our patient complies with MRI features of Alexander disease. Prust et al. correlated the genotype-phenotype of 215 Alexander disease patients.1 The authors defined a 2-class model based on age of onset. Patients with type I Alexander disease presented with macrocephaly, failure to thrive, encephalopathy, seizures, motor and cognitive delay, and paroxysmal deterioration within the first 4 years of life. Patients with type II presented with bulbar symptoms, autonomic dysfunction, ataxia, dysarthria, dysphonia, ocular movement abnormalities, and palatal myoclonus across the lifespan.1 The radiologic features in type I patients were the typical MRI features defined by van der Knaap et al.9 Type II patients had atypical MRI features characterized most commonly by a predominance of posterior fossa white matter abnormalities, brainstem, cerebellar, and spinal cord atrophy.1 Despite the high quality fit, this model did not allow for type classification in all the cases: the authors confirmed genotype-phenotype correlation for the R79 and R239 mutations but failed to find typical clinical features associated with R88 or R416 mutations.1 Our patient was found to harbor the R416W, a c-terminus mutation in the GFAP gene. This mutation, the 4th most common, was found to be causative in 5.6% of cases.1 The R416W mutation is located in the tail domain of GFAP and may be a hot spot mutation that presents in multiple forms of Alexander disease.3 Der Perng et al.14 demonstrated that the R416W mutation significantly perturbs in vitro filament assembly. Consistent with the heterozygosity of the mutation, this effect was found to be dominant over wild-type GFAP in coassembly experiments. The R416W mutation changes the assembly process in a way that encourages aberrant filamentefilament interactions that then lead to
protein aggregation and chaperone sequestration.14 Yoshida et al.3 found that most of the astrocytes with R416W gene mutation were capable of cell division. The authors speculated that the alteration of R416W GFAP function in astrocytes may depend on other elements interacting with GFAP. This may support the notion that the heterogeneous phenotypes caused by GFAP mutations in the tail domain are influenced by other genetic or environmental factors. The relationship between GFAP mutations and aberrant mitochondrial function seems too frequent to be incidental. The mitochondria are known to take a pivotal role in the pathophysiology of various neuro-degenerative disorders, either directly, via proteins participating in known mitochondrial biochemical pathways and structure, or indirectly, via proteins that are not necessarily targeted to mitochondria, but affect their function secondarily. Impaired sub-cellular mitochondrial trafficking is one the mechanisms accounting for neurodegenerative disorders. The mitochondria are transported on cytoskeletal elements (microtubules and actin cables) often in association with intermediate filaments.15 Hence, disruption of the intermediate filaments’ frame may cause mitochondrial mal-distribution and disruption of the normal energetic function of the cell, causing accumulation of lactic acid. The increased urinary 3-methylglutaconic acid and Krebs cycle intermediates as well as elevated blood lactic acid found in our patient, may reflect a secondary mitochondrial dysfunction or be a marker of another, unknown mutation in a mitochondrial-related gene, modifying the overall phenotype. Most infants with Alexander disease develop a rapidly increasing head circumference; however, this feature is not found in all cases.2,6 The explanation for a normal head circumference in patients with Alexander disease and prominent mitochondrial features is not clear. It has been suggested that the acquired macrocephaly, seen in Alexander disease patients, results from a proliferation of atypical astrocytes.6 It has been reported that the astrocytes in Alexander disease may demonstrate oxidative stress and ultrastructural mitochondrial abnormalities.16 It is possible that disruption of the normal energetic equilibrium of astrocytes in certain cases causes a decrease in their proliferation and early death thereby preventing rapid acceleration of head growth. Why certain patients with Alexander disease manifest overt mitochondrial dysfunction is not clear and requires further research.
6.
Conclusions
Alexander disease should be considered in the differential diagnosis of an infant presenting in the first year of life with a clinical picture suggesting a mitochondrial disorder, even without macrocephaly and when typical white matter changes are not yet present. Screening for the four most common mutations in the GFAP gene is warranted in these cases. When they are negative, next generation sequencing is a useful tool in unraveling the molecular etiology of these rare genetic neurodegenerative disorders.
Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009
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Acknowledgments This work has been supported by a trilateral grant from the German Research Foundation (Ga354/9-1).
references
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Please cite this article in press as: Nishri D, et al., Diagnosis by whole exome sequencing of atypical infantile onset Alexander disease masquerading as a mitochondrial disorder, European Journal of Paediatric Neurology (2014), http://dx.doi.org/10.1016/ j.ejpn.2014.03.009