- Email: [email protected]
Proximal myopathy with focal depletion of mitochondria and megaconial congenital muscular dystrophy are allelic conditions caused by mutations in CHKB
Accepted Manuscript Title: Proximal myopathy with focal depletion of mitochondria and megaconial congenital muscular dystrophy are allelic conditions caused by mutations in CHKB Author: L. Brady, M. Giri, J. Provias, E. Hoffman, M. Tarnopolsky PII: DOI: Reference:
S0960-8966(15)00771-3 http://dx.doi.org/doi: 10.1016/j.nmd.2015.11.002 NMD 3117
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
4-4-2015 13-10-2015 9-11-2015
Please cite this article as: L. Brady, M. Giri, J. Provias, E. Hoffman, M. Tarnopolsky, Proximal myopathy with focal depletion of mitochondria and megaconial congenital muscular dystrophy are allelic conditions caused by mutations in CHKB, Neuromuscular Disorders (2015), http://dx.doi.org/doi: 10.1016/j.nmd.2015.11.002. 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.
Proximal Myopathy with Focal Depletion of Mitochondria and Megaconial Congenital Muscular Dystrophy Are Allelic Conditions Caused by Mutations in CHKB L. Brady1, M. Giri2, J. Provias3, E. Hoffman2, M. Tarnopolsky1* 1
Department of Pediatrics, McMaster University Medical Centre, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada 2
Children's National Medical Center, Research Center for Genetic Medicine, 111 Michigan Ave, Washington DC 20010, USA 3
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada *Corresponding Author: fax 905 521 2638 email [email protected] Highlights
Proximal Myopathy with Focal Depletion of Mitochondria and Megaconial Congenital Muscular Dystrophy are allelic conditions.
Phenotype expanded to include rhabdomyolysis and normal cognition
Muscle biopsy shows focal oligomitodondria and megaconial mitochondria
Sequencing of CHKB recommended for patients with this muscle biopsy finding
We recently evaluated two of the original three patients (siblings) diagnosed with Proximal Myopathy with Focal Depletion of Mitochondria. The condition was named for the distinctive pattern of enlarged mitochondria around the periphery of muscle fibres with a complete absence in the middle. These siblings, ages 37 and 40, are cognitively normal with mild non-progressive muscle weakness and a susceptibility to rhabdomyolysis. Both were shown to be compound heterozygotes for novel mutations (c.263C>T + c.950T>A) in CHKB, the gene currently associated with Megaconial Congenital Muscular Dystrophy. Individuals with this condition have early-onset muscle weakness and profound intellectual disability but share the same unique pattern on muscle biopsy as was noted in Proximal Myopathy with Focal Depletion of Mitochondria; focal depletion of mitochondria surrounded by abnormally large “megaconial” mitochondria. Thus the phenotypic spectrum of CHKB mutations ranges from a congenital muscular dystrophy with intellectual disability to a later-onset non-progressive muscular weakness with normal cognition.
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Abstract Word Count: 157 Keywords: megaconial muscular dystrophy; mitochondrial depletion; oligomitochondria; CHKB; rhabdomyolysis; myalgia
*Corresponding Author: Dr. Mark Tarnopolsky Department of Pediatrics, McMaster University Medical Centre, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada Fax: (905) 521-2638 Email: [email protected]
Introduction A distinctive muscle pathology showing a peculiar absence of intermyofibrillar mitochondria and enlarged “megaconial” mitochondria around the periphery was first linked to a novel congenital muscular dystrophy in four patients by Nishino et al. 1998 [1]. While the phenotypes of these patients were similar to merosin-positive congenital muscular dystrophy all of Nishino et al.’s patients had pronounced intellectual disability in the absence of brain malformations [1]. Genetic analysis of these original patients, and eleven others with an analogous phenotype, found that all had homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB) (involved in the biosynthesis of phosphatidylcholine/phosphatidylethanolamine) [2, 3]. The clinical spectrum of this condition, now known as “Megaconial Congenital Muscular Dystrophy” (MDCMC) (OMIM #602541), includes hypotonia in the neonatal/infantile period followed by profound muscle weakness, muscle wasting, and intellectual disability. Some patients also have ichthyosis, microcephaly,
