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Use of Next-Generation Sequencing as a Diagnostic Tool for Congenital Myasthenic Syndrome
Pediatric Neurology 51 (2014) 717e720
Contents lists available at ScienceDirect
Pediatric Neurology journal homepage: www.elsevier.com/locate/pnu
Clinical Observations
Use of Next-Generation Sequencing as a Diagnostic Tool for Congenital Myasthenic Syndrome Alvin S. Das MD a, Dimitri P. Agamanolis MD b, c, Bruce H. Cohen MD d, e, * a
Northeast Ohio Medical University, Rootstown, Ohio Department of Pathology and Laboratory Medicine, Akron Children’s Hospital, Akron, Ohio c Department of Pathology, Northeast Ohio Medical University, Rootstown, Ohio d Division of Neurology, NeuroDevelopmental Science Center, Akron Children’s Hospital, Akron, Ohio e Department of Pediatrics, Northeast Ohio Medical University, Rootstown, Ohio b
abstract BACKGROUND: The clinical presentation of congenital myasthenic syndromes is similar to many other neuromus-
cular disorders of infancy, and with 12 known discrete genetic forms of congenital myasthenic syndromes, both the diagnosis and treatment decisions present clinical challenges. PATIENT DESCRIPTION: We report a 20-month-old boy with rapsyn deficiency. At birth, he presented with a weak cry, hypotonia, joint contractures, and facial deformity. Because of respiratory difficulty associated with muscle fatigue, he spent a total of 71 days in the neonatal intensive care unit and 47 days in the pediatric intensive care unit. Imaging study results were normal, along with a battery of metabolic tests and electrodiagnostic studies. A limited genetic evaluation for reversible cytochrome c oxidase deficiency was negative, as was the oligonucleotide microarray. A muscle biopsy demonstrated myofiber atrophy in a pattern consistent with early denervation. Based on nonspecific and nondiagnostic results, whole-exome (next generation) sequencing was performed. This study identified two confirmed pathogenic mutations in the RAPSN gene that are associated with congenital myasthenic syndrome (OMIM 608931). The patient was treated with pyridostigmine at 16 months of age, which resulted in a dramatic improvement in muscle tone and strength and a steady resolution of joint contractures. Four months after treatment was initiated, he was beginning to bear weight and was able to sit unsupported and vocalize full words. CONCLUSIONS: This patient serves to highlight next-generation sequencing as an important diagnostic tool that can result in life-saving treatment. Keywords: congenital myasthenic syndrome, rapsyn, next-generation sequencing, pyridostigmine
Pediatr Neurol 2014; 51: 717-720 Ó 2014 Elsevier Inc. All rights reserved.
Introduction
Congenital myasthenic syndromes (CMS) comprise a spectrum of at least a dozen genetic disorders characterized by abnormal synaptic transmission at the neuromuscular junction. Mutations in genes encoding the proteins that make up the presynaptic, synaptic basal lamina, and postsynaptic neuromuscular junction have been identified in
Article History: Received May 20, 2014; Accepted in final form July 31, 2014 * Communications should be addressed to: Dr. Cohen; Department of Neurology; NeuroDevelopmental Science Center; Akron Children’s Hospital; 215 W. Bowery Street; Suite 4400; Akron, Ohio 44308. E-mail address: [email protected] 0887-8994/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pediatrneurol.2014.07.032
patients with CMS. Genetic inheritance patterns include both autosomal dominant and recessive expressions. Specifically, mutations in RAPSN, CHAT, AGRN, COLQ, and DOK7 genes (among others) are inherited in an autosomal recessive pattern, and genes encoding the acetylcholine receptor (AChR) subunits (CHRNA1, CHRNB1, CHRND, and CHRNE) are inherited in an autosomal dominant pattern.1 Clinical signs of the disease are usually evident at or soon after birth and include muscle weakness, hypotonia, bulbar paresis, facial paresis, hypoventilation or episodic apnea, and ptosis. Because of the vast number of mutations resulting in CMS, clinical manifestations of the disease vary greatly with signs presenting at birth or delayed until adolescence or even adulthood.2 In addition, the presentation is not specific and is mimicked by other genetic diseases and disorders of
718
A.S. Das et al. / Pediatric Neurology 51 (2014) 717e720
