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Pontocerebellar hypoplasia type 2D and optic nerve atrophy further expand the spectrum associated with selenoprotein biosynthesis deficiency
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 2 0 ( 2 0 1 6 ) 4 8 3 e4 8 8
Official Journal of the European Paediatric Neurology Society
Case Study
Pontocerebellar hypoplasia type 2D and optic nerve atrophy further expand the spectrum associated with selenoprotein biosynthesis deficiency Efterpi Pavlidou a, Vincenzo Salpietro a,b, Rahul Phadke b, Iain P. Hargreaves b, Leigh Batten c, Kenneth McElreavy d, Matthew Pitt e,f, Kshitij Mankad e,f, Clare Wilson g, Maria Concetta Cutrupi h, Martino Ruggieri i, David McCormick e,j, Anand Saggar e,k, Maria Kinali a,e,* a
Department of Paediatric Neurology, Chelsea and Westminster NHS Foundation Trust, 369 Fulham Road, London, SW10 9NH, United Kingdom b Department of Molecular Neurosciences, University College of London, Gower Street, London, WC1E 6BT, United Kingdom c The Doctors Laboratory, Bupa Cromwell Hospital Pathology Department, 1-3 Pennant Mews, London, SW5 0TU, United Kingdom d Human Developmental Genetics, Institute Pasteur, 25-28 Rue du Docteur Roux, 75015, Paris, France e The Portland Hospital for Women and Children, 205-209 Great Portland St, London, W1W 5AH, United Kingdom f Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond St, London, WC1N 3JH, United Kingdom g Department of Paediatric Ophthalmology, Chelsea and Westminster NHS Foundation Trust, 369 Fulham Road, London, SW10 9NH, United Kingdom h Unit of Genetics and Paediatric Immunology, Department of Paediatrics, University of Messina, Via Consolare Valeria 1, 98125, Messina, Italy i Department of Clinical and Experimental Medicine, University of Catania, Ospedale Garibaldi “Nesima” e Via Palermo, 636, I-95122, Catania, Italy j Department of Paediatrics, King's College Hospital, Denmark Hill, London, SE5 9RS, United Kingdom k St George's Hospital, NHS Foundation Trust, Blackshaw Rd, Tooting, SW17 0QT, London, United Kingdom
List of Abbreviations: MRI, Magnetic resonance imaging; PCH, Pontocerebellar hypoplasia; WES, Whole exome sequencing; RCE, Respiratory chain enzyme analysis. * Corresponding author. Department of Paediatric Neurology, Chelsea and Westminster NHS Foundation Trust, London, United Kingdom. Tel.: þ44 20 3315 8645; fax: þ44 20 3315 7998. E-mail addresses: [email protected] (E. Pavlidou), [email protected] (V. Salpietro), [email protected] (R. Phadke), [email protected] (I.P. Hargreaves), [email protected] (L. Batten), [email protected] (K. McElreavy), [email protected] (M. Pitt), [email protected] (K. Mankad), [email protected] (C. Wilson), mcutrupi@ libero.it (M.C. Cutrupi), [email protected] (M. Ruggieri), [email protected] (D. McCormick), [email protected] (A. Saggar), m. [email protected] (M. Kinali). http://dx.doi.org/10.1016/j.ejpn.2015.12.016 1090-3798/© 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
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article info
abstract
Article history:
Background: The term Pontocerebellar hypoplasias collectively refers to a group of rare,
Received 6 November 2015
heterogeneous and progressive disorders, which are frequently inherited in an autosomal
Received in revised form
recessive manner and usually have a prenatal onset. Mutations in the SEPSECS gene,
29 December 2015
leading to deficiency in selenoprotein biosynthesis, have been identified in recent times as
Accepted 30 December 2015
the molecular etiology of different pre/perinatal onset neurological phenotypes, including cerebello-cerebral atrophy, Pontocerebellar hypoplasia type 2D and progressive encepha-
Keywords:
lopathy with elevated lactate. These disorders share a similar spectrum of central (e.g.,
Pontocerebellar hypoplasia
brain neurodegeneration with grey and white matter both involved) and peripheral (e.g.,
SEPSECS
spasticity due to axonal neuropathy) nervous system impairment.