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and seizures [2, 4, 5]. Approximately half develop dilated cardiomyopathy [2, 4, 5]. Over two dozen cases of MDCMC have been published to date [1, 2, 4-11]. Three years prior to the Nishino et al. 1998 report [1], Genge et al. reported on three patients (two of them siblings) with almost identical muscle pathology and a juvenile/adult onset myopathy with myalgias [12]. This condition was named Proximal Myopathy with Focal Depletion of Mitochondria (PMFDM) (OMIM #600706) but is sometimes referred to as “Mitochondrial Depletion” syndrome [12]. No genetic locus was associated with this condition. We recently performed genetic testing on the original siblings in the latter paper [12], and identified two novel mutations in CHKB (c.263C>T + c.950T>A). These siblings, ages 37 and 40 years, are cognitively normal with mild non-progressive muscle weakness and a susceptibility to rhabdomyolysis. Neither has signs of ichthyosis or other skin findings. Although the presenting phenotypes of PMFDM and MDCMC are very different, our evidence shows these conditions are allelic. The focal oligomitochondria and peripheral “megaconial” mitochondria pattern of muscle biopsy seen in both PMFDM and MDCMC appears to be pathognomonic for homozygous or compound heterozygous mutations in CHKB. This report expands the clinical spectrum of CHKB mutations from a severe congenital muscular dystrophy with intellectual disability to include a later-onset non-progressive phenotype with normal cognition. Case Report Case 1 This patient was originally reported by Genge et al. 1995 [12]. She presented at 16y with unresolved muscle fatigue, generalized myalgias, and a consistently elevated CK (400-1000 U/L,
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N < 220) following a febrile possible upper respiratory infection. This worsened to rapidly progressive muscle weakness leading to wheelchair requirement. There was gradual improvement of muscle strength over the following years where she no longer needed any assistance with ambulation but was unable to do strenuous activities [12]. The patient is currently 40y old and continues to experience myalgias during and following exercise and has had multiple episodes of rhabdomyolysis with myoglobinuria requiring hospitalization (highest measured CK 32,000 U/L . She is ambulatory but reports frequent episodes of weakness where she has difficulty walking, climbing stairs, and is not able to raise her arms above her head. These episodes are often precipitated by stress or illness. In the non-stressed state her neurological examination is normal with the exception of mild shoulder abduction and knee extension weakness (4/5, MRC scale) and mild scapular winging. Cardiac work-up (echocardiogram and electrocardiogram) was normal. She does not report any abnormal skin changes. She has had several muscle biopsies; 16y (left quadriceps), 17y (left bicep), 23y (right bicep), 34y (left deltoid), and 37y (right vastus lateralis). Her biopsy done at 17y (bicep) was the first to show the distinctive “oligomitochondrial” pattern and enlarged mitochondria around the periphery (Fig 1,2,4 in Reference 12). Subsequent biopsies from both biceps (17y, 23y) and deltoid (34y) (Figure 1d) also reported this pattern. Her most recent biopsy from the vastus lateralis (37y) showed paucity of mitochondria in focal regions of some sarcomeres (Figure 1e). EM from the most recent biopsy did not identify any enlarged mitochondria but showed regions of relative depletion (Figure 1f). Myopathic changes were reported in all biopsies (Figure 1a-c). Additional fibre degeneration with central loss of sarcoplasm was seen in the right
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bicep biopsy (Figure 1a). Some slight dystrophic changes were seen in the biopsies taken at 34y (left deltoid) and 37y (right vastus lateralis). Immunohistochemistry from the right vastus lateralis (37y) was grossly normal with normal expression of alpha, beta, gamma, and delta sarcoglycans, spectrin, and beta dystroglycan. Dystrophin 2 and 3 were grossly normal. Dystrophin 1 showed a weak pattern of positivity but this was also seen in the control. Southern blot for mitochondrial DNA (mtDNA) depletion was reported to be normal by Genge et al. 1995 (Patient age 17y – left bicep) [12] and confirmed by us in 2011 on a new muscle sample with qPCR (Patient age 37y – right vastus lateralis). Electron transport chain enzyme activity was robustly within normal limits (NADH cytochrome c reductase = 2.21 (control = 1.15±0.13); succinate cytochrome c reductase = 1.88 (control = 1.77±0.2); Cytochrome c oxidase = 2.31 (control = 2.12 ± 0.23); citrate synthase = 8.89 (control = 6.09±0.29) (nanomoles/min/g wet weight) [13].
Case 2 The younger brother of Case 1 whose history was also outlined by Genge et al. 1995 [12]. He presented in his early teenage years with similar complaints of post-exertional myalgias. At age 15y he was shown to have proximal muscle weakness. His basal CK activity was elevated at 900 U/L with increases to 9,000 U/L with the post-exertional myalgias (N < 220)). Muscle biopsy showed the same oligomitochondrial pattern seen in his sister without any additional dystrophic changes (Fig 6, 7 in Reference 12). No subsequent muscle biopsies were performed. His significant myalgias continued during his teenage years until he reported improvement in his symptoms around 18y.