muscle or collagen. The primary treatment for some of these disorders is pyridostigmine or 3,4-diaminopyridine. However, the effectiveness of therapy is heavily dependent on the underlying mutation resulting in the disease, and the manifestations of some forms of CMS can be worsened by pyridostigmine.2,3 In this case report, a patient was diagnosed using next-generation (NextGen) sequencing (NGS) technology, which identified compound heterozygote mutations in RAPSN, the gene-encoding receptor-associated protein of the synapse (rapsyn). Rapsyn is a protein involved in aggregation, assembly, and stabilization of the postsynaptic AChRs at the neuromuscular junction. NextGen sequencing refers to several different methodologies that have evolved since the 1990s. NGS cannot detect all genetic disorders and is currently not an appropriate technology for detecting trinucleotide repeats, mutations in the noncoding regions, or copy number variants.4 Whole-exome sequencing (WES) uses NGS technology and provides sequencing of the approximately 23,000 proteincoding genes. The protein-coding region (termed exome) constitutes a small percentage (roughly 1.5%) of the genome, although estimates show that mutations in the exome account for approximately 85% of the mutations linked to human disease, and first performed in humans in 2009.5,6 Whole exome sequencing allowed us to definitively diagnose an infant with a treatable disorder so this report emphasizes the importance of WES in current clinical practice because its use can result in a crucial diagnosis that would otherwise go undiagnosed or misdiagnosed. Patient Description This boy was born at 33 weeks of gestation to a 30-year-old G2P1101 woman and delivered by Cesarean section because of severe oligohydramnios. Fetal ultrasonography revealed a choroid plexus cyst along with hydronephrosis, both of which resolved with no apparent sequelae. The mother had previously delivered a dysmorphic term female infant who died 2 hours after birth from complications of meconium aspiration. The postmortem examination revealed arthrogryposis, and the muscle histology was suspicious for denervation atrophy, but spinal muscular atrophy gene testing was normal. His parents are of English, Irish, Scottish, and German ancestry. The Apgar scores were 4 and 7 at 1 and 5 minutes, respectively. Birth weight was 2050 g (fiftieth percentile), length was 42 cm (twentieth percentile), and head circumference was 33.5 cm (greater than ninetieth percentile). Initial laboratory studies were unremarkable demonstrating normal amino acid, fatty acid, and organic acid profiles and a thyroid stimulating hormone of 4.5 mIU/mL (normal, <34 mIU/ mL). Hemoglobin electrophoresis and newborn screening study results were normal for biotinidase deficiency, galactosemia, congenial adrenal hyperplasia, and cystic fibrosis. The Ohio Department of Health newborn screening evaluation was normal. At delivery, he had a weak cry, hypotonia, and joint contractures. He failed continuous positive airway pressure (CPAP) and was intubated after treatment with poractant alfa. He remained intubated for 1 day and was then weaned to nasal CPAP, which was used intermittently during his 71-day neonatal intensive care unit course. His course was complicated by several aspiration pneumonias. Eventually, arterial blood gases demonstrated stable carbon dioxide measurements between 40 and 44 Hg, obviating the need for CPAP. The patient had been started on caffeine initially for apnea of prematurity and was continued on caffeine as it resulted in improvements in respiratory function and drive. Multiple congenital abnormalities were observed including a prominent occiput, bilaterally low-set ears, hypertelorism with small palpebral fissures, micrognathia, a broad nasal bridge, a high-arched palate,
cortical thumbs, and multiple limb contractures. Physical examination revealed coarse breath sounds, normal heart sounds and heart rhythm, bounding femoral pulses, no abdominal masses, an undescended left testicle and a fully descended right testicle, decreased motor tone, limited spontaneous movement, decreased muscle stretch reflexes, and weak, but present, Moro and grasp reflexes. Audiologic and ophthalmologic evaluations were normal. Muscle and nerve biopsies were obtained during his gastrostomy tube placement and Nissen fundoplication. The nerve biopsy was normal, but the muscle biopsy revealed mild myofiber atrophy (without fiber-type grouping) consistent with early nerve denervation and was similar to findings from the patient’s older sibling. Testing for Cockayne syndrome was negative for mutations in CSB/ERCC6 and CSA/ERCC8 genes by multiplex ligation-dependent probe amplification. A magnetic resonance imaging was performed at 1 month of life, which revealed a nonspecific triangular focus of mildly increased signal in the right putamen (more prominent on T2-weighted image). Serum studies demonstrated a lactate level of 1.4 mmol/L (normal, 0.6-2.4 mmol/L) and a creatine kinase level of 88 U/L (normal, 24-195 U/L). The patient was then discharged to home with multivitamin supplementation (1 mL/day) and caffeine (40 mg/day). After 17 days at home, the patient developed an apneic episode and was evident to have aspiration pneumonia and sepsis resulting in a 47-day hospitalization. Echocardiogram and electrocardiogram study results were normal. A host of metabolic study results were normal including organic acids, carnitine, oligosaccharides, and guanidinoacetic acid, and serum very long-chain fatty acids, carbohydrate-deficient transferrin, and coenzyme Q10. Furthermore, Prader-Willi methylation, reversible cytochrome c oxidase deficiency (mitochondrial DNA m.14674 T), and electroencephalography study results were normal. The chromosomal microarray was normal as well. At his 3-week follow-up