Selenoprotein biosynthesis
Case presentation: We hereby describe a 9-year-old boy with (i) a typical Pontocerebellar
deficiency
hypoplasia type 2D phenotype (e.g. profound mental retardation, spastic quadriplegia,
Optic nerve atrophy
ponto-cerebellar hypoplasia and progressive cerebral atrophy); (ii) optic nerve atrophy and
Mitochondrial myopathy
(iii) mild secondary mitochondrial myopathy detected by muscle biopsy and respiratory chain enzyme analysis. We performed whole exome sequencing which identified a homozygous mutation of the SEPSECS gene (c.1001T > C), confirming the clinical suspect of Pontocerebellar hypoplasia type 2D. Conclusion: This report further corroborates the notion of a potential secondary mitochondrial dysfunction in the context of selenoprotein biosynthesis deficiency and also adds optic nerve atrophy as a new potential clinical feature within the SEPSECS-associated clinical spectrum. These findings suggest the presence of a possible shared genetic etiology among similar clinical entities characterized by the combination of progressive cerebellocerebral and optic nerve atrophy and also stress the biological importance of selenoproteins in the regulation of neuronal and metabolic homeostasis. © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The term Pontocerebellar hypoplasias (PCHs) collectively refers to a group of rare, heterogeneous and progressive disorders, which are inherited in an autosomal recessive manner and usually have a prenatal onset.1 PCHs are generally characterized by an abnormally small cerebellum and ventral pons. Supra-tentorial structures are also affected, although less prominently. The estimated incidence is calculated less than 1:200.000 within the general paediatric population.2 The main clinical features are characterised by severe psychomotor delay and microcephaly. In most cases, PCHs have a severe prognosis and are fatal in early childhood. At least ten types of PCHs have been described so far, with PCH Type 2 (PCH2) being the most frequently reported. Children with PCH2 have microcephaly since birth, associated with extrapyramidal dyskinesia and spasticity.3 PCH2 is usually suspected on the basis of specific magnetic resonance imaging (MRI) features, genetic and neuropathological findings. The understanding of the natural history of this disorder is extremely important in prenatal counselling of affected families and also in management and prognosis of these children. Most of typical PCH2 cases so far reported are due to missense mutations in the TSEN54 (MIM 608755) gene on chromosome 17.4 Less frequently reported PCH2 sub-types, namely PCH2B, PCH2C and PCH2D are due to mutations in the genes TSEN2 (MIM 608753), TSEN34 (MIM 608754) and SEPSECS (MIM 613009), respectively.5,6 Mutation in SEPSECS
impair the cytoplasmic transfer RNA (tRNA)-charging in the selenoprotein biosynthesis pathway. We hereby report on a child diagnosed with a clinical and radiological diagnosis of PCH2 who underwent whole exome sequencing (WES), which disclosed a pathogenic mutation in the SEPSECS gene. The child also had a severe visual impairment due to optic atrophy and a mild secondary mitochondrial dysfunction in his muscles showed by muscle biopsy and respiratory chain enzyme analysis. We described on the present boy as these latter clinical features (i.e., mitochondrial myopathy, optic nerve atrophy) contribute in further expanding the clinical spectrum associated with SEPSECS mutation, highlighting the potentially crucial role of selenoproteins.
2.
Case study
An 8-year-old boy was referred to our Department due to profound psychomotor delay, quadriplegia and microcephaly. He was the third child of two consanguineous Arabian parents. He had two elder and one younger brothers. His prenatal ultrasound was normal and his birth was via uncomplicated vaginal delivery with no need for resuscitation or admission to special care. Additionally, his feeding was normal at birth. Birth weight was 2700 gr (10th). Shortly after birth he was noticed to have generalized hypotonia. At 2 months his head circumference was 36.5 cm (<10th). Brain MRI was performed at 18 months of age and showed a large collection of
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 2 0 ( 2 0 1 6 ) 4 8 3 e4 8 8
cerebrospinal fluid (CSF) inferiorly to the cerebellum communicating with the 4th ventricle. There were no focal lesions within the brain parenchyma. The child initially presented to our clinic at the age of 6.5 years old with spastic quadriplegia, microcephaly, severe mental retardation and inability to swallow normally. Clinical examination revealed severe hypertonia probably since birth, neurogenic muscle atrophy and decreased deep tendon reflexes, especially at his lower limbs. Nerve conduction studies demonstrated borderline reductions of the sensory nerve action potentials from the medial plantar and superficial peroneal nerves with normal sensory nerve conduction velocities. Motor conduction studies showed normal compound motor action potential (CMAP) amplitude and velocity. Electromyography (EMG) of a leg muscle showed no indication of either motor nerve involvement or myopathy. Ophthalmological examination revealed optic atrophy with impaired vision. The child also had bilateral femoral derotation varus osteotomies and scoliosis. The child experienced intense pain due to the severe neuropathy and for this reason he was taking carbamazepine 100 mg twice daily and gabapentin 500 mg twice daily. He was also started on melatonin for sleep disorder and ranitidine and omeprazole as anti-reflux medication. At the age of 7 the patient had suffered severe bronchopneumonia complicated by pneumothorax and required ventilation in intensive care. He had poor feeding, trouble managing secretions and had
485
marked cachexia. Therefore, gastrostomy and Nissen fundoplication were mandatory. A second brain MRI was performed at the age of 7 years old showing progressive pontocerebellar atrophy with deficiency of the cerebellar vermis (Fig. 1). There was also progressive global brain atrophy. Repeat nerve conduction studies, 11 months after the first studies, showed absence of sensory responses not only in the leg but also in the arm. Motor conduction studies showed preservation of the CMAP amplitude and normal conduction velocity. EMG of a leg muscle showed a mixed pattern containing both neurogenic and myopathic features. Blood tests revealed an elevated CPK (1091 IU/L, normal values 26-140 IU/L), attributed to his marked hypertonia. Thyroid function was normal. Full blood count, lipids, lysosomal enzymes and carnitine profile were also all normal. Urine organic acids revealed a mildly increased 3-hydroxyisovaleric acid with mild increased TCA cycle intermediate excretion although a lactate and fumarate excretion were not deranged. CSF studies including metabolic investigations were also within normal limits. CSF neurotransmitters where indicative of a methyltetrahydrofolate deficiency (CSF 5methyltetrahydrofolate was 43 nmol/l [72e150]) and for that reason the child was started on 15 mg of Folinic Acid once daily. He was also started on various supplements to include coenzyme Q10, vitamin C, riboflavin, carnitine, biotin and thiamine.
