- Email: [email protected]
Late-Onset Zellweger Spectrum Disorder Caused by PEX6 Mutations Mimicking X-Linked Adrenoleukodystrophy
Accepted Manuscript Late onset Zellweger spectrum disorder caused by PEX6 mutations mimicking Xlinked adrenoleukodystrophy C. Tran, S. Hewson, S.J. Steinberg, S. Mercimek-Mahmutoglu PII:
S0887-8994(14)00197-0
DOI:
10.1016/j.pediatrneurol.2014.03.020
Reference:
PNU 8317
To appear in:
Pediatric Neurology
Received Date: 18 January 2014 Revised Date:
16 March 2014
Accepted Date: 22 March 2014
Please cite this article as: Tran C, Hewson S, Steinberg S, Mercimek-Mahmutoglu S, Late onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked adrenoleukodystrophy, Pediatric Neurology (2014), doi: 10.1016/j.pediatrneurol.2014.03.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT 1
Late onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked
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adrenoleukodystrophy
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Tran C1, Hewson S 1, Steinberg SJ 2, Mercimek-Mahmutoglu S 1,3*
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1
6
Canada
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2
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School of Medicine, Baltimore, MD, USA
9
3
Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto,
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Institute of Genetic Medicine and Department of Neurology, Johns Hopkins University
Genetics & Genome Biology, Research Institute, The Hospital for Sick Children, Toronto,
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Canada
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*Corresponding Author:
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Saadet Mercimek-Mahmutoglu, MD, FCCMG, PhD
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The Hospital for Sick Children
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Division of Clinical and Metabolic Genetics
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Department of Pediatrics, University of Toronto
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525 University Avenue, Suite 935, 9th Floor
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Toronto, ON, M5G 1X8, Canada
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Phone: 416-813-6390
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Fax: 416-813-5345
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Email: [email protected]
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Submission: Pediatric Neurology as Clinical Observations, Resident-Fellow Authorship
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Pathway
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Word count: 2019 words (including introduction, results, discussion).
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ACCEPTED MANUSCRIPT Abstract
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Background: Zellweger Spectrum Disorders (ZSD) are autosomal recessively inherited
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multisystem disorders caused by one of the 13 different PEX gene defects resulting in
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defective peroxisomal assembly and multiple peroxisomal enzyme deficiencies. We report a
30
new patient with late onset ZSD mimicking X-linked adrenoleukodystrophy (X-ALD).
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Case report and results: This 8.5-year-old boy with normal development until 6.5 years of
32
age presented with bilateral sensorial-neural-hearing loss during school hearing test. He then
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developed acute onset diplopia, clumsiness and cognitive dysfunction at age 7 years. Brain
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MRI showed symmetrical leukodystrophy, although without gadolinium enhancement.
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Elevated plasma very long chain fatty acids were suggestive of X-ALD, but his ABCD1 gene
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had normal coding sequence and dosage. We performed additional studies in cultured skin
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fibroblasts that were consistent with ZSD. Molecular testing identified disease causing
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compound heterozygous mutations in the PEX6 gene supporting the ZSD diagnosis in this
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patient.
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Conclusions: We report a new patient with late onset ZSD caused by PEX6 mutations who
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presented with acute neurodegenerative disease course mimicking X-ALD. This provides an
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additional reason that molecular confirmation is important for the genetic counseling and
43
management of patients with a clinical and biochemical diagnosis of X-ALD.
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Key words: peroxisome biogenesis disorder, Zellweger spectrum disorder, X-linked
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adrenoleukodystrophy, PEX6 gene, late onset
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ACCEPTED MANUSCRIPT Introduction
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Peroxisome biogenesis disorders are autosomal-recessive disorders characterized by defective
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peroxisomal assembly resulting in multiple peroxisomal enzyme deficiencies and is divided
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into two subgroups: 1) Zellweger spectrum disorders (ZSD) (OMIM #601539); 2) rhizomelic
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chondrodysplasia punctate type I (OMIM #215100). ZSD is a continuum of three phenotypes:
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Zellweger syndrome (OMIM #214100); neonatal adrenoleukodystrophy (OMIM #202370);
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and infantile Refsum disease (OMIM # 266510)1. ZSD are caused by mutations in one of 13
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PEX genes that encode peroxins, required for normal peroxisome assembly2. The X-linked
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adrenoleukodystrophy (X-ALD) (OMIM # 300100) is an X-linked disorder of peroxisomal β-
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oxidation caused by ABCD1 genetic defect3.
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ZSD are usually severe neonatal onset multisystem disorders resulting in deaths in early
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childhood. Later onset and milder phenotypes of ZSD have also been reported with or without
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cerebral white matter involvement. Zellweger syndrome is characterized by craniofacial
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abnormalities (high forehead, large anterior fontanel, hypoplastic supraorbital ridges,
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epicanthal folds, midface hypoplasia), eye abnormalities, neuronal migration defects,
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hepatomegaly, and chondrodysplasia punctata. Zellweger syndrome usually presents in the
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neonatal period with profound hypotonia, seizures and feeding difficulties. Neonatal
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adrenoleukodystrophy and infantile Refsum disease have similar but less pronounced clinical
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features than Zellweger syndrome. Most patients with latter two have hypotonia, but acquire
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certain developmental milestones including head control, unsupported sitting or walking1,2,4.