after discharge (at 5 months of age), he was evaluated by a neurologist (B.H.C.) as he continued to have a paucity of movements and remained hypotonic with joint contractures of the thumb, knee, and elbow. Additionally there was decreased motor bulk in all muscle groups with pronounced weakness in the jaw; his mouth was triangular shaped because of weakness of the muscles of facial expression. The patient was only able to elevate his head for very brief periods. He was able to move all his extremities spontaneously but had more difficulty when he was in an upright posture than when he was supine. Cognitive function was progressing appropriately. Visual fields were normal by confrontation; however, mild ptosis was observed. Based on clinical signs, consideration of Ullrich congenital muscular dystrophy was entertained, but sequencing of COL6A1, COL6A2, and COL6A3 genes was normal. At his 8-month visit, he had developed modest head control and was able to extend his trunk. Furthermore, an overall increase in limb movement was observed with increased elbow flexion and/or extension and active wrist flexion. At his 10-month visit, the patient was able to say a few words and was able to turn his head side-to-side in the supine position, as he was still not able to hold his head unassisted. His breathing and secretions were positional, and his cough was getting stronger, although he still required a great deal of suctioning. At this point in the care of the patient, as all the tests and diagnostic imaging provided minimal leads, WES (next generation) was performed on a clinical basis (GeneDx Labs). Results from this study, confirmed by Sanger sequencing, identified two in trans mutations (c.264 C >A and c.1083_1084dupCT) in the RAPSN gene leading to the diagnosis of CMS. At 16 months of age, the patient was administered 1.8 mg (60 mg/5 mL) of pyridostigmine (Mestinon). Within 7 minutes, the patient started kicking and his ability to sit unsupported had improved to a greater extent than witnessed before. Additionally within the next few hours, his drooling had ceased as he began swallowing saliva. Three months after starting pyridostigmine, a dramatic improvement in clinical impairments was observed. He was able to sit unsupported and began using his hands for signing and manipulating objects. His fine motor skills were appropriate for age level; however, gross motor skills were still delayed. At his last examination, he was sitting independently and was beginning to bear weight and walk in a gait trainer. The patient demonstrated a great deal of babbling and was able to say approximately seven to eight words. His motor bulk was still reduced but was
A.S. Das et al. / Pediatric Neurology 51 (2014) 717e720 improving in all muscle groups. Additionally his hypotonia had decreased to near normal and his joint contractures had significantly improved. Muscle strength was still globally weak, but his trunk and upper extremity strength had greatly improved. Before treatment, he had frequent intensive care unit admissions for respiratory failure; these essentially resolved after therapy.
Discussion
Historically, the diagnosis of CMS is based on the clinical and laboratory findings present in Table. However, many of the clinical features overlap with the congenital myopathies and muscular dystrophies. In some instances, a histologic analysis or electromyogram (EMG) studies are sufficient for diagnosis; however, in other types of CMS, a much more thorough investigation using data from electrophysiological, ultrastructural, and immunocytochemical studies is needed.7 Single muscle fiber EMG is an excellent diagnostic modality8 but requires considerable experience and is often difficult or impossible to obtain in a child with minimal muscle bulk. Furthermore, infant EMG services are also not available in many medical centers. Therefore, in spite of considerable discussion about the possible diagnoses, neither an EMG nor a trial of an acetylcholinesterase inhibitor was performed in this case prior to receiving the results of WES. WES provided a rapid diagnosis of RAPSN-associated CMS based on complex heterozygote mutations inherited in trans. Both disease-associated confirmed missense mutation at c.264 C>A resulting in a lysine substitution for asparagine at codon 88 (N88K) in exon 2 and a previously confirmed frameshift mutation at (p.Tyr362SerfsX10) resulting in generation of a premature stop codon at 371 in exon 7 of RAPSN were identified. The parents were tested and were evident to each be a carrier of one variant allele. This heterozygous combination (N88K and c.1083_ 1084dupCT) has been previously described in two patients with similar phenotypes (arthrogryposis and respiratory distress) and similar clinical courses as the patient described in this report.9,10 Both cases improved with anticholinesterase treatment, and interestingly, one of the patients had an older sister with arthrogryposis.11 TABLE. Clinical and Laboratory Features of Congenital Myasthenic Syndromes3,7