Fig. 1 e Brain MRI. Coronal T2 and Flair weighted and Sagittal T1 weighted sequences showing progressive ponto-cerebellar atrophy with deficiency of the cerebellar vermis. Note also the progressive global brain atrophy.
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Fig. 2 e Muscle Biopsy. Right thigh biopsy taken at 6 years and 10 months of age. There is increased perimysial fat and connective tissue separating the fascicles, with increased variation in fibre size within fascicles (A, B, Haematoxylin and Eosin). Several fibres of varying size show abnormal fetal myosin heavy chain expression (C, inset). Increased lipid staining in the form of punctate large lipid droplets within myofibres is seen (D, Oil Red O). Sequential staining for COX-SDH shows preserved fibre typing, but there is a prominent punctate internal mitochondrial staining pattern with a paucity of sub-sarcolemmal mitochondrial aggregates (E), compared to an age-matched histologically minimal-change control (F). (Scale bar ¼ 100 microns; AeF). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Muscle biopsy was performed at 7 years of age and showed unequivocal myopathic features including increased lipid accumulation and punctate mitochondria aggregation in many fibres compared with an age-matched control (Fig. 2). Additionally, several fibres contained cytoplasmic bodies, which is a rare finding in children. However, there were no other features to suggest a protein aggregation myopathy. Muscle respiratory chain enzyme analysis (RCE) also indicated evidence of a mitochondrial respiratory chain complex IV borderline deficiency in the muscle biopsy provided as the ratio of Muscle RCE complex IV was 0.01 (normal range 0.014e0.034), while the other complexes (I, II, III) were normal. Mitochondrial DNA whole genome sequencing on blood testing for 37 genes including two ribosomal RNA genes, 22 transfer RNA genes and 13 protein coding genes were found to be negative. mtDNA on muscle was borderline low as 58% of mean normal level.
2.1.
Whole exome sequencing (WES)
At this point we decided to perform WES to identify the molecular cause of his phenotype. Exon enrichment was
performed using Agilent Sure Select Human All Exon V4. Paired-end sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry. Read files (Fastq) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were mapped using the Burrows-Wheeler Aligner and local realignment of the mapped reads around potential insertion/deletion (indel) sites was carried out with the GATK version 1.6. Duplicate reads were marked using Picard version 1.62. Additional BAM file manipulations were performed with Samtools 0.1.18. Single nucleotide polymorphism (SNP) and indel variants were called using the GATK Unified Genotyper for each sample. SNP novelty was determined against dbSNP138. Novel variants were analyzed by a range of web-based bioinformatics tools using the EnsEMBL SNP Effect Predictor (http://www.ensembl.org/homosapiens/userdata/ uploadvariations). Analyses of the variant datasets from these patients using a panel of gene prioritization software (http://homes.esat. kuleuven.be/~bioiuser/gpp/tools.php). All variants were screened manually against the Human Gene Mutation Database Professional [Biobase] (http://www.biobase-international.
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 2 0 ( 2 0 1 6 ) 4 8 3 e4 8 8
com/product/hgmd). In silico analysis was performed to determine the potential pathogenicity of the variants. Potentially pathogenic mutations were verified using classic Sanger sequencing. The exome sequencing revealed a homozygous missense mutation 1001T > C in exon 8 of the SEPSECS (ENSG00000109618) gene. The identified mutation was not present in dbSNP and it has been not detected in 400 unrelated control exome studies, nor observed by the 1000 Genomes Project. No other gene mutations/variants that could be linked to the phenotype were identified in the proband. The mutation was confirmed by traditional sanger sequencing and both parents carried the heterozygous mutation. The 1001T > C mutation led to substitution of the conserved tyrosine 334 residue with histidine in the Sepsecs protein, impairing the selenoprotein biosynthesis according to some studies in which patients carried missense mutation (c.1001A > G) in the same codon 334.6
3.