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At 36y of age he is healthy and has no intellectual disability. He reports limited exercise tolerance and has had one major episode of rhabdomyolysis with myoglobinuria following activity which required hospitalization (CK > 20,000 U/L). His CK remains mildly elevated at baseline (361 U/L, N < 220 IU). A complete neurological exam was normal including normal strength (MRC 5/5) for all major muscle groups in the upper and lower extremities. Cardiac work-up (echocardiogram, ECG, and 24h Holter) were normal. Genomic DNA extracted from whole blood using Qiagen DNAeasy kit was sheered in 5kb long fragments on the Covaris system (Covaris, Woburn, MA) using manufacturer’s recommended protocol. The DNA fragment was used as input for the targeted amplification of the 81 genes on the Raindance RDT1000 system (RainDance Technologies, Lexington, MA) using manufacturer’s protocol followed by sequencing using Illumina Trueseq kit (Illumina, CA, USA) adding a unique adapter to each sample on the Illumina Miseq system (Table S1, S2). The fastq data generated was checked for quality using fastQC and aligned and analyzed using the NextGene software (SoftGenetics, State College, PA). The variants observed were filtered based on in-house errors, polymorphisms found in dbSNP, 1000genomes, and NHLBI exome variant server database. The 81 gene Next Generation Sequencing myopathy panel uncovered two variants in the CHKB gene in both cases; c.263C>T in exon 2 causing p.Pro88Leu and c.950T>A in exon 9 causing p.Leu317Gln. Both mutations are rare; c.263C>T is reported with an allele frequency of 0.0004 and dnSNP (rs146163970), c.950T>A is reported with an allele frequency of 8.243e-06 [14]. The c.950T>A substitution creates an ApG dinucleotide that resembles the consensus eukaryotic splice acceptor signal though this was not tested through in vitro analysis. SIFT,
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Mutation-Taster, and PolyPhen2 predicted both mutations to be deleterious, disease causing, and probably damaging, respectively. Family studies confirmed that the mutations are in trans.
Discussion Here we provide the results of genetic investigations done in two of the original three patients reported with easy fatigability and post-exercise myalgias diagnosed with PMFDM (OMIM #600706) by Genge et al. [12]. This condition, also known as oligomitochondrial myopathy or mitochondrial depletion syndrome, was named to describe the abnormally enlarged mitochondria located around the periphery of muscle fibres with a complete absence in the middle of the sarcoplasm [12]. An almost identical muscle biopsy pattern as to what was seen in the original report of PMFDM was also described in four patients by Nishino et al. [1]. These patients were later diagnosed with MDCMC due to homozygous and compound heterozygous mutations in CHKB [1, 2]. The pathologic similarities were noted yet the phenotypic differences and lack of dystrophic changes in the PMFDM biopsies led them to consider them as separate conditions [1, 12]. Although dystrophic changes were not appreciated in the muscle biopsy of Case 1 at the time of the original publication, mild dystrophic changes consisting of increased internalized nuclei and fibrosis were seen in the biopsies taken at 34y and 37y of age. It appears that these dystrophic changes may not be an early feature of the milder presentation of this myopathy. Unlike the dystrophic changes, the pathognomonic pattern of focal mitochondrial depletion and megaconial mitochondria appears to be present at the beginning of the disease process regardless of phenotype. This pattern was most apparent in 4 of the 6 muscle biopsies
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performed on Case 1; all 4 being taken from the arm (biceps or deltoid). There was no mention of abnormal distribution or size from the earliest (16y – quadriceps). For the most recent biopsy (37 – vastus lateralis) a paucity of mitochondria was seen in a minority of sarcomeres. The pattern of mitochondrial depletion/megaconial mitochondria has been reported in the quadriceps biopsies of several MDCMC patients indicating that it does appear in this proximal muscles [2, 4, 5, 7, 8, 10]. It is not clear if this discrepancy between muscle types is a feature of patients with the milder presentation of this myopathy or if it is just due to limited sampling. Case 1 in this report only presented with her first symptoms of this condition immediately following an upper respiratory infection. She continues to have episodes of worsening symptoms during acute illnesses and requires extended periods of recovery. Her brother (Case 2) has not reported similar episodes of symptom worsening outside of activity induced rhabdomyolysis and fatigue with long periods of walking. Other case reports have hinted that there may be a correlation between acute infections and illnesses, general anaesthesia and/or vaccination and worsening muscle weakness and/or cardiac function [5]. Two of the patients in one case series were reported to have periods of increased falling following an episode of chicken pox and an upper respiratory tract infection, respectively [5]. One of these patients also experiences lethargy, anorexia, and severe hypoxia following a surgery requiring general anaesthesia and was identified to have a severe dilated cardiomyopathy in the absence of other signs of cardiac failure [5]. Other case reports have also reported similar events of transient muscle weakness following acute illnesses [7]. The clinical history of Case 1 further suggests that fluctuations in muscle strength may be affected by
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external factors such as illness or exposure to anaesthesia, though further data is needed to confirm this as part of the natural history. Severe intellectual disability (ID) in the absence of CNS malformations is currently considered to be one of the primary phenotype features of MDCMC [6]. Both patients reported here are cognitively normal and completed post-secondary education. There has only been two cases that have been reported with less significant ID [5, 8]. Both patients were reported to require support in school; one with an IQ of 80 and described as having a “very slight” ID [8], while the other had speech delay in childhood followed by more significant learning difficulties that became apparent around the age of 12 [5]. Interestingly, both were reported to have a milder phenotype more similar to a limb girdle muscular dystrophy (LGMD) [5, 8]. Collectively, our data and that previously published does suggest that there may be a correlation between degree of ID and severity of muscle weakness. There are a few other cases in the literature with the same muscle pathology but a similarly mild phenotype (LGMD-like phenotype and no ID) for which it would be of interest to investigate with CHKB studies. This includes the third PMFDM case [12], a patient with myalgias and proximal muscle pain unrelated to exercise [15], and a myopathy described to be similar to polymyositis [16]. Nonsense, frameshift, missense, and splice-site mutations have been reported in almost all exons of CHKB (1, 4, 5, 6, 7, 8, 9, and 11) [1, 2, 4-11]. Both CHKB mutations reported here (c.263C>T + c.950T>A) are novel. No in vitro analyses of residual choline kinase beta activity were performed as the shared unique pattern of the muscle biopsies seen in the patients of this case report, MDCMC patients [1, 2, 4-11] and CHKB knockout mice [3] support the pathogenicity of these novel CHKB mutations.