Clinical features Weakness of bulbar, ocular, or limb muscles most often at birth High-arched palate (variable) Contractures (variable) Positive family history for the disease (in autosomal dominant cases) Positive motor response to edrophonium Laboratory features Decremental electromyographic (EMG) response of the compound muscle action potential on 2- to 3-Hz stimulation Increased jitter on single-fiber EMG Normal or slightly elevated creatine kinase Absence of overt pathologic findings in a muscle biopsy Negative serum antibodies for ACh receptors (found in myasthenia gravis), muscle specific kinase, and P/Q-type calcium channels Abbreviation: ACh ¼ Acetylcholine
719
Illness caused by RAPSN mutations exhibits an autosomal recessive inheritance pattern and accounts for approximately 15-20% of all CMS. RAPSN, located on 11p11.2, encodes for a 43-kDA cyclic adenosine monophosphate-dependent protein kinase, rapsyn. Rapsyn functions as the link between the AChR and the agrin-binding dystrophin-associated glycoprotein complex and functions to maintain the correct position and orientation of the AChR in the postsynaptic junction. In vitro studies suggest N88K rapsyn results in reduced colocalization with the AChR and causes diminished postsynaptic receptor clustering and receptor stability.12 In patients with RAPSN-associated CMS, signs manifest early in infancy but can present in the second and third decades.13 Furthermore, arthrogryposis (as was observed in this family), ptosis, and multiple joint contractures present at birth are characteristic findings of this specific type of CMS.14 In addition, a decremental EMG response to lowfrequency stimulation can be mild or absent.3 As observed with our patient, respiratory infections, stress, and other febrile illnesses exacerbate muscle fatigability and can precipitate respiratory arrest and anoxic encephalopathy.14 Fortunately, most of the patients demonstrate a favorable response to pyridostigmine, and case reports reveal patients benefiting from 3,4-diaminopyridine (an orphan drug), as well as ephedrine or albuterol.13,15 In the current case, after we provided the appropriate treatment with anticholinesterase therapy, our patient displayed marked improvements in muscle strength, bulbar function, and fine motor control. Conclusions
CMS are often misdiagnosed or underdiagnosed. These conditions closely resemble other neuromuscular disorders, physicians are less familiar with them than with other conditions, some patients exhibit a paucity of classic clinical signs that point to the diagnosis of CMS, and lastly, not all institutions have the EMG sophistication to accurately diagnose CMS.3 The use of NGS in diagnosis underscores the importance of this technology and significance of this case. Furthermore, identification of the specific mutation is crucial in this disease as treatment outcomes are dependent on the specific type of CMS; moreover, acetylcholine esterase inhibitor therapy can have deleterious consequences if used to treat some forms of CMS.3 As is evident from our patient’s clinical improvement, NGS played a pivotal role in his diagnosis and treatment. The cost of this technology fares well considering the saved future costs of additional diagnostic testing and averted hospitalizations. It should be considered in patients with an undiagnosed disease with a suspected genetic etiology. B.H.C. has received compensation from the American Academy of Neurology for his activities as an Assistant Advisor to Current Procedural Terminology and speakers honoraria, from Motive Medical Intelligence for his activities as a content editor and medical writer, and as a speaker for Transgenomic Labs and Courtagen Labs. He has received travel reimbursement for clinical research activities from Edison Pharmaceutical and Raptor Pharmaceutical, and his primary institution receives financial compensation for research conducted by him. He has a consulting agreement with Stealth Peptides and intends to conduct clinical research with this company. A.S.D. and D.P.A. have no conflicts to report.
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References 1. Abicht A, Müller JS, Lochmüller H. Congenital myasthenic syndromes [Updated 2012 Jun 28]. In: Pagon RA, Adam MP, Bird TD, et al., eds. GeneReviewsÔ [Internet]. Seattle (WA): University of Washington, Seattle; 2003:1993-2013. 2. Lorenzoni PJ, Scola RH, Kay CS, Werneck LC. Congenital myasthenic syndrome: a brief review. Pediatr Neurol. 2012;46:141-148. 3. Engel AG. The therapy of congenital myasthenic syndromes. Neurotherapeutics. 2007;4:252-257. 4. Bamshad MJ, Ng SB, Bigham AW, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 2011;12:745-755. 5. Foo JN, Liu JJ, Tan EK. Whole-genome and whole-exome sequencing in neurological diseases. Nat Rev Neurol. 2012;8:508-517. 6. Ng SB, Turner EH, Robertson PD, et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009; 461:272-276. 7. Engel AG, Ohno K, Sine SM. Congenital myasthenic syndromes: progress over the past decade. Muscle Nerve. 2003;27:4-25. 8. Rabie M, Jossiphov J, Nevo Y. Electromyography (EMG) accuracy compared to muscle biopsy in childhood. J Child Neurol. 2007;22: 803-808.