Discussion
The inheritance of PCH2 follows an autosomal recessive pattern.4 Defects in gene SEPSECS are the main cause of pontocerebellar hypoplasia type 2D (PCH2D). This disorder, described previously as progressive cerebello-cerebral atrophy (PCCA), features an autosomal recessive progressive microcephaly with spasticity and profound mental impairment as well as cerebellar (and neocortical) atrophy rather than hypoplasia.7 It has been identified as a mutation disrupting selenocysteine formation by affecting synthesis of the enzyme SEPSECS, which converts Sep-tRNA to SectRNA.6 Mutations in the selenocysteine synthase gene, SEPSECS, were demonstrated to abolish enzyme activity resulting in the disruption of the sole route to selenocysteine biosynthesis and the generation of essential selenoenzymes.6,8 Numerous findings indicated that selenium and selenoproteins are critical to brain development and function, while functional and neuropathologic studies showed that selenoproteins are required for correct hippocampal and cortical interneuron development and the function of neurons in the developing cerebellum.6,9 Treatment of PCH2D is symptomatic and palliative, while prognosis is guarded, since most of patients sadly die in early childhood.2 Our patient showed clinical signs of a mild mitochondrial myopathy, and the mitochondrial investigations performed were borderline abnormal. Interestingly, a decreased level of CSF methyltetrahydrofolate was also determined in the patient which has been associated with RCE dysfunction.10 The brain MRI findings and the clinical semiology in our patient strongly suggest pontocerebellar hypoplasia type 2D. The clinical phenotype of our patient was in accordance with PCH2D and accordingly WES showed a SEPSECS homozygous mutation. The present case has clinical, neuroradiological and genetic evidence of pontocerebellar hypoplasia type 2D associated with two additional features which have never been reported in PCH2D, consisting myopathy with mild mitochondrial abnormalities and optic nerve atrophy. Secondary mitochondria abnormalities have been documented in muscle biopsies of children with muscular
487
dystrophies, SMA and Zellweger syndrome.11,12 A potential secondary mitochondrial involvement has also been recently reported by Anttonen et al. in association with a SEPSECS defect in four Finnish children who presented progressive cerebello-cerebral atrophy and axonal neuropathy associated with lactate elevation in the blood and some (indirect) evidence of mitochondrial dysfunction in their brain samples.8 However, the muscle biopsy in these children did not show any mitochondrial morphologic abnormality and even the RCE analysis on brain samples were reported as normal. On the basis of these findings, the present case further highlights the occurrence of potential secondary mitochondrial involvement in the PCH2D-associated clinical spectrum and also describe for the first time mitochondrial myopathy and borderline RCE deficiency in association with mutation in SEPSECS. Secondary mitochondrial dysfunction in selenoprotein disorders could be explained by the role that selenoproteins have in maintaining the cellular redox potential and the antioxidant defence.13 Many indirect signs of oxidative stress have been also identified in brain samples of patients with SEPSECS deficiency.8 Notably, the Finnish patients with SEPSECS deficiency described by Anttonen et al. were clinically characterised by progressive encephalopathy, oedema of face, hands and feet, dysmorphic features and infantile spasms.8 The combination of these clinical features resemble Progressive encephalopathy with Edema, Hypsarrhythmia, and Optic atrophy (PEHO, MIM 260565) syndrome, although the patients lacked any optic nerve involvement, which has been regarded as one of the major clinical diagnostic criteria. Interestingly, our patient had progressive encephalopathy with cerebello-cerebral atrophy and optic nerve atrophy, although he did not present any EEG abnormalities, hypotonia or oedema. Consequently, one might speculate PEHO syndrome on the presence of a possible continuum clinical spectrum among some patients who carry a homozygous defect of the SEPSECS gene. Previously, a connection between PCHs and PEHO syndrome has been proposed in PCH6 that is caused by RARS mutations.14 The genetic cause of PEHO syndrome still remains undetermined and, on the basis of present and previous findings, our advice is to test patients who present PEHO or PEHO-like features for the SEPSECS gene. Our report further corroborates the notion of a potential secondary mitochondrial dysfunction in the context of selenoprotein biosynthesis deficiency and also adds optic nerve atrophy as a new potential clinical feature within the SEPSECS-associated clinical spectrum. These findings suggest the presence of a possible shared genetic etiology among similar clinical entities characterized by the combination of progressive cerebello-cerebral and optic nerve atrophy and also stress the biological importance of selenoproteins in the regulation of neuronal and metabolic homeostasis.
Consent Written informed consent was obtained from the patient's family for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
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Competing interests The authors declare that they have no competing interests.
Authors' contributions EP and VS analysed and interpreted the case, conducted the literature search and wrote the manuscript. MK was the leading clinician in the clinical care and diagnosis of the patient, provided clinical information; she supervised the writing of the manuscript and critically reviewed it. Various investigations were led by different authors. All authors read and approved the final version of the submitted manuscript.
Conflict of interest With this statement all authors of the submitted manuscript declare that they have no Conflict of Interest.
references
1. Namavar Y, Barth PG, Kasher PR, et al. Clinical neuroradiological and genetic findings in pontocerebellar hypoplasia. Brain 2011b;134:143e56. 2. Namavar Y, Barth PG, Poll-The BT, Baas F. Classification, diagnosis and potential mechanisms in pontocerebellar hypoplasia. Orphanet J Rare Dis 2011a;6:50.