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In conclusion, the conditions known as PMFDM and MCMDC are allelic and result from homozygous or compound heterozygous mutations in CHKB. PMFDM was originally considered an autosomal dominant condition with incomplete penetrance based on a transiently increased CK noted in the father of Cases 1 and 2 [12]. With the discovery of two CHKB mutations in trans it is now clear that the inheritance is autosomal recessive in PMFDM, as it is in MDCMC. With this report the clinical spectrum has expanded from a congenital muscular dystrophy with intellectual disability to include a later-onset non-progressive muscle condition with normal cognition. Sequencing of CHKB should be considered for patients with muscle biopsies showing complete absence of intermyofibrillar mitochondria and enlarged “megaconial” mitochondria along the periphery, even in the absence of intellectual disability.
References [1]
Nishino I, Kobayashi O, Goto Y-i, et al. A new congenital muscular dystrophy with mitochondrial structural abnormalities. Muscle Nerve 1998;21:40-7.
[2]
Mitsuhashi S, Ohkuma A, Talim B, et al. A Congenital Muscular Dystrophy with Mitochondrial Structural Abnormalities Caused by Defective De Novo Phosphatidylcholine Biosynthesis. Am J Hum Genet 2011;88:845-51.
[3]
Sher RB, Aoyama C, Huebsch KA, et al. A Rostrocaudal Muscular Dystrophy Caused by a Defect in Choline Kinase Beta, the First Enzyme in Phosphatidylcholine Biosynthesis. J Biol Chem 2006;281:4938-48.
[4]
Mitsuhashi S, Nishino I. Megaconial congenital muscular dystrophy due to loss-of-function mutations in choline kinase β. Curr Opin Neurol 2013;26:536-43.
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[5]
Quinlivan R, Mitsuahashi S, Sewry C, et al. Muscular dystrophy with large mitochondria associated with mutations in the CHKB gene in three British patients: Extending the clinical and pathological phenotype. Neuromuscul Disord 2013;23:549-56.
[6]
Haliloglu G, Talim B, Sel CG, Topaloglu H. Clinical characteristics of megaconial congenital muscular dystrophy due to choline kinase beta gene defects in a series of 15 patients. J Inherit Metab Dis 2015.
[7]
Cabrera-Serrano M, Junckerstorff RC, Atkinson V, et al. Novel CHKB mutation expands the megaconial muscular dystrophy phenotype. Muscle Nerve 2015;51:140-43.
[8]
Castro-Gago M, Dacruz-Alvarez D, Pintos-Martínez E, et al. Exome sequencing identifies a CHKB mutation in Spanish patient with Megaconial Congenital Muscular Dystrophy and mtDNA depletion. Eur J Paediatr Neurol 2014;18:796-800.
[9]
Castro-Gago M, Dacruz-Alvarez D, Pintos-Martínez E, et al. Congenital neurogenic muscular atrophy in megaconial myopathy due to a mutation in CHKB gene. Brain Dev 2015.
[10]
Gutierrez Rios P, Kalra AA, Wilson JD, et al. Congenital megaconial myopathy due to a novel defect in the choline kinase Beta gene. Arch Neurol 2012;69:657-61.
[11]
Oliveira J, Negrão L, Fineza I, et al. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing. J Hum Genet 2015;6:30512.
[12]
Genge A, Karpati G, Arnold D, Shoubridge EA, Carpenter S. Familial myopathy with conspicuous depletion of mitochondria in muscle fibers: a morphologically distinct disease. Neuromuscul Disord 1995;5:139-44.
[13]
McKenzie S, Phillips SM, Carter SL, Lowther S, Gibala MJ, Tarnopolsky MA. Endurance exercise training attenuates leucine oxidation and BCOAD activation during exercise in humans. Am J Physiol Endocrinol Metab 2000;278:580-87.
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[14]
Exome Aggregation Consortium (ExAC), Cambridge, MA (URL: http://exac.broadinstitute.org) [March 30 2015].
[15]
Osaki Y, Nishino I, Murakami N, et al. Mitochondrial abnormalities in selenium-deficient myopathy. Muscle Nerve 1998;21:637-9.