9. Richard P, Gaudon K, Andreux F, et al. Possible founder effect of rapsyn N88K mutation and identification of novel rapsyn mutations in congenital myasthenic syndromes. J Med Genet. 2003; 40:e81. 10. Ioos C, Barois A, Richard P, Eymard B, Hantaï D, EstournetMathiaud B. Congenital myasthenic syndrome due to rapsyn deficiency: three cases with arthrogryposis and bulbar symptoms. Neuropediatrics. 2004;35:246-249. 11. Cossins J, Burke G, Maxwell S, et al. Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations. Brain. 2006; 129(Pt 10):2773-2783. 12. Milone M, Shen XM, Selcen D, et al. Myasthenic syndrome due to defects in rapsyn: clinical and molecular findings in 39 patients. Neurology. 2009;73:228-235. 13. Engel AG. Congenital myasthenic syndromes in 2012. Curr Neurol Neurosci Rep. 2012;12:92-101. 14. Engel AG. Current status of the congenital myasthenic syndromes. Neuromuscul Disord. 2012;22:99-111. 15. Banwell BL, Ohno K, Sieb JP, Engel AG. Novel truncating RAPSN mutations causing congenital myasthenic syndrome responsive to 3,4-diaminopyridine. Neuromuscul Disord. 2004; 14:202-207.
Contents lists available at ScienceDirect
Pediatric Neurology journal homepage: www.elsevier.com/locate/pnu
Clinical Observations
Use of Next-Generation Sequencing as a Diagnostic Tool for Congenital Myasthenic Syndrome Alvin S. Das MD a, Dimitri P. Agamanolis MD b, c, Bruce H. Cohen MD d, e, * a
Northeast Ohio Medical University, Rootstown, Ohio Department of Pathology and Laboratory Medicine, Akron Children’s Hospital, Akron, Ohio c Department of Pathology, Northeast Ohio Medical University, Rootstown, Ohio d Division of Neurology, NeuroDevelopmental Science Center, Akron Children’s Hospital, Akron, Ohio e Department of Pediatrics, Northeast Ohio Medical University, Rootstown, Ohio b
abstract BACKGROUND: The clinical presentation of congenital myasthenic syndromes is similar to many other neuromus-
cular disorders of infancy, and with 12 known discrete genetic forms of congenital myasthenic syndromes, both the diagnosis and treatment decisions present clinical challenges. PATIENT DESCRIPTION: We report a 20-month-old boy with rapsyn deficiency. At birth, he presented with a weak cry, hypotonia, joint contractures, and facial deformity. Because of respiratory difficulty associated with muscle fatigue, he spent a total of 71 days in the neonatal intensive care unit and 47 days in the pediatric intensive care unit. Imaging study results were normal, along with a battery of metabolic tests and electrodiagnostic studies. A limited genetic evaluation for reversible cytochrome c oxidase deficiency was negative, as was the oligonucleotide microarray. A muscle biopsy demonstrated myofiber atrophy in a pattern consistent with early denervation. Based on nonspecific and nondiagnostic results, whole-exome (next generation) sequencing was performed. This study identified two confirmed pathogenic mutations in the RAPSN gene that are associated with congenital myasthenic syndrome (OMIM 608931). The patient was treated with pyridostigmine at 16 months of age, which resulted in a dramatic improvement in muscle tone and strength and a steady resolution of joint contractures. Four months after treatment was initiated, he was beginning to bear weight and was able to sit unsupported and vocalize full words. CONCLUSIONS: This patient serves to highlight next-generation sequencing as an important diagnostic tool that can result in life-saving treatment. Keywords: congenital myasthenic syndrome, rapsyn, next-generation sequencing, pyridostigmine
Pediatr Neurol 2014; 51: 717-720 Ó 2014 Elsevier Inc. All rights reserved.