3. Barth PG, Aronica A, De Vries L, et al. Pontocerebellar hypoplasia type 2: a neuropathological update. Acta Neuropathol 2007;114:373e86. 4. Battini R, D'Arrigo S, Cassandrini D, et al. Novel mutations in TSEN54 in pontocerebelar hypoplasia type 2. J Child Neurol 2014;29:520e5. nchez-Albisua I, Fro € lich S, Barth PG, Steinlin M, Kra € geloh5. Sa Mann I. Natural course of pontocerebellar hypoplasia type 2A. Orphanet J Rare Dis 2014;9:70. 6. Agamy O, Ben Zeev B, Lev D, et al. Mutationsdisruptingselenocysteineformation cause progressive cerebello-cerebralatrophy. Am J Hum Genet 2010;87:538e44. 7. Ben-Zeev B, Hoffman C, Lev D, et al. Progressive cerebellocerebral atrophy: a new syndrome with microcephaly, mental retardation, and spastic quadriplegia. J Med Genet 2003;40:e96. 8. Anttonen AK, Hilander T, Linnankivi T, et al. Selenoprotein biosynthesis defect causes progressive encephalopathy with elevated lactate. Neurology 2015;85:306e15. 9. Wirth EK, Bharathi BS, Hatfield D, et al. Cerebellar hypoplasia in mice lacking selenoprotein biosynthesis in neurons. Biol Trace Elem Res 2014;158:203e10. 10. Ormazabal A, Casado M, Molero-Luis M, et al. Can folic acid have a role in mitochondrial disorders? Drug Discov Today 2015;14:1359e70. 11. Katsetos CD, Koutzaki S, Melvin JJ. Mitochondrial dysfunction in neuromuscular disorders. Semin Pediatr Neurol 2013;20:202e15. 12. Salpietro V, Phadke R, Saggar A, et al. Zellweger syndrome and secondary mitochondrial myopathy. Eur J Pediatr 2015;174:557e63. 13. Bellinger FP, Raman AV, Reeves MA, Berry MJ. Regulation and function of selenoproteins in human disease. Biochem J 2009;422:11e22. 14. Rankin J, Brown R, Dobyns WB, et al. Pontocerebellar hypoplasia type 6: a British case with PEHO-like features. Am J Med Genet A 2010;152A:2079e84.
Official Journal of the European Paediatric Neurology Society
Case Study
Pontocerebellar hypoplasia type 2D and optic nerve atrophy further expand the spectrum associated with selenoprotein biosynthesis deficiency Efterpi Pavlidou a, Vincenzo Salpietro a,b, Rahul Phadke b, Iain P. Hargreaves b, Leigh Batten c, Kenneth McElreavy d, Matthew Pitt e,f, Kshitij Mankad e,f, Clare Wilson g, Maria Concetta Cutrupi h, Martino Ruggieri i, David McCormick e,j, Anand Saggar e,k, Maria Kinali a,e,* a
Department of Paediatric Neurology, Chelsea and Westminster NHS Foundation Trust, 369 Fulham Road, London, SW10 9NH, United Kingdom b Department of Molecular Neurosciences, University College of London, Gower Street, London, WC1E 6BT, United Kingdom c The Doctors Laboratory, Bupa Cromwell Hospital Pathology Department, 1-3 Pennant Mews, London, SW5 0TU, United Kingdom d Human Developmental Genetics, Institute Pasteur, 25-28 Rue du Docteur Roux, 75015, Paris, France e The Portland Hospital for Women and Children, 205-209 Great Portland St, London, W1W 5AH, United Kingdom f Great Ormond Street Hospital for Children NHS Foundation Trust, Great Ormond St, London, WC1N 3JH, United Kingdom g Department of Paediatric Ophthalmology, Chelsea and Westminster NHS Foundation Trust, 369 Fulham Road, London, SW10 9NH, United Kingdom h Unit of Genetics and Paediatric Immunology, Department of Paediatrics, University of Messina, Via Consolare Valeria 1, 98125, Messina, Italy i Department of Clinical and Experimental Medicine, University of Catania, Ospedale Garibaldi “Nesima” e Via Palermo, 636, I-95122, Catania, Italy j Department of Paediatrics, King's College Hospital, Denmark Hill, London, SE5 9RS, United Kingdom k St George's Hospital, NHS Foundation Trust, Blackshaw Rd, Tooting, SW17 0QT, London, United Kingdom
List of Abbreviations: MRI, Magnetic resonance imaging; PCH, Pontocerebellar hypoplasia; WES, Whole exome sequencing; RCE, Respiratory chain enzyme analysis. * Corresponding author. Department of Paediatric Neurology, Chelsea and Westminster NHS Foundation Trust, London, United Kingdom. Tel.: þ44 20 3315 8645; fax: þ44 20 3315 7998. E-mail addresses: [email protected] (E. Pavlidou), [email protected] (V. Salpietro), [email protected] (R. Phadke), [email protected] (I.P. Hargreaves), [email protected] (L. Batten), [email protected] (K. McElreavy), [email protected] (M. Pitt), [email protected] (K. Mankad), [email protected] (C. Wilson), mcutrupi@ libero.it (M.C. Cutrupi), [email protected] (M. Ruggieri), [email protected] (D. McCormick), [email protected] (A. Saggar), m. [email protected] (M. Kinali). http://dx.doi.org/10.1016/j.ejpn.2015.12.016 1090-3798/© 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
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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 2 0 ( 2 0 1 6 ) 4 8 3 e4 8 8
article info
abstract
Article history:
Background: The term Pontocerebellar hypoplasias collectively refers to a group of rare,
Received 6 November 2015
heterogeneous and progressive disorders, which are frequently inherited in an autosomal
Received in revised form
recessive manner and usually have a prenatal onset. Mutations in the SEPSECS gene,
29 December 2015
leading to deficiency in selenoprotein biosynthesis, have been identified in recent times as
Accepted 30 December 2015
the molecular etiology of different pre/perinatal onset neurological phenotypes, including cerebello-cerebral atrophy, Pontocerebellar hypoplasia type 2D and progressive encepha-
Keywords:
lopathy with elevated lactate. These disorders share a similar spectrum of central (e.g.,
Pontocerebellar hypoplasia
brain neurodegeneration with grey and white matter both involved) and peripheral (e.g.,
SEPSECS
spasticity due to axonal neuropathy) nervous system impairment.