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The X-ALD mainly affects the nervous system and adrenal cortex. Its clinical phenotype is
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divided into three main subgroups: 1) cerebral form (childhood, adolescent and adult form) 2)
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adrenomyeloneuropathy (AMN) with or without cerebral disease and 3) Addison disease
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only3. The central nervous system involvement in X-ALD is characterized by cerebral white
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matter demyelination as a result of inflammatory process caused by accumulation of very long
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ACCEPTED MANUSCRIPT chain fatty acids (VLCFA). Uniform ring-shaped gadolinium enhancement occurs on T1-
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weigthed brain magnetic resonance imaging (MRI) corresponding to regions of active
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demyelination and inflammation3.
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Elevated VLCFA, phytanic acid, pristanic acid, bile acid intermediates (di-and
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trihydroxycholestanoic acid) and decreased erythrocytes plasmalogens are the biochemical
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hallmarks of ZSD4. Up to 80% of all ZSD are caused by mutations in the PEX1 (70%) and
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PEX6 (10%) genes. The phenotype is variable and is a continuum from severe to mild forms5.
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This phenotypic variability is also found in patients with PEX10, PEX12, and PEX26
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mutations. Elevated VLCFA are the biochemical hallmark of X-ALD. Mutations in the
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ABCD1 gene have been identified in patients with X-ALD3.
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We report a ZSD patient harboring previously reported disease-causing compound
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heterozygous mutations in the PEX6 gene who presented with late onset, acute rapidly
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progressive leukodystrophy mimicking a childhood cerebral X-ALD clinical phenotype and
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neuroimaging features at the age of 7 years.
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Case report and results
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This 8.5-year-old boy was born at term by spontaneous vaginal delivery after an uneventful
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pregnancy to non-consanguine parents as second child. There was nuchal cord two times
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around the neck and he was grey for few seconds, but recovered immediately with some
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positive pressure ventilation and had no further perinatal concerns.
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History of infantile and early childhood neurodevelopmental and cognitive functions were
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normal. He had excellent grades in a regular grade 1 classroom. He was diagnosed with
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bilateral high frequency sensorial-neural hearing loss at the age of 6.5 years after not passing
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a hearing test performed at school. Between 6.5-7 years of age, he was reviewed by an
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otolaryngologist and was treated with hearing aids. In addition, he had a low pitch gruff voice
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and underwent flexible nasal laryngoscopy, which identified bilateral vocal cord nodules. At
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age 6 years 10 months, he began complaining of diplopia and was prescribed eye patch and
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glasses and scheduled for a brain imaging study. He also started showing cognitive decline
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and poor attention span based on reports by his parents and schoolteachers. His school performance declined significantly compared to the previous school year. He also developed
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significant balance problems and incoordination in the upper extremities and was referred to
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neurology clinic for further investigations. On his initial physical examination at the age of 7
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years 4 months, his weight and height were at the 75th percentile and his head circumference
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was greater than the 98th percentile. There were no clinical signs of adrenal insufficiency. He
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had no dysmorphic features except macrocephaly. He had abnormal neurological examination
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findings including exotropia of his left eye, increased deep tendon reflexes in the lower
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extremities, dysmetria and abnormal rapid alternating movements. He was not able to perform
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tandem gait and had an ataxic gait. He had decreased vibration sense. Mini Mental Status
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exam showed slow response to questions.
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Brain MRI revealed symmetrical increased signal intensity in T2-weigthed images in
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periventricular and deep white matter, extending to posterior limbs of the internal capsules,
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cerebral peduncles, pons and pyramids (figure 1). There was no diffusion restriction and no
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gadolinium enhancement within the brain parenchyma. Cranial magnetic resonance
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spectroscopy (TE of 35 and 144 milliseconds) revealed low N-acetylaspartate, elevated
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phosphoryl-choline, myoinositol and lipid peak in the basal ganglia and white matter. These
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neuroimaging features together with the clinical findings raised the suspicion of
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metachromatic leukodystrophy or X-linked adrenoleukodystrophy (X-ALD). Investigations
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performed at presentation revealed normal results for galactocerebrosidase enzyme activity
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(excluding Krabbe’s disease), arylsulfatse enzyme activity (excluding metachromatic
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leukodystrophy), blood count, creatine kinase, gamma glutamyl transpeptidase, conjugated
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ACCEPTED MANUSCRIPT and unconjugated bilirubin levels, lactate, ammonia, kidney functions, homocysteine,
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acylcarnitine profile, plasma amino acid analysis, urine mucopolysaccharides and
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oligosaccharides and urine organic acid analysis. Liver enzymes, alanine transaminase
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(48U/L; reference range 0-40) and aspartate transaminase (61U/L; reference range 0-45) were
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marginally elevated. Kidney ultrasound was normal with no cystic changes. Plasma VLCFAs
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levels were markedly elevated and were suggestive of X-ALD (table 1). However, sequencing
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and multiplex ligation-dependent probe amplification of the ABCD1 gene did not reveal any
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mutations to confirm X-ALD on a molecular genetic basis.