[16]
Jeffree RL, Wills EJ, Harper C. An unusual myopathy: speckled muscle fibers due to enlarged mitochondria. Muscle Nerve 2007;36:118-22.
FIGURE 1. Muscle sections of Case 1. (a-c) modified Gomori-trichrome (mGT, 400x) staining showing myopathic changes. (d-e) COX staining showing focal absence in muscle fibers (400x). (f) Electron microscopy (EM) showing zones of relative mitochondrial depletion (Bar = 2μm Mag 6000x). A highresolution version of this slide for use with the Virtual Microscope is available as eSlide: VM02070
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S0960-8966(15)00771-3 http://dx.doi.org/doi: 10.1016/j.nmd.2015.11.002 NMD 3117
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
4-4-2015 13-10-2015 9-11-2015
Please cite this article as: L. Brady, M. Giri, J. Provias, E. Hoffman, M. Tarnopolsky, Proximal myopathy with focal depletion of mitochondria and megaconial congenital muscular dystrophy are allelic conditions caused by mutations in CHKB, Neuromuscular Disorders (2015), http://dx.doi.org/doi: 10.1016/j.nmd.2015.11.002. 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.
Proximal Myopathy with Focal Depletion of Mitochondria and Megaconial Congenital Muscular Dystrophy Are Allelic Conditions Caused by Mutations in CHKB L. Brady1, M. Giri2, J. Provias3, E. Hoffman2, M. Tarnopolsky1* 1
Department of Pediatrics, McMaster University Medical Centre, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada 2
Children's National Medical Center, Research Center for Genetic Medicine, 111 Michigan Ave, Washington DC 20010, USA 3
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada *Corresponding Author: fax 905 521 2638 email [email protected] Highlights
Proximal Myopathy with Focal Depletion of Mitochondria and Megaconial Congenital Muscular Dystrophy are allelic conditions.
Phenotype expanded to include rhabdomyolysis and normal cognition
Muscle biopsy shows focal oligomitodondria and megaconial mitochondria
Sequencing of CHKB recommended for patients with this muscle biopsy finding
We recently evaluated two of the original three patients (siblings) diagnosed with Proximal Myopathy with Focal Depletion of Mitochondria. The condition was named for the distinctive pattern of enlarged mitochondria around the periphery of muscle fibres with a complete absence in the middle. These siblings, ages 37 and 40, are cognitively normal with mild non-progressive muscle weakness and a susceptibility to rhabdomyolysis. Both were shown to be compound heterozygotes for novel mutations (c.263C>T + c.950T>A) in CHKB, the gene currently associated with Megaconial Congenital Muscular Dystrophy. Individuals with this condition have early-onset muscle weakness and profound intellectual disability but share the same unique pattern on muscle biopsy as was noted in Proximal Myopathy with Focal Depletion of Mitochondria; focal depletion of mitochondria surrounded by abnormally large “megaconial” mitochondria. Thus the phenotypic spectrum of CHKB mutations ranges from a congenital muscular dystrophy with intellectual disability to a later-onset non-progressive muscular weakness with normal cognition.
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Abstract Word Count: 157 Keywords: megaconial muscular dystrophy; mitochondrial depletion; oligomitochondria; CHKB; rhabdomyolysis; myalgia
*Corresponding Author: Dr. Mark Tarnopolsky Department of Pediatrics, McMaster University Medical Centre, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada Fax: (905) 521-2638 Email: [email protected]
Introduction A distinctive muscle pathology showing a peculiar absence of intermyofibrillar mitochondria and enlarged “megaconial” mitochondria around the periphery was first linked to a novel congenital muscular dystrophy in four patients by Nishino et al. 1998 [1]. While the phenotypes of these patients were similar to merosin-positive congenital muscular dystrophy all of Nishino et al.’s patients had pronounced intellectual disability in the absence of brain malformations [1]. Genetic analysis of these original patients, and eleven others with an analogous phenotype, found that all had homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB) (involved in the biosynthesis of phosphatidylcholine/phosphatidylethanolamine) [2, 3]. The clinical spectrum of this condition, now known as “Megaconial Congenital Muscular Dystrophy” (MDCMC) (OMIM #602541), includes hypotonia in the neonatal/infantile period followed by profound muscle weakness, muscle wasting, and intellectual disability. Some patients also have ichthyosis, microcephaly,
Page 2 of 12