Selenoprotein biosynthesis
Case presentation: We hereby describe a 9-year-old boy with (i) a typical Pontocerebellar
deficiency
hypoplasia type 2D phenotype (e.g. profound mental retardation, spastic quadriplegia,
Optic nerve atrophy
ponto-cerebellar hypoplasia and progressive cerebral atrophy); (ii) optic nerve atrophy and
Mitochondrial myopathy
(iii) mild secondary mitochondrial myopathy detected by muscle biopsy and respiratory chain enzyme analysis. We performed whole exome sequencing which identified a homozygous mutation of the SEPSECS gene (c.1001T > C), confirming the clinical suspect of Pontocerebellar hypoplasia type 2D. Conclusion: This report further corroborates the notion of a potential secondary mitochondrial dysfunction in the context of selenoprotein biosynthesis deficiency and also adds optic nerve atrophy as a new potential clinical feature within the SEPSECS-associated clinical spectrum. These findings suggest the presence of a possible shared genetic etiology among similar clinical entities characterized by the combination of progressive cerebellocerebral and optic nerve atrophy and also stress the biological importance of selenoproteins in the regulation of neuronal and metabolic homeostasis. © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The term Pontocerebellar hypoplasias (PCHs) collectively refers to a group of rare, heterogeneous and progressive disorders, which are inherited in an autosomal recessive manner and usually have a prenatal onset.1 PCHs are generally characterized by an abnormally small cerebellum and ventral pons. Supra-tentorial structures are also affected, although less prominently. The estimated incidence is calculated less than 1:200.000 within the general paediatric population.2 The main clinical features are characterised by severe psychomotor delay and microcephaly. In most cases, PCHs have a severe prognosis and are fatal in early childhood. At least ten types of PCHs have been described so far, with PCH Type 2 (PCH2) being the most frequently reported. Children with PCH2 have microcephaly since birth, associated with extrapyramidal dyskinesia and spasticity.3 PCH2 is usually suspected on the basis of specific magnetic resonance imaging (MRI) features, genetic and neuropathological findings. The understanding of the natural history of this disorder is extremely important in prenatal counselling of affected families and also in management and prognosis of these children. Most of typical PCH2 cases so far reported are due to missense mutations in the TSEN54 (MIM 608755) gene on chromosome 17.4 Less frequently reported PCH2 sub-types, namely PCH2B, PCH2C and PCH2D are due to mutations in the genes TSEN2 (MIM 608753), TSEN34 (MIM 608754) and SEPSECS (MIM 613009), respectively.5,6 Mutation in SEPSECS
impair the cytoplasmic transfer RNA (tRNA)-charging in the selenoprotein biosynthesis pathway. We hereby report on a child diagnosed with a clinical and radiological diagnosis of PCH2 who underwent whole exome sequencing (WES), which disclosed a pathogenic mutation in the SEPSECS gene. The child also had a severe visual impairment due to optic atrophy and a mild secondary mitochondrial dysfunction in his muscles showed by muscle biopsy and respiratory chain enzyme analysis. We described on the present boy as these latter clinical features (i.e., mitochondrial myopathy, optic nerve atrophy) contribute in further expanding the clinical spectrum associated with SEPSECS mutation, highlighting the potentially crucial role of selenoproteins.
2.
Case study
An 8-year-old boy was referred to our Department due to profound psychomotor delay, quadriplegia and microcephaly. He was the third child of two consanguineous Arabian parents. He had two elder and one younger brothers. His prenatal ultrasound was normal and his birth was via uncomplicated vaginal delivery with no need for resuscitation or admission to special care. Additionally, his feeding was normal at birth. Birth weight was 2700 gr (10th). Shortly after birth he was noticed to have generalized hypotonia. At 2 months his head circumference was 36.5 cm (<10th). Brain MRI was performed at 18 months of age and showed a large collection of
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 2 0 ( 2 0 1 6 ) 4 8 3 e4 8 8
cerebrospinal fluid (CSF) inferiorly to the cerebellum communicating with the 4th ventricle. There were no focal lesions within the brain parenchyma. The child initially presented to our clinic at the age of 6.5 years old with spastic quadriplegia, microcephaly, severe mental retardation and inability to swallow normally. Clinical examination revealed severe hypertonia probably since birth, neurogenic muscle atrophy and decreased deep tendon reflexes, especially at his lower limbs. Nerve conduction studies demonstrated borderline reductions of the sensory nerve action potentials from the medial plantar and superficial peroneal nerves with normal sensory nerve conduction velocities. Motor conduction studies showed normal compound motor action potential (CMAP) amplitude and velocity. Electromyography (EMG) of a leg muscle showed no indication of either motor nerve involvement or myopathy. Ophthalmological examination revealed optic atrophy with impaired vision. The child also had bilateral femoral derotation varus osteotomies and scoliosis. The child experienced intense pain due to the severe neuropathy and for this reason he was taking carbamazepine 100 mg twice daily and gabapentin 500 mg twice daily. He was also started on melatonin for sleep disorder and ranitidine and omeprazole as anti-reflux medication. At the age of 7 the patient had suffered severe bronchopneumonia complicated by pneumothorax and required ventilation in intensive care. He had poor feeding, trouble managing secretions and had