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At the time of the diagnosis of X-ALD, he underwent extensive neuropsychological
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assessments to investigate if he would be eligible for bone marrow transplantation. Wechsler
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Intelligence Scale for Children-Fourth Edition (WISC-IV) revealed extremely low verbal and
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perceptual intellectual functioning, and borderline impaired working memory skills. The
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Beery-Buktenica Test of Visual Motor Integration (Beery VMI 6th Ed) revealed impaired
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visual motor skills. A measure of Expressive Vocabulary (EVT-2) was well below age
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expected levels, and rote verbal memory (CMS list learning) was borderline impaired. Short-
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term simple verbal recall (digit span) and oral math were relatively well preserved (low
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average to average range).
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He also underwent investigations of adrenal functions using random morning cortisol and
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exogenous ACTH (cosyntropin) stimulation test, both of which were consistent with normal
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adrenal function. Three months later, he received G-tube insertion for feeding difficulties and
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risk of aspiration. He was in vegetative state 10 months later, and passed away at 16 months
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after clinical and biochemical diagnosis of X-ALD.
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His mother and older healthy brother had normal plasma VLCFA levels. Further
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investigations were undertaken due to the discrepancy between the failure to identify an
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ABCD1 defect and the desire of the family to have the possibility of prenatal diagnosis in
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ACCEPTED MANUSCRIPT future pregnancies. Blood dot spot C26:0 lyso-phosphorylcholine was markedly elevated
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(3.03pmole per 1/8th inch spot; reference range= 0.06-0.51). In cultured skin fibroblasts, total
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lipid VLCFAs were normal and cells were immunopositive after staining with an ABCD1
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antibody. However, there were fewer peroxisomes detected than normal. Enzymatic testing
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revealed a partial reduction in phytanic acid oxidation (41% activity of the simultaneous
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normal control) and a significant elevation in the percentage of cytosolic catalase in the
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patient’s fibroblasts compared to a simultaneous normal control (75% versus 17%,
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respectively). Overall, these results are consistent with ZSD. Molecular genetic testing
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following the PEX Gene Screen algorithm2 identified two previously reported PEX6
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mutations (c.1802G>A; p.Arg601Gln6 and c.2356C>T; p.Arg786Trp7) in the heterozygous
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state. The parents of the index case were provided genetic counseling after identification of
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these PEX6 mutations, but they declined parental testing to confirm that the mutations existed
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in a trans orientation.
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Discussion
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We present the case of an 8 years 10 months old boy with late onset ZSD caused by
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previously reported heterozygous disease-causing mutations in the PEX6 gene. He presented
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with acute onset progressive cognitive dysfunction and typical brain MRI findings of
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symmetrical leukodystrophy suggestive of X-ALD. Indeed, elevated VLCFAs were consistent
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with the biochemical diagnosis of X-ALD in this patient. Interestingly, he did not have
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adrenal insufficiency and there was no gadolinium enhancement on brain MRI at the time of
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the diagnosis. Furthermore, no pathogenic mutation was identified in the ABCD1 gene.
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Further investigations using cultured skin fibroblasts revealed normal total lipid VLCFAs,
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presence of ABCD1 protein, a decreased number of peroxisomes and elevated cytosolic
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catalase. Overall these results were suggestive of a peroxisome assembly defect. Molecular
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ACCEPTED MANUSCRIPT genetic testing of PEX genes confirmed the diagnosis of ZSD caused by a PEX6 defect after
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his death.
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Late onset mild ZSD has been reported in patients with mutations in various PEX genes8-11.
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Childhood onset slowly progressive ataxia caused by PEX2 and PEX10 mutations was
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reported in few patients ages between 20-50 years8,9,11. Age of onset of ataxia was between 3-
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18 years. Brain MRI revealed cerebellar atrophy with no white matter changes in all patients.
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Infantile onset lower limb spasticity and ataxia were reported in 6 patients with PEX16
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mutations. All patients showed progressive cognitive dysfunction from early childhood and
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symmetrical slowly progressive leukodystrophy on brain MRI10. Our patient presented with
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ataxia, but he developed acute progressive neurodegenerative disease course leading to death
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in 1.5 years of initial presentation following a neurologically normal appearance within the
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first 6-7 years of life. Taken together, to the best of our knowledge our patient is the first
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patient with PEX6 mutations and acute progressive neurodegenerative disease course
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mimicking phenotype and neuroimaging features of X-ALD.
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So far more than 100 mutations have been identified in the PEX6 gene (www.dbpex.org).