and seizures [2, 4, 5]. Approximately half develop dilated cardiomyopathy [2, 4, 5]. Over two dozen cases of MDCMC have been published to date [1, 2, 4-11]. Three years prior to the Nishino et al. 1998 report [1], Genge et al. reported on three patients (two of them siblings) with almost identical muscle pathology and a juvenile/adult onset myopathy with myalgias [12]. This condition was named Proximal Myopathy with Focal Depletion of Mitochondria (PMFDM) (OMIM #600706) but is sometimes referred to as “Mitochondrial Depletion” syndrome [12]. No genetic locus was associated with this condition. We recently performed genetic testing on the original siblings in the latter paper [12], and identified two novel mutations in CHKB (c.263C>T + c.950T>A). These siblings, ages 37 and 40 years, are cognitively normal with mild non-progressive muscle weakness and a susceptibility to rhabdomyolysis. Neither has signs of ichthyosis or other skin findings. Although the presenting phenotypes of PMFDM and MDCMC are very different, our evidence shows these conditions are allelic. The focal oligomitochondria and peripheral “megaconial” mitochondria pattern of muscle biopsy seen in both PMFDM and MDCMC appears to be pathognomonic for homozygous or compound heterozygous mutations in CHKB. This report expands the clinical spectrum of CHKB mutations from a severe congenital muscular dystrophy with intellectual disability to include a later-onset non-progressive phenotype with normal cognition. Case Report Case 1 This patient was originally reported by Genge et al. 1995 [12]. She presented at 16y with unresolved muscle fatigue, generalized myalgias, and a consistently elevated CK (400-1000 U/L,
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N < 220) following a febrile possible upper respiratory infection. This worsened to rapidly progressive muscle weakness leading to wheelchair requirement. There was gradual improvement of muscle strength over the following years where she no longer needed any assistance with ambulation but was unable to do strenuous activities [12]. The patient is currently 40y old and continues to experience myalgias during and following exercise and has had multiple episodes of rhabdomyolysis with myoglobinuria requiring hospitalization (highest measured CK 32,000 U/L . She is ambulatory but reports frequent episodes of weakness where she has difficulty walking, climbing stairs, and is not able to raise her arms above her head. These episodes are often precipitated by stress or illness. In the non-stressed state her neurological examination is normal with the exception of mild shoulder abduction and knee extension weakness (4/5, MRC scale) and mild scapular winging. Cardiac work-up (echocardiogram and electrocardiogram) was normal. She does not report any abnormal skin changes. She has had several muscle biopsies; 16y (left quadriceps), 17y (left bicep), 23y (right bicep), 34y (left deltoid), and 37y (right vastus lateralis). Her biopsy done at 17y (bicep) was the first to show the distinctive “oligomitochondrial” pattern and enlarged mitochondria around the periphery (Fig 1,2,4 in Reference 12). Subsequent biopsies from both biceps (17y, 23y) and deltoid (34y) (Figure 1d) also reported this pattern. Her most recent biopsy from the vastus lateralis (37y) showed paucity of mitochondria in focal regions of some sarcomeres (Figure 1e). EM from the most recent biopsy did not identify any enlarged mitochondria but showed regions of relative depletion (Figure 1f). Myopathic changes were reported in all biopsies (Figure 1a-c). Additional fibre degeneration with central loss of sarcoplasm was seen in the right
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bicep biopsy (Figure 1a). Some slight dystrophic changes were seen in the biopsies taken at 34y (left deltoid) and 37y (right vastus lateralis). Immunohistochemistry from the right vastus lateralis (37y) was grossly normal with normal expression of alpha, beta, gamma, and delta sarcoglycans, spectrin, and beta dystroglycan. Dystrophin 2 and 3 were grossly normal. Dystrophin 1 showed a weak pattern of positivity but this was also seen in the control. Southern blot for mitochondrial DNA (mtDNA) depletion was reported to be normal by Genge et al. 1995 (Patient age 17y – left bicep) [12] and confirmed by us in 2011 on a new muscle sample with qPCR (Patient age 37y – right vastus lateralis). Electron transport chain enzyme activity was robustly within normal limits (NADH cytochrome c reductase = 2.21 (control = 1.15±0.13); succinate cytochrome c reductase = 1.88 (control = 1.77±0.2); Cytochrome c oxidase = 2.31 (control = 2.12 ± 0.23); citrate synthase = 8.89 (control = 6.09±0.29) (nanomoles/min/g wet weight) [13].
Case 2 The younger brother of Case 1 whose history was also outlined by Genge et al. 1995 [12]. He presented in his early teenage years with similar complaints of post-exertional myalgias. At age 15y he was shown to have proximal muscle weakness. His basal CK activity was elevated at 900 U/L with increases to 9,000 U/L with the post-exertional myalgias (N < 220)). Muscle biopsy showed the same oligomitochondrial pattern seen in his sister without any additional dystrophic changes (Fig 6, 7 in Reference 12). No subsequent muscle biopsies were performed. His significant myalgias continued during his teenage years until he reported improvement in his symptoms around 18y.