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marked cachexia. Therefore, gastrostomy and Nissen fundoplication were mandatory. A second brain MRI was performed at the age of 7 years old showing progressive pontocerebellar atrophy with deficiency of the cerebellar vermis (Fig. 1). There was also progressive global brain atrophy. Repeat nerve conduction studies, 11 months after the first studies, showed absence of sensory responses not only in the leg but also in the arm. Motor conduction studies showed preservation of the CMAP amplitude and normal conduction velocity. EMG of a leg muscle showed a mixed pattern containing both neurogenic and myopathic features. Blood tests revealed an elevated CPK (1091 IU/L, normal values 26-140 IU/L), attributed to his marked hypertonia. Thyroid function was normal. Full blood count, lipids, lysosomal enzymes and carnitine profile were also all normal. Urine organic acids revealed a mildly increased 3-hydroxyisovaleric acid with mild increased TCA cycle intermediate excretion although a lactate and fumarate excretion were not deranged. CSF studies including metabolic investigations were also within normal limits. CSF neurotransmitters where indicative of a methyltetrahydrofolate deficiency (CSF 5methyltetrahydrofolate was 43 nmol/l [72e150]) and for that reason the child was started on 15 mg of Folinic Acid once daily. He was also started on various supplements to include coenzyme Q10, vitamin C, riboflavin, carnitine, biotin and thiamine.
Fig. 1 e Brain MRI. Coronal T2 and Flair weighted and Sagittal T1 weighted sequences showing progressive ponto-cerebellar atrophy with deficiency of the cerebellar vermis. Note also the progressive global brain atrophy.
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Fig. 2 e Muscle Biopsy. Right thigh biopsy taken at 6 years and 10 months of age. There is increased perimysial fat and connective tissue separating the fascicles, with increased variation in fibre size within fascicles (A, B, Haematoxylin and Eosin). Several fibres of varying size show abnormal fetal myosin heavy chain expression (C, inset). Increased lipid staining in the form of punctate large lipid droplets within myofibres is seen (D, Oil Red O). Sequential staining for COX-SDH shows preserved fibre typing, but there is a prominent punctate internal mitochondrial staining pattern with a paucity of sub-sarcolemmal mitochondrial aggregates (E), compared to an age-matched histologically minimal-change control (F). (Scale bar ¼ 100 microns; AeF). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Muscle biopsy was performed at 7 years of age and showed unequivocal myopathic features including increased lipid accumulation and punctate mitochondria aggregation in many fibres compared with an age-matched control (Fig. 2). Additionally, several fibres contained cytoplasmic bodies, which is a rare finding in children. However, there were no other features to suggest a protein aggregation myopathy. Muscle respiratory chain enzyme analysis (RCE) also indicated evidence of a mitochondrial respiratory chain complex IV borderline deficiency in the muscle biopsy provided as the ratio of Muscle RCE complex IV was 0.01 (normal range 0.014e0.034), while the other complexes (I, II, III) were normal. Mitochondrial DNA whole genome sequencing on blood testing for 37 genes including two ribosomal RNA genes, 22 transfer RNA genes and 13 protein coding genes were found to be negative. mtDNA on muscle was borderline low as 58% of mean normal level.
2.1.
Whole exome sequencing (WES)
At this point we decided to perform WES to identify the molecular cause of his phenotype. Exon enrichment was
performed using Agilent Sure Select Human All Exon V4. Paired-end sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry. Read files (Fastq) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were mapped using the Burrows-Wheeler Aligner and local realignment of the mapped reads around potential insertion/deletion (indel) sites was carried out with the GATK version 1.6. Duplicate reads were marked using Picard version 1.62. Additional BAM file manipulations were performed with Samtools 0.1.18. Single nucleotide polymorphism (SNP) and indel variants were called using the GATK Unified Genotyper for each sample. SNP novelty was determined against dbSNP138. Novel variants were analyzed by a range of web-based bioinformatics tools using the EnsEMBL SNP Effect Predictor (http://www.ensembl.org/homosapiens/userdata/ uploadvariations). Analyses of the variant datasets from these patients using a panel of gene prioritization software (http://homes.esat. kuleuven.be/~bioiuser/gpp/tools.php). All variants were screened manually against the Human Gene Mutation Database Professional [Biobase] (http://www.biobase-international.
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com/product/hgmd). In silico analysis was performed to determine the potential pathogenicity of the variants. Potentially pathogenic mutations were verified using classic Sanger sequencing. The exome sequencing revealed a homozygous missense mutation 1001T > C in exon 8 of the SEPSECS (ENSG00000109618) gene. The identified mutation was not present in dbSNP and it has been not detected in 400 unrelated control exome studies, nor observed by the 1000 Genomes Project. No other gene mutations/variants that could be linked to the phenotype were identified in the proband. The mutation was confirmed by traditional sanger sequencing and both parents carried the heterozygous mutation. The 1001T > C mutation led to substitution of the conserved tyrosine 334 residue with histidine in the Sepsecs protein, impairing the selenoprotein biosynthesis according to some studies in which patients carried missense mutation (c.1001A > G) in the same codon 334.6
3.