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Less than 20 patients have been reported with severe ZSD phenotype12-14. A pathogenic
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mutation (c.802_815del, p.[Val207_Gln294del, Val76_Gln294del) in the PEX6 gene was
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reported in 5 French-Canadian patients with ZSD12. A patient with sensorial-neural hearing
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loss, retinopathy, dysmorphic features, developmental delay, hepatomegaly and
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hypsarrhythmia was reported with ZSD who died at the age of 17 months. Interestingly, his
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non-consanguine parents had diagnosis of Usher syndrome due to sensorial-neural hearing
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loss and retinitis pigmentosa. Patient and both parents had elevated VLCFAs and mutations in
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the PEX6 gene. Patient was compound heterozygous for IVS10+2T>C (splice-site mutation)
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and c.1715C>T (p.T572I) mutations; father was homozygous for .1715C>T (p.T572I) and
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mother was compound heterozygous for IVS10+2T>C and two missense mutations
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ACCEPTED MANUSCRIPT c.2426C>T and c.2534T>C) confirming ZSD in this patient and attenuated ZSD in the
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parents15. Two previously reported mutations identified in our patient was reported in ZSD
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patients: c.1802G>A missense mutation in 3 patients6 and c.2356C>T missense mutation in
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one patient7, but there was no clinical or outcome information of the patients from both
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studies to compare with our patient. Both previously reported mutations in our patient resulted
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in late onset acute progressive neurodegenerative phenotype of ZSD, which was a unique
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PEX6 phenotype, compared to the patients reported in the literature with PEX6 mutations.
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In summary, we report a new patient with late onset ZSD mimicking X-ALD. This case
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highlights the importance of molecular genetic confirmation in X-ALD, as the genetic
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counseling and prenatal diagnosis varies between X-ALD and ZSD. As historically X-ALD
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patients were diagnosed based on the clinical, neuroimaging and biochemical features,
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probably additional patients who actually have ZSD have been misdiagnosed with X-ALD.
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This is the corollary to patients harboring contiguous gene deletions involving ABCD1 and
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BCAP31; those patients have an early onset clinical phenotype more similar to ZSD and may
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not be recognized as having an X-linked disorder without comprehensive biochemical and
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molecular testing. No identifiable mutations in the ABCD1 gene warrants peroxisomal studies
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in cultured skin fibroblasts, which is an invasive procedure. Non-invasive whole exome
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sequencing is a very recent addition to our diagnostic work-up, which is currently not
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available on the clinical basis (except United States), but applied to the selected patients with
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diagnostic dilemma on the research basis. We would have confirmed the diagnosis of ZSD
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earlier in our patient by using whole exome sequencing. Based on the experience we acquired
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from this patient, we would recommend molecular genetic studies in every patient with
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clinical, neuroimaging and biochemical diagnosis of X-ALD.
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ACCEPTED MANUSCRIPT Acknowledgements
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We would like to thank to Dr. Susan Blaser for brain MRI reviews on the clinical basis. We
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would like to thank to Dr. Eva Mamak for performing neuropsychological assessments. We
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would like to thank to Dr. Bradsema and Dr. Donner for referring this interesting patient.
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Finally, we would like to thank to the parents for allowing us to report their child’s results.
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References
1. Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW.
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Peroxisome biogenesis disorders. Biochim Biophys Acta. 2006; 1763:1733-1748.
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2. Steinberg S, Chen L, Wei L, et al. The PEX Gene Screen: molecular diagnosis of
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peroxisome biogenesis disorders in the Zellweger syndrome spectrum. Mol Genet
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Metab. 2004; 83:252-263.
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3. Melhem ER, Barker PB, Raymond GV, Moser HW. X-linked adrenoleukodystrophy in children: review of genetic, clinical, and MR imaging characteristics. Am J
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Roentgenol. 1999; 173:1575-1581.
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4. Wanders RJ. Peroxisomal disorders: clinical, biochemical, and molecular aspects. Neurochem Res. 1999; 24:565-580.
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5. Ebberink MS, Mooijer PA, Gootjes J, Koster J, Wanders RJ, Waterham HR. Genetic
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classification and mutational spectrum of more than 600 patients with a Zellweger
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syndrome spectrum disorder. Hum Mutat. 2011; 32:59-69.
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6. Yik WY, Steinberg SJ, Moser AB, Moser HW, Hacia JG. Identification of novel
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mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome
243
biogenesis disorders. Hum Mutat. 2009; 30:E467-80.
244 245
7. Ebberink MS, Koster J, Wanders RJ, Waterham HR. Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients. Hum Mutat. 2010; 31(1):E1058-70.
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8. Sevin C, Ferdinandusse S, Waterham HR, Wanders RJ, Aubourg P. Autosomal
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recessive cerebellar ataxia caused by mutations in the PEX2 gene. Orphanet J Rare
248
Dis. 2011; 10:6-8.
250 251
9. Mignarri A, Vinciguerra C, Giorgio A, et al. Zellweger Spectrum Disorder with Mild Phenotype Caused by PEX2 Gene Mutations. JIMD Rep. 2012; 6:43-46.
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10. Ebberink MS, Csanyi B, Chong WK, et al. Identification of an unusual variant
peroxisome biogenesis disorder caused by mutations in the PEX16 gene. J Med Genet.
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2010; 47:608-615.
255 256
11. Régal L, Ebberink MS, Goemans N, et al. Mutations in PEX10 are a cause of autosomal recessive ataxia. Ann Neurol. 2010; 68:259-263.
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12. Levesque S, Morin C, Guay SP, et al. A founder mutation in the PEX6 gene is
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responsible for increased incidence of Zellweger syndrome in a French Canadian
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population. BMC Med Genet. 2012; 13:72-79.
13. Krause C, Rosewich H, Thanos M, Gärtner J. Identification of novel mutations in
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PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger spectrum patients. Hum
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Mutat. 2006; 27:1157-1164.