Introduction
Congenital myasthenic syndromes (CMS) comprise a spectrum of at least a dozen genetic disorders characterized by abnormal synaptic transmission at the neuromuscular junction. Mutations in genes encoding the proteins that make up the presynaptic, synaptic basal lamina, and postsynaptic neuromuscular junction have been identified in
Article History: Received May 20, 2014; Accepted in final form July 31, 2014 * Communications should be addressed to: Dr. Cohen; Department of Neurology; NeuroDevelopmental Science Center; Akron Children’s Hospital; 215 W. Bowery Street; Suite 4400; Akron, Ohio 44308. E-mail address: [email protected] 0887-8994/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pediatrneurol.2014.07.032
patients with CMS. Genetic inheritance patterns include both autosomal dominant and recessive expressions. Specifically, mutations in RAPSN, CHAT, AGRN, COLQ, and DOK7 genes (among others) are inherited in an autosomal recessive pattern, and genes encoding the acetylcholine receptor (AChR) subunits (CHRNA1, CHRNB1, CHRND, and CHRNE) are inherited in an autosomal dominant pattern.1 Clinical signs of the disease are usually evident at or soon after birth and include muscle weakness, hypotonia, bulbar paresis, facial paresis, hypoventilation or episodic apnea, and ptosis. Because of the vast number of mutations resulting in CMS, clinical manifestations of the disease vary greatly with signs presenting at birth or delayed until adolescence or even adulthood.2 In addition, the presentation is not specific and is mimicked by other genetic diseases and disorders of
718
A.S. Das et al. / Pediatric Neurology 51 (2014) 717e720
muscle or collagen. The primary treatment for some of these disorders is pyridostigmine or 3,4-diaminopyridine. However, the effectiveness of therapy is heavily dependent on the underlying mutation resulting in the disease, and the manifestations of some forms of CMS can be worsened by pyridostigmine.2,3 In this case report, a patient was diagnosed using next-generation (NextGen) sequencing (NGS) technology, which identified compound heterozygote mutations in RAPSN, the gene-encoding receptor-associated protein of the synapse (rapsyn). Rapsyn is a protein involved in aggregation, assembly, and stabilization of the postsynaptic AChRs at the neuromuscular junction. NextGen sequencing refers to several different methodologies that have evolved since the 1990s. NGS cannot detect all genetic disorders and is currently not an appropriate technology for detecting trinucleotide repeats, mutations in the noncoding regions, or copy number variants.4 Whole-exome sequencing (WES) uses NGS technology and provides sequencing of the approximately 23,000 proteincoding genes. The protein-coding region (termed exome) constitutes a small percentage (roughly 1.5%) of the genome, although estimates show that mutations in the exome account for approximately 85% of the mutations linked to human disease, and first performed in humans in 2009.5,6 Whole exome sequencing allowed us to definitively diagnose an infant with a treatable disorder so this report emphasizes the importance of WES in current clinical practice because its use can result in a crucial diagnosis that would otherwise go undiagnosed or misdiagnosed. Patient Description This boy was born at 33 weeks of gestation to a 30-year-old G2P1101 woman and delivered by Cesarean section because of severe oligohydramnios. Fetal ultrasonography revealed a choroid plexus cyst along with hydronephrosis, both of which resolved with no apparent sequelae. The mother had previously delivered a dysmorphic term female infant who died 2 hours after birth from complications of meconium aspiration. The postmortem examination revealed arthrogryposis, and the muscle histology was suspicious for denervation atrophy, but spinal muscular atrophy gene testing was normal. His parents are of English, Irish, Scottish, and German ancestry. The Apgar scores were 4 and 7 at 1 and 5 minutes, respectively. Birth weight was 2050 g (fiftieth percentile), length was 42 cm (twentieth percentile), and head circumference was 33.5 cm (greater than ninetieth percentile). Initial laboratory studies were unremarkable demonstrating normal amino acid, fatty acid, and organic acid profiles and a thyroid stimulating hormone of 4.5 mIU/mL (normal, <34 mIU/ mL). Hemoglobin electrophoresis and newborn screening study results were normal for biotinidase deficiency, galactosemia, congenial adrenal hyperplasia, and cystic fibrosis. The Ohio Department of Health newborn screening evaluation was normal. At delivery, he had a weak cry, hypotonia, and joint contractures. He failed continuous positive airway pressure (CPAP) and was intubated after treatment with poractant alfa. He remained intubated for 1 day and was then weaned to nasal CPAP, which was used intermittently during his 71-day neonatal intensive care unit course. His course was complicated by several aspiration pneumonias. Eventually, arterial blood gases demonstrated stable carbon dioxide measurements between 40 and 44 Hg, obviating the need for CPAP. The patient had been started on caffeine initially for apnea of prematurity and was continued on caffeine as it resulted in improvements in respiratory function and drive. Multiple congenital abnormalities were observed including a prominent occiput, bilaterally low-set ears, hypertelorism with small palpebral fissures, micrognathia, a broad nasal bridge, a high-arched palate,