Discussion
The inheritance of PCH2 follows an autosomal recessive pattern.4 Defects in gene SEPSECS are the main cause of pontocerebellar hypoplasia type 2D (PCH2D). This disorder, described previously as progressive cerebello-cerebral atrophy (PCCA), features an autosomal recessive progressive microcephaly with spasticity and profound mental impairment as well as cerebellar (and neocortical) atrophy rather than hypoplasia.7 It has been identified as a mutation disrupting selenocysteine formation by affecting synthesis of the enzyme SEPSECS, which converts Sep-tRNA to SectRNA.6 Mutations in the selenocysteine synthase gene, SEPSECS, were demonstrated to abolish enzyme activity resulting in the disruption of the sole route to selenocysteine biosynthesis and the generation of essential selenoenzymes.6,8 Numerous findings indicated that selenium and selenoproteins are critical to brain development and function, while functional and neuropathologic studies showed that selenoproteins are required for correct hippocampal and cortical interneuron development and the function of neurons in the developing cerebellum.6,9 Treatment of PCH2D is symptomatic and palliative, while prognosis is guarded, since most of patients sadly die in early childhood.2 Our patient showed clinical signs of a mild mitochondrial myopathy, and the mitochondrial investigations performed were borderline abnormal. Interestingly, a decreased level of CSF methyltetrahydrofolate was also determined in the patient which has been associated with RCE dysfunction.10 The brain MRI findings and the clinical semiology in our patient strongly suggest pontocerebellar hypoplasia type 2D. The clinical phenotype of our patient was in accordance with PCH2D and accordingly WES showed a SEPSECS homozygous mutation. The present case has clinical, neuroradiological and genetic evidence of pontocerebellar hypoplasia type 2D associated with two additional features which have never been reported in PCH2D, consisting myopathy with mild mitochondrial abnormalities and optic nerve atrophy. Secondary mitochondria abnormalities have been documented in muscle biopsies of children with muscular
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dystrophies, SMA and Zellweger syndrome.11,12 A potential secondary mitochondrial involvement has also been recently reported by Anttonen et al. in association with a SEPSECS defect in four Finnish children who presented progressive cerebello-cerebral atrophy and axonal neuropathy associated with lactate elevation in the blood and some (indirect) evidence of mitochondrial dysfunction in their brain samples.8 However, the muscle biopsy in these children did not show any mitochondrial morphologic abnormality and even the RCE analysis on brain samples were reported as normal. On the basis of these findings, the present case further highlights the occurrence of potential secondary mitochondrial involvement in the PCH2D-associated clinical spectrum and also describe for the first time mitochondrial myopathy and borderline RCE deficiency in association with mutation in SEPSECS. Secondary mitochondrial dysfunction in selenoprotein disorders could be explained by the role that selenoproteins have in maintaining the cellular redox potential and the antioxidant defence.13 Many indirect signs of oxidative stress have been also identified in brain samples of patients with SEPSECS deficiency.8 Notably, the Finnish patients with SEPSECS deficiency described by Anttonen et al. were clinically characterised by progressive encephalopathy, oedema of face, hands and feet, dysmorphic features and infantile spasms.8 The combination of these clinical features resemble Progressive encephalopathy with Edema, Hypsarrhythmia, and Optic atrophy (PEHO, MIM 260565) syndrome, although the patients lacked any optic nerve involvement, which has been regarded as one of the major clinical diagnostic criteria. Interestingly, our patient had progressive encephalopathy with cerebello-cerebral atrophy and optic nerve atrophy, although he did not present any EEG abnormalities, hypotonia or oedema. Consequently, one might speculate PEHO syndrome on the presence of a possible continuum clinical spectrum among some patients who carry a homozygous defect of the SEPSECS gene. Previously, a connection between PCHs and PEHO syndrome has been proposed in PCH6 that is caused by RARS mutations.14 The genetic cause of PEHO syndrome still remains undetermined and, on the basis of present and previous findings, our advice is to test patients who present PEHO or PEHO-like features for the SEPSECS gene. Our report further corroborates the notion of a potential secondary mitochondrial dysfunction in the context of selenoprotein biosynthesis deficiency and also adds optic nerve atrophy as a new potential clinical feature within the SEPSECS-associated clinical spectrum. These findings suggest the presence of a possible shared genetic etiology among similar clinical entities characterized by the combination of progressive cerebello-cerebral and optic nerve atrophy and also stress the biological importance of selenoproteins in the regulation of neuronal and metabolic homeostasis.
Consent Written informed consent was obtained from the patient's family for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
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Competing interests The authors declare that they have no competing interests.
Authors' contributions EP and VS analysed and interpreted the case, conducted the literature search and wrote the manuscript. MK was the leading clinician in the clinical care and diagnosis of the patient, provided clinical information; she supervised the writing of the manuscript and critically reviewed it. Various investigations were led by different authors. All authors read and approved the final version of the submitted manuscript.
Conflict of interest With this statement all authors of the submitted manuscript declare that they have no Conflict of Interest.
references
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