14. Zhang Z, Suzuki Y, Shimozawa N, et al. Genomic structure and identification of 11
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novel mutations of the PEX6 (peroxisome assembly factor-2) gene in patients with
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peroxisome biogenesis disorders. Hum Mutat. 1999; 13:487-496.
265
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15. Raas-Rothschild A, Wanders RJ, Mooijer PA, et al. A PEX6-defective peroxisomal
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biogenesis disorder with severe phenotype in an infant, versus mild phenotype
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resembling Usher syndrome in the affected parents. Am J Hum Genet. 2002; 70:1062-
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1068.
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ACCEPTED MANUSCRIPT Legends to figure:
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Figure 1: Brain MRI showing symmetrical abnormal signal in the periventricular and deep
272
white matter of both hemispheres with sparing of the subcortical U fibers. 1a) 3-D axial T2
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1b) 3-D axial T2 showing basal ganglia and corpus callosum; 1c) 3-D sagittal T1 showing
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normal size cerebellum.
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ACCEPTED MANUSCRIPT Legends to table:
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Table 1: Elevated VLCFA levels in three occasions were given in the current patient with
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ZSD caused by PEX6 mutations.
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ACCEPTED MANUSCRIPT Table 1: Elevated VLCFA levels in three occasions were given in the current patient with ZSD caused by PEX6 mutations. At diagnosis
(reference range) C26:0
Repeat 2 months
Repeat 6 months
later
later
5.626
2.391
0.54
0.649
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VLCFA
2.513
(0.247-1.095µ µmol/L) 0.088
C26:C22
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(0.005-0.017µ µmol/L) 0.929
C24:C22
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(0.543-0.941µ µmol/L)
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(0.26-0.92µ µmol/L)
0.969
0.053
0.039
1.289
0.999
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ACCEPTED MANUSCRIPT
S0887-8994(14)00197-0
DOI:
10.1016/j.pediatrneurol.2014.03.020
Reference:
PNU 8317
To appear in:
Pediatric Neurology
Received Date: 18 January 2014 Revised Date:
16 March 2014
Accepted Date: 22 March 2014
Please cite this article as: Tran C, Hewson S, Steinberg S, Mercimek-Mahmutoglu S, Late onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked adrenoleukodystrophy, Pediatric Neurology (2014), doi: 10.1016/j.pediatrneurol.2014.03.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT 1
Late onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked
2
adrenoleukodystrophy
3
Tran C1, Hewson S 1, Steinberg SJ 2, Mercimek-Mahmutoglu S 1,3*
5
1
6
Canada
7
2
8
School of Medicine, Baltimore, MD, USA
9
3
Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto,
SC
Institute of Genetic Medicine and Department of Neurology, Johns Hopkins University
Genetics & Genome Biology, Research Institute, The Hospital for Sick Children, Toronto,
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10
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4
Canada
11
*Corresponding Author:
13
Saadet Mercimek-Mahmutoglu, MD, FCCMG, PhD
14
The Hospital for Sick Children
15
Division of Clinical and Metabolic Genetics
16
Department of Pediatrics, University of Toronto
17
525 University Avenue, Suite 935, 9th Floor
18
Toronto, ON, M5G 1X8, Canada
19
Phone: 416-813-6390
20
Fax: 416-813-5345
21
Email: [email protected]
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22 23
Submission: Pediatric Neurology as Clinical Observations, Resident-Fellow Authorship
24
Pathway
25
Word count: 2019 words (including introduction, results, discussion).
2
ACCEPTED MANUSCRIPT Abstract
27
Background: Zellweger Spectrum Disorders (ZSD) are autosomal recessively inherited
28
multisystem disorders caused by one of the 13 different PEX gene defects resulting in
29
defective peroxisomal assembly and multiple peroxisomal enzyme deficiencies. We report a
30
new patient with late onset ZSD mimicking X-linked adrenoleukodystrophy (X-ALD).
31
Case report and results: This 8.5-year-old boy with normal development until 6.5 years of
32
age presented with bilateral sensorial-neural-hearing loss during school hearing test. He then
33
developed acute onset diplopia, clumsiness and cognitive dysfunction at age 7 years. Brain
34
MRI showed symmetrical leukodystrophy, although without gadolinium enhancement.
35
Elevated plasma very long chain fatty acids were suggestive of X-ALD, but his ABCD1 gene
36
had normal coding sequence and dosage. We performed additional studies in cultured skin
37
fibroblasts that were consistent with ZSD. Molecular testing identified disease causing
38
compound heterozygous mutations in the PEX6 gene supporting the ZSD diagnosis in this
39
patient.
40
Conclusions: We report a new patient with late onset ZSD caused by PEX6 mutations who
41
presented with acute neurodegenerative disease course mimicking X-ALD. This provides an
42
additional reason that molecular confirmation is important for the genetic counseling and
43
management of patients with a clinical and biochemical diagnosis of X-ALD.