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At 36y of age he is healthy and has no intellectual disability. He reports limited exercise tolerance and has had one major episode of rhabdomyolysis with myoglobinuria following activity which required hospitalization (CK > 20,000 U/L). His CK remains mildly elevated at baseline (361 U/L, N < 220 IU). A complete neurological exam was normal including normal strength (MRC 5/5) for all major muscle groups in the upper and lower extremities. Cardiac work-up (echocardiogram, ECG, and 24h Holter) were normal. Genomic DNA extracted from whole blood using Qiagen DNAeasy kit was sheered in 5kb long fragments on the Covaris system (Covaris, Woburn, MA) using manufacturer’s recommended protocol. The DNA fragment was used as input for the targeted amplification of the 81 genes on the Raindance RDT1000 system (RainDance Technologies, Lexington, MA) using manufacturer’s protocol followed by sequencing using Illumina Trueseq kit (Illumina, CA, USA) adding a unique adapter to each sample on the Illumina Miseq system (Table S1, S2). The fastq data generated was checked for quality using fastQC and aligned and analyzed using the NextGene software (SoftGenetics, State College, PA). The variants observed were filtered based on in-house errors, polymorphisms found in dbSNP, 1000genomes, and NHLBI exome variant server database. The 81 gene Next Generation Sequencing myopathy panel uncovered two variants in the CHKB gene in both cases; c.263C>T in exon 2 causing p.Pro88Leu and c.950T>A in exon 9 causing p.Leu317Gln. Both mutations are rare; c.263C>T is reported with an allele frequency of 0.0004 and dnSNP (rs146163970), c.950T>A is reported with an allele frequency of 8.243e-06 [14]. The c.950T>A substitution creates an ApG dinucleotide that resembles the consensus eukaryotic splice acceptor signal though this was not tested through in vitro analysis. SIFT,
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Mutation-Taster, and PolyPhen2 predicted both mutations to be deleterious, disease causing, and probably damaging, respectively. Family studies confirmed that the mutations are in trans.
Discussion Here we provide the results of genetic investigations done in two of the original three patients reported with easy fatigability and post-exercise myalgias diagnosed with PMFDM (OMIM #600706) by Genge et al. [12]. This condition, also known as oligomitochondrial myopathy or mitochondrial depletion syndrome, was named to describe the abnormally enlarged mitochondria located around the periphery of muscle fibres with a complete absence in the middle of the sarcoplasm [12]. An almost identical muscle biopsy pattern as to what was seen in the original report of PMFDM was also described in four patients by Nishino et al. [1]. These patients were later diagnosed with MDCMC due to homozygous and compound heterozygous mutations in CHKB [1, 2]. The pathologic similarities were noted yet the phenotypic differences and lack of dystrophic changes in the PMFDM biopsies led them to consider them as separate conditions [1, 12]. Although dystrophic changes were not appreciated in the muscle biopsy of Case 1 at the time of the original publication, mild dystrophic changes consisting of increased internalized nuclei and fibrosis were seen in the biopsies taken at 34y and 37y of age. It appears that these dystrophic changes may not be an early feature of the milder presentation of this myopathy. Unlike the dystrophic changes, the pathognomonic pattern of focal mitochondrial depletion and megaconial mitochondria appears to be present at the beginning of the disease process regardless of phenotype. This pattern was most apparent in 4 of the 6 muscle biopsies
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performed on Case 1; all 4 being taken from the arm (biceps or deltoid). There was no mention of abnormal distribution or size from the earliest (16y – quadriceps). For the most recent biopsy (37 – vastus lateralis) a paucity of mitochondria was seen in a minority of sarcomeres. The pattern of mitochondrial depletion/megaconial mitochondria has been reported in the quadriceps biopsies of several MDCMC patients indicating that it does appear in this proximal muscles [2, 4, 5, 7, 8, 10]. It is not clear if this discrepancy between muscle types is a feature of patients with the milder presentation of this myopathy or if it is just due to limited sampling. Case 1 in this report only presented with her first symptoms of this condition immediately following an upper respiratory infection. She continues to have episodes of worsening symptoms during acute illnesses and requires extended periods of recovery. Her brother (Case 2) has not reported similar episodes of symptom worsening outside of activity induced rhabdomyolysis and fatigue with long periods of walking. Other case reports have hinted that there may be a correlation between acute infections and illnesses, general anaesthesia and/or vaccination and worsening muscle weakness and/or cardiac function [5]. Two of the patients in one case series were reported to have periods of increased falling following an episode of chicken pox and an upper respiratory tract infection, respectively [5]. One of these patients also experiences lethargy, anorexia, and severe hypoxia following a surgery requiring general anaesthesia and was identified to have a severe dilated cardiomyopathy in the absence of other signs of cardiac failure [5]. Other case reports have also reported similar events of transient muscle weakness following acute illnesses [7]. The clinical history of Case 1 further suggests that fluctuations in muscle strength may be affected by
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external factors such as illness or exposure to anaesthesia, though further data is needed to confirm this as part of the natural history. Severe intellectual disability (ID) in the absence of CNS malformations is currently considered to be one of the primary phenotype features of MDCMC [6]. Both patients reported here are cognitively normal and completed post-secondary education. There has only been two cases that have been reported with less significant ID [5, 8]. Both patients were reported to require support in school; one with an IQ of 80 and described as having a “very slight” ID [8], while the other had speech delay in childhood followed by more significant learning difficulties that became apparent around the age of 12 [5]. Interestingly, both were reported to have a milder phenotype more similar to a limb girdle muscular dystrophy (LGMD) [5, 8]. Collectively, our data and that previously published does suggest that there may be a correlation between degree of ID and severity of muscle weakness. There are a few other cases in the literature with the same muscle pathology but a similarly mild phenotype (LGMD-like phenotype and no ID) for which it would be of interest to investigate with CHKB studies. This includes the third PMFDM case [12], a patient with myalgias and proximal muscle pain unrelated to exercise [15], and a myopathy described to be similar to polymyositis [16]. Nonsense, frameshift, missense, and splice-site mutations have been reported in almost all exons of CHKB (1, 4, 5, 6, 7, 8, 9, and 11) [1, 2, 4-11]. Both CHKB mutations reported here (c.263C>T + c.950T>A) are novel. No in vitro analyses of residual choline kinase beta activity were performed as the shared unique pattern of the muscle biopsies seen in the patients of this case report, MDCMC patients [1, 2, 4-11] and CHKB knockout mice [3] support the pathogenicity of these novel CHKB mutations.