cortical thumbs, and multiple limb contractures. Physical examination revealed coarse breath sounds, normal heart sounds and heart rhythm, bounding femoral pulses, no abdominal masses, an undescended left testicle and a fully descended right testicle, decreased motor tone, limited spontaneous movement, decreased muscle stretch reflexes, and weak, but present, Moro and grasp reflexes. Audiologic and ophthalmologic evaluations were normal. Muscle and nerve biopsies were obtained during his gastrostomy tube placement and Nissen fundoplication. The nerve biopsy was normal, but the muscle biopsy revealed mild myofiber atrophy (without fiber-type grouping) consistent with early nerve denervation and was similar to findings from the patient’s older sibling. Testing for Cockayne syndrome was negative for mutations in CSB/ERCC6 and CSA/ERCC8 genes by multiplex ligation-dependent probe amplification. A magnetic resonance imaging was performed at 1 month of life, which revealed a nonspecific triangular focus of mildly increased signal in the right putamen (more prominent on T2-weighted image). Serum studies demonstrated a lactate level of 1.4 mmol/L (normal, 0.6-2.4 mmol/L) and a creatine kinase level of 88 U/L (normal, 24-195 U/L). The patient was then discharged to home with multivitamin supplementation (1 mL/day) and caffeine (40 mg/day). After 17 days at home, the patient developed an apneic episode and was evident to have aspiration pneumonia and sepsis resulting in a 47-day hospitalization. Echocardiogram and electrocardiogram study results were normal. A host of metabolic study results were normal including organic acids, carnitine, oligosaccharides, and guanidinoacetic acid, and serum very long-chain fatty acids, carbohydrate-deficient transferrin, and coenzyme Q10. Furthermore, Prader-Willi methylation, reversible cytochrome c oxidase deficiency (mitochondrial DNA m.14674 T), and electroencephalography study results were normal. The chromosomal microarray was normal as well. At his 3-week follow-up after discharge (at 5 months of age), he was evaluated by a neurologist (B.H.C.) as he continued to have a paucity of movements and remained hypotonic with joint contractures of the thumb, knee, and elbow. Additionally there was decreased motor bulk in all muscle groups with pronounced weakness in the jaw; his mouth was triangular shaped because of weakness of the muscles of facial expression. The patient was only able to elevate his head for very brief periods. He was able to move all his extremities spontaneously but had more difficulty when he was in an upright posture than when he was supine. Cognitive function was progressing appropriately. Visual fields were normal by confrontation; however, mild ptosis was observed. Based on clinical signs, consideration of Ullrich congenital muscular dystrophy was entertained, but sequencing of COL6A1, COL6A2, and COL6A3 genes was normal. At his 8-month visit, he had developed modest head control and was able to extend his trunk. Furthermore, an overall increase in limb movement was observed with increased elbow flexion and/or extension and active wrist flexion. At his 10-month visit, the patient was able to say a few words and was able to turn his head side-to-side in the supine position, as he was still not able to hold his head unassisted. His breathing and secretions were positional, and his cough was getting stronger, although he still required a great deal of suctioning. At this point in the care of the patient, as all the tests and diagnostic imaging provided minimal leads, WES (next generation) was performed on a clinical basis (GeneDx Labs). Results from this study, confirmed by Sanger sequencing, identified two in trans mutations (c.264 C >A and c.1083_1084dupCT) in the RAPSN gene leading to the diagnosis of CMS. At 16 months of age, the patient was administered 1.8 mg (60 mg/5 mL) of pyridostigmine (Mestinon). Within 7 minutes, the patient started kicking and his ability to sit unsupported had improved to a greater extent than witnessed before. Additionally within the next few hours, his drooling had ceased as he began swallowing saliva. Three months after starting pyridostigmine, a dramatic improvement in clinical impairments was observed. He was able to sit unsupported and began using his hands for signing and manipulating objects. His fine motor skills were appropriate for age level; however, gross motor skills were still delayed. At his last examination, he was sitting independently and was beginning to bear weight and walk in a gait trainer. The patient demonstrated a great deal of babbling and was able to say approximately seven to eight words. His motor bulk was still reduced but was
A.S. Das et al. / Pediatric Neurology 51 (2014) 717e720 improving in all muscle groups. Additionally his hypotonia had decreased to near normal and his joint contractures had significantly improved. Muscle strength was still globally weak, but his trunk and upper extremity strength had greatly improved. Before treatment, he had frequent intensive care unit admissions for respiratory failure; these essentially resolved after therapy.