44
Key words: peroxisome biogenesis disorder, Zellweger spectrum disorder, X-linked
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adrenoleukodystrophy, PEX6 gene, late onset
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Peroxisome biogenesis disorders are autosomal-recessive disorders characterized by defective
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peroxisomal assembly resulting in multiple peroxisomal enzyme deficiencies and is divided
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into two subgroups: 1) Zellweger spectrum disorders (ZSD) (OMIM #601539); 2) rhizomelic
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chondrodysplasia punctate type I (OMIM #215100). ZSD is a continuum of three phenotypes:
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Zellweger syndrome (OMIM #214100); neonatal adrenoleukodystrophy (OMIM #202370);
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and infantile Refsum disease (OMIM # 266510)1. ZSD are caused by mutations in one of 13
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PEX genes that encode peroxins, required for normal peroxisome assembly2. The X-linked
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adrenoleukodystrophy (X-ALD) (OMIM # 300100) is an X-linked disorder of peroxisomal β-
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oxidation caused by ABCD1 genetic defect3.
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ZSD are usually severe neonatal onset multisystem disorders resulting in deaths in early
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childhood. Later onset and milder phenotypes of ZSD have also been reported with or without
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cerebral white matter involvement. Zellweger syndrome is characterized by craniofacial
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abnormalities (high forehead, large anterior fontanel, hypoplastic supraorbital ridges,
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epicanthal folds, midface hypoplasia), eye abnormalities, neuronal migration defects,
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hepatomegaly, and chondrodysplasia punctata. Zellweger syndrome usually presents in the
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neonatal period with profound hypotonia, seizures and feeding difficulties. Neonatal
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adrenoleukodystrophy and infantile Refsum disease have similar but less pronounced clinical
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features than Zellweger syndrome. Most patients with latter two have hypotonia, but acquire
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certain developmental milestones including head control, unsupported sitting or walking1,2,4.
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The X-ALD mainly affects the nervous system and adrenal cortex. Its clinical phenotype is
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divided into three main subgroups: 1) cerebral form (childhood, adolescent and adult form) 2)
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adrenomyeloneuropathy (AMN) with or without cerebral disease and 3) Addison disease
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only3. The central nervous system involvement in X-ALD is characterized by cerebral white
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matter demyelination as a result of inflammatory process caused by accumulation of very long
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ACCEPTED MANUSCRIPT chain fatty acids (VLCFA). Uniform ring-shaped gadolinium enhancement occurs on T1-
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weigthed brain magnetic resonance imaging (MRI) corresponding to regions of active
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demyelination and inflammation3.
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Elevated VLCFA, phytanic acid, pristanic acid, bile acid intermediates (di-and
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trihydroxycholestanoic acid) and decreased erythrocytes plasmalogens are the biochemical
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hallmarks of ZSD4. Up to 80% of all ZSD are caused by mutations in the PEX1 (70%) and
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PEX6 (10%) genes. The phenotype is variable and is a continuum from severe to mild forms5.
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This phenotypic variability is also found in patients with PEX10, PEX12, and PEX26
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mutations. Elevated VLCFA are the biochemical hallmark of X-ALD. Mutations in the
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ABCD1 gene have been identified in patients with X-ALD3.
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We report a ZSD patient harboring previously reported disease-causing compound
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heterozygous mutations in the PEX6 gene who presented with late onset, acute rapidly
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progressive leukodystrophy mimicking a childhood cerebral X-ALD clinical phenotype and
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neuroimaging features at the age of 7 years.
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Case report and results
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This 8.5-year-old boy was born at term by spontaneous vaginal delivery after an uneventful
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pregnancy to non-consanguine parents as second child. There was nuchal cord two times
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around the neck and he was grey for few seconds, but recovered immediately with some
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positive pressure ventilation and had no further perinatal concerns.
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History of infantile and early childhood neurodevelopmental and cognitive functions were
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normal. He had excellent grades in a regular grade 1 classroom. He was diagnosed with
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bilateral high frequency sensorial-neural hearing loss at the age of 6.5 years after not passing
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a hearing test performed at school. Between 6.5-7 years of age, he was reviewed by an
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otolaryngologist and was treated with hearing aids. In addition, he had a low pitch gruff voice
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and underwent flexible nasal laryngoscopy, which identified bilateral vocal cord nodules. At
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age 6 years 10 months, he began complaining of diplopia and was prescribed eye patch and
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glasses and scheduled for a brain imaging study. He also started showing cognitive decline
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and poor attention span based on reports by his parents and schoolteachers. His school performance declined significantly compared to the previous school year. He also developed
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significant balance problems and incoordination in the upper extremities and was referred to
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neurology clinic for further investigations. On his initial physical examination at the age of 7
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years 4 months, his weight and height were at the 75th percentile and his head circumference
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was greater than the 98th percentile. There were no clinical signs of adrenal insufficiency. He
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had no dysmorphic features except macrocephaly. He had abnormal neurological examination
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findings including exotropia of his left eye, increased deep tendon reflexes in the lower
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extremities, dysmetria and abnormal rapid alternating movements. He was not able to perform
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tandem gait and had an ataxic gait. He had decreased vibration sense. Mini Mental Status
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exam showed slow response to questions.
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Brain MRI revealed symmetrical increased signal intensity in T2-weigthed images in
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periventricular and deep white matter, extending to posterior limbs of the internal capsules,
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cerebral peduncles, pons and pyramids (figure 1). There was no diffusion restriction and no
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gadolinium enhancement within the brain parenchyma. Cranial magnetic resonance
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spectroscopy (TE of 35 and 144 milliseconds) revealed low N-acetylaspartate, elevated
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phosphoryl-choline, myoinositol and lipid peak in the basal ganglia and white matter. These
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neuroimaging features together with the clinical findings raised the suspicion of
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metachromatic leukodystrophy or X-linked adrenoleukodystrophy (X-ALD). Investigations
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performed at presentation revealed normal results for galactocerebrosidase enzyme activity
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(excluding Krabbe’s disease), arylsulfatse enzyme activity (excluding metachromatic
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leukodystrophy), blood count, creatine kinase, gamma glutamyl transpeptidase, conjugated
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ACCEPTED MANUSCRIPT and unconjugated bilirubin levels, lactate, ammonia, kidney functions, homocysteine,
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acylcarnitine profile, plasma amino acid analysis, urine mucopolysaccharides and
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oligosaccharides and urine organic acid analysis. Liver enzymes, alanine transaminase
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(48U/L; reference range 0-40) and aspartate transaminase (61U/L; reference range 0-45) were
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marginally elevated. Kidney ultrasound was normal with no cystic changes. Plasma VLCFAs
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levels were markedly elevated and were suggestive of X-ALD (table 1). However, sequencing
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and multiplex ligation-dependent probe amplification of the ABCD1 gene did not reveal any
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mutations to confirm X-ALD on a molecular genetic basis.
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At the time of the diagnosis of X-ALD, he underwent extensive neuropsychological
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assessments to investigate if he would be eligible for bone marrow transplantation. Wechsler
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Intelligence Scale for Children-Fourth Edition (WISC-IV) revealed extremely low verbal and
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perceptual intellectual functioning, and borderline impaired working memory skills. The
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Beery-Buktenica Test of Visual Motor Integration (Beery VMI 6th Ed) revealed impaired
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visual motor skills. A measure of Expressive Vocabulary (EVT-2) was well below age
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expected levels, and rote verbal memory (CMS list learning) was borderline impaired. Short-
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term simple verbal recall (digit span) and oral math were relatively well preserved (low
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average to average range).
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He also underwent investigations of adrenal functions using random morning cortisol and
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exogenous ACTH (cosyntropin) stimulation test, both of which were consistent with normal
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adrenal function. Three months later, he received G-tube insertion for feeding difficulties and
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risk of aspiration. He was in vegetative state 10 months later, and passed away at 16 months
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after clinical and biochemical diagnosis of X-ALD.
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His mother and older healthy brother had normal plasma VLCFA levels. Further
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investigations were undertaken due to the discrepancy between the failure to identify an
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ABCD1 defect and the desire of the family to have the possibility of prenatal diagnosis in
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ACCEPTED MANUSCRIPT future pregnancies. Blood dot spot C26:0 lyso-phosphorylcholine was markedly elevated
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(3.03pmole per 1/8th inch spot; reference range= 0.06-0.51). In cultured skin fibroblasts, total
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lipid VLCFAs were normal and cells were immunopositive after staining with an ABCD1
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antibody. However, there were fewer peroxisomes detected than normal. Enzymatic testing
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revealed a partial reduction in phytanic acid oxidation (41% activity of the simultaneous
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normal control) and a significant elevation in the percentage of cytosolic catalase in the
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patient’s fibroblasts compared to a simultaneous normal control (75% versus 17%,
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respectively). Overall, these results are consistent with ZSD. Molecular genetic testing
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following the PEX Gene Screen algorithm2 identified two previously reported PEX6
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mutations (c.1802G>A; p.Arg601Gln6 and c.2356C>T; p.Arg786Trp7) in the heterozygous
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state. The parents of the index case were provided genetic counseling after identification of
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these PEX6 mutations, but they declined parental testing to confirm that the mutations existed
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in a trans orientation.
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Discussion
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We present the case of an 8 years 10 months old boy with late onset ZSD caused by
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previously reported heterozygous disease-causing mutations in the PEX6 gene. He presented
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with acute onset progressive cognitive dysfunction and typical brain MRI findings of
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symmetrical leukodystrophy suggestive of X-ALD. Indeed, elevated VLCFAs were consistent
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with the biochemical diagnosis of X-ALD in this patient. Interestingly, he did not have
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adrenal insufficiency and there was no gadolinium enhancement on brain MRI at the time of
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the diagnosis. Furthermore, no pathogenic mutation was identified in the ABCD1 gene.
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Further investigations using cultured skin fibroblasts revealed normal total lipid VLCFAs,
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presence of ABCD1 protein, a decreased number of peroxisomes and elevated cytosolic
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catalase. Overall these results were suggestive of a peroxisome assembly defect. Molecular
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ACCEPTED MANUSCRIPT genetic testing of PEX genes confirmed the diagnosis of ZSD caused by a PEX6 defect after
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his death.
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Late onset mild ZSD has been reported in patients with mutations in various PEX genes8-11.