Discussion
Historically, the diagnosis of CMS is based on the clinical and laboratory findings present in Table. However, many of the clinical features overlap with the congenital myopathies and muscular dystrophies. In some instances, a histologic analysis or electromyogram (EMG) studies are sufficient for diagnosis; however, in other types of CMS, a much more thorough investigation using data from electrophysiological, ultrastructural, and immunocytochemical studies is needed.7 Single muscle fiber EMG is an excellent diagnostic modality8 but requires considerable experience and is often difficult or impossible to obtain in a child with minimal muscle bulk. Furthermore, infant EMG services are also not available in many medical centers. Therefore, in spite of considerable discussion about the possible diagnoses, neither an EMG nor a trial of an acetylcholinesterase inhibitor was performed in this case prior to receiving the results of WES. WES provided a rapid diagnosis of RAPSN-associated CMS based on complex heterozygote mutations inherited in trans. Both disease-associated confirmed missense mutation at c.264 C>A resulting in a lysine substitution for asparagine at codon 88 (N88K) in exon 2 and a previously confirmed frameshift mutation at (p.Tyr362SerfsX10) resulting in generation of a premature stop codon at 371 in exon 7 of RAPSN were identified. The parents were tested and were evident to each be a carrier of one variant allele. This heterozygous combination (N88K and c.1083_ 1084dupCT) has been previously described in two patients with similar phenotypes (arthrogryposis and respiratory distress) and similar clinical courses as the patient described in this report.9,10 Both cases improved with anticholinesterase treatment, and interestingly, one of the patients had an older sister with arthrogryposis.11 TABLE. Clinical and Laboratory Features of Congenital Myasthenic Syndromes3,7
Clinical features Weakness of bulbar, ocular, or limb muscles most often at birth High-arched palate (variable) Contractures (variable) Positive family history for the disease (in autosomal dominant cases) Positive motor response to edrophonium Laboratory features Decremental electromyographic (EMG) response of the compound muscle action potential on 2- to 3-Hz stimulation Increased jitter on single-fiber EMG Normal or slightly elevated creatine kinase Absence of overt pathologic findings in a muscle biopsy Negative serum antibodies for ACh receptors (found in myasthenia gravis), muscle specific kinase, and P/Q-type calcium channels Abbreviation: ACh ¼ Acetylcholine
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Illness caused by RAPSN mutations exhibits an autosomal recessive inheritance pattern and accounts for approximately 15-20% of all CMS. RAPSN, located on 11p11.2, encodes for a 43-kDA cyclic adenosine monophosphate-dependent protein kinase, rapsyn. Rapsyn functions as the link between the AChR and the agrin-binding dystrophin-associated glycoprotein complex and functions to maintain the correct position and orientation of the AChR in the postsynaptic junction. In vitro studies suggest N88K rapsyn results in reduced colocalization with the AChR and causes diminished postsynaptic receptor clustering and receptor stability.12 In patients with RAPSN-associated CMS, signs manifest early in infancy but can present in the second and third decades.13 Furthermore, arthrogryposis (as was observed in this family), ptosis, and multiple joint contractures present at birth are characteristic findings of this specific type of CMS.14 In addition, a decremental EMG response to lowfrequency stimulation can be mild or absent.3 As observed with our patient, respiratory infections, stress, and other febrile illnesses exacerbate muscle fatigability and can precipitate respiratory arrest and anoxic encephalopathy.14 Fortunately, most of the patients demonstrate a favorable response to pyridostigmine, and case reports reveal patients benefiting from 3,4-diaminopyridine (an orphan drug), as well as ephedrine or albuterol.13,15 In the current case, after we provided the appropriate treatment with anticholinesterase therapy, our patient displayed marked improvements in muscle strength, bulbar function, and fine motor control. Conclusions
CMS are often misdiagnosed or underdiagnosed. These conditions closely resemble other neuromuscular disorders, physicians are less familiar with them than with other conditions, some patients exhibit a paucity of classic clinical signs that point to the diagnosis of CMS, and lastly, not all institutions have the EMG sophistication to accurately diagnose CMS.3 The use of NGS in diagnosis underscores the importance of this technology and significance of this case. Furthermore, identification of the specific mutation is crucial in this disease as treatment outcomes are dependent on the specific type of CMS; moreover, acetylcholine esterase inhibitor therapy can have deleterious consequences if used to treat some forms of CMS.3 As is evident from our patient’s clinical improvement, NGS played a pivotal role in his diagnosis and treatment. The cost of this technology fares well considering the saved future costs of additional diagnostic testing and averted hospitalizations. It should be considered in patients with an undiagnosed disease with a suspected genetic etiology. B.H.C. has received compensation from the American Academy of Neurology for his activities as an Assistant Advisor to Current Procedural Terminology and speakers honoraria, from Motive Medical Intelligence for his activities as a content editor and medical writer, and as a speaker for Transgenomic Labs and Courtagen Labs. He has received travel reimbursement for clinical research activities from Edison Pharmaceutical and Raptor Pharmaceutical, and his primary institution receives financial compensation for research conducted by him. He has a consulting agreement with Stealth Peptides and intends to conduct clinical research with this company. A.S.D. and D.P.A. have no conflicts to report.
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