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Childhood onset slowly progressive ataxia caused by PEX2 and PEX10 mutations was
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reported in few patients ages between 20-50 years8,9,11. Age of onset of ataxia was between 3-
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18 years. Brain MRI revealed cerebellar atrophy with no white matter changes in all patients.
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Infantile onset lower limb spasticity and ataxia were reported in 6 patients with PEX16
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mutations. All patients showed progressive cognitive dysfunction from early childhood and
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symmetrical slowly progressive leukodystrophy on brain MRI10. Our patient presented with
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ataxia, but he developed acute progressive neurodegenerative disease course leading to death
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in 1.5 years of initial presentation following a neurologically normal appearance within the
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first 6-7 years of life. Taken together, to the best of our knowledge our patient is the first
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patient with PEX6 mutations and acute progressive neurodegenerative disease course
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mimicking phenotype and neuroimaging features of X-ALD.
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So far more than 100 mutations have been identified in the PEX6 gene (www.dbpex.org).
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Less than 20 patients have been reported with severe ZSD phenotype12-14. A pathogenic
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mutation (c.802_815del, p.[Val207_Gln294del, Val76_Gln294del) in the PEX6 gene was
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reported in 5 French-Canadian patients with ZSD12. A patient with sensorial-neural hearing
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loss, retinopathy, dysmorphic features, developmental delay, hepatomegaly and
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hypsarrhythmia was reported with ZSD who died at the age of 17 months. Interestingly, his
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non-consanguine parents had diagnosis of Usher syndrome due to sensorial-neural hearing
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loss and retinitis pigmentosa. Patient and both parents had elevated VLCFAs and mutations in
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the PEX6 gene. Patient was compound heterozygous for IVS10+2T>C (splice-site mutation)
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and c.1715C>T (p.T572I) mutations; father was homozygous for .1715C>T (p.T572I) and
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mother was compound heterozygous for IVS10+2T>C and two missense mutations
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ACCEPTED MANUSCRIPT c.2426C>T and c.2534T>C) confirming ZSD in this patient and attenuated ZSD in the
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parents15. Two previously reported mutations identified in our patient was reported in ZSD
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patients: c.1802G>A missense mutation in 3 patients6 and c.2356C>T missense mutation in
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one patient7, but there was no clinical or outcome information of the patients from both
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studies to compare with our patient. Both previously reported mutations in our patient resulted
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in late onset acute progressive neurodegenerative phenotype of ZSD, which was a unique
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PEX6 phenotype, compared to the patients reported in the literature with PEX6 mutations.
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In summary, we report a new patient with late onset ZSD mimicking X-ALD. This case
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highlights the importance of molecular genetic confirmation in X-ALD, as the genetic
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counseling and prenatal diagnosis varies between X-ALD and ZSD. As historically X-ALD
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patients were diagnosed based on the clinical, neuroimaging and biochemical features,
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probably additional patients who actually have ZSD have been misdiagnosed with X-ALD.
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This is the corollary to patients harboring contiguous gene deletions involving ABCD1 and
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BCAP31; those patients have an early onset clinical phenotype more similar to ZSD and may
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not be recognized as having an X-linked disorder without comprehensive biochemical and
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molecular testing. No identifiable mutations in the ABCD1 gene warrants peroxisomal studies
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in cultured skin fibroblasts, which is an invasive procedure. Non-invasive whole exome
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sequencing is a very recent addition to our diagnostic work-up, which is currently not
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available on the clinical basis (except United States), but applied to the selected patients with
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diagnostic dilemma on the research basis. We would have confirmed the diagnosis of ZSD
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earlier in our patient by using whole exome sequencing. Based on the experience we acquired
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from this patient, we would recommend molecular genetic studies in every patient with
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clinical, neuroimaging and biochemical diagnosis of X-ALD.
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ACCEPTED MANUSCRIPT Acknowledgements
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We would like to thank to Dr. Susan Blaser for brain MRI reviews on the clinical basis. We
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would like to thank to Dr. Eva Mamak for performing neuropsychological assessments. We
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would like to thank to Dr. Bradsema and Dr. Donner for referring this interesting patient.
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Finally, we would like to thank to the parents for allowing us to report their child’s results.
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1068.
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Figure 1: Brain MRI showing symmetrical abnormal signal in the periventricular and deep
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white matter of both hemispheres with sparing of the subcortical U fibers. 1a) 3-D axial T2
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1b) 3-D axial T2 showing basal ganglia and corpus callosum; 1c) 3-D sagittal T1 showing
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normal size cerebellum.
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ACCEPTED MANUSCRIPT Legends to table:
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Table 1: Elevated VLCFA levels in three occasions were given in the current patient with
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ZSD caused by PEX6 mutations.
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ACCEPTED MANUSCRIPT Table 1: Elevated VLCFA levels in three occasions were given in the current patient with ZSD caused by PEX6 mutations. At diagnosis
(reference range) C26:0
Repeat 2 months
Repeat 6 months
later
later
5.626
2.391
0.54
0.649
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VLCFA
2.513
(0.247-1.095µ µmol/L) 0.088
C26:C22
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(0.005-0.017µ µmol/L) 0.929
C24:C22
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(0.543-0.941µ µmol/L)
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(0.26-0.92µ µmol/L)
0.969
0.053
0.039
1.289
0.999
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