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In conclusion, the conditions known as PMFDM and MCMDC are allelic and result from homozygous or compound heterozygous mutations in CHKB. PMFDM was originally considered an autosomal dominant condition with incomplete penetrance based on a transiently increased CK noted in the father of Cases 1 and 2 [12]. With the discovery of two CHKB mutations in trans it is now clear that the inheritance is autosomal recessive in PMFDM, as it is in MDCMC. With this report the clinical spectrum has expanded from a congenital muscular dystrophy with intellectual disability to include a later-onset non-progressive muscle condition with normal cognition. Sequencing of CHKB should be considered for patients with muscle biopsies showing complete absence of intermyofibrillar mitochondria and enlarged “megaconial” mitochondria along the periphery, even in the absence of intellectual disability.
References [1]
Nishino I, Kobayashi O, Goto Y-i, et al. A new congenital muscular dystrophy with mitochondrial structural abnormalities. Muscle Nerve 1998;21:40-7.
[2]
Mitsuhashi S, Ohkuma A, Talim B, et al. A Congenital Muscular Dystrophy with Mitochondrial Structural Abnormalities Caused by Defective De Novo Phosphatidylcholine Biosynthesis. Am J Hum Genet 2011;88:845-51.
[3]
Sher RB, Aoyama C, Huebsch KA, et al. A Rostrocaudal Muscular Dystrophy Caused by a Defect in Choline Kinase Beta, the First Enzyme in Phosphatidylcholine Biosynthesis. J Biol Chem 2006;281:4938-48.
[4]
Mitsuhashi S, Nishino I. Megaconial congenital muscular dystrophy due to loss-of-function mutations in choline kinase β. Curr Opin Neurol 2013;26:536-43.
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[5]
Quinlivan R, Mitsuahashi S, Sewry C, et al. Muscular dystrophy with large mitochondria associated with mutations in the CHKB gene in three British patients: Extending the clinical and pathological phenotype. Neuromuscul Disord 2013;23:549-56.
[6]
Haliloglu G, Talim B, Sel CG, Topaloglu H. Clinical characteristics of megaconial congenital muscular dystrophy due to choline kinase beta gene defects in a series of 15 patients. J Inherit Metab Dis 2015.
[7]
Cabrera-Serrano M, Junckerstorff RC, Atkinson V, et al. Novel CHKB mutation expands the megaconial muscular dystrophy phenotype. Muscle Nerve 2015;51:140-43.
[8]
Castro-Gago M, Dacruz-Alvarez D, Pintos-Martínez E, et al. Exome sequencing identifies a CHKB mutation in Spanish patient with Megaconial Congenital Muscular Dystrophy and mtDNA depletion. Eur J Paediatr Neurol 2014;18:796-800.
[9]
Castro-Gago M, Dacruz-Alvarez D, Pintos-Martínez E, et al. Congenital neurogenic muscular atrophy in megaconial myopathy due to a mutation in CHKB gene. Brain Dev 2015.
[10]
Gutierrez Rios P, Kalra AA, Wilson JD, et al. Congenital megaconial myopathy due to a novel defect in the choline kinase Beta gene. Arch Neurol 2012;69:657-61.
[11]
Oliveira J, Negrão L, Fineza I, et al. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing. J Hum Genet 2015;6:30512.
[12]
Genge A, Karpati G, Arnold D, Shoubridge EA, Carpenter S. Familial myopathy with conspicuous depletion of mitochondria in muscle fibers: a morphologically distinct disease. Neuromuscul Disord 1995;5:139-44.
[13]
McKenzie S, Phillips SM, Carter SL, Lowther S, Gibala MJ, Tarnopolsky MA. Endurance exercise training attenuates leucine oxidation and BCOAD activation during exercise in humans. Am J Physiol Endocrinol Metab 2000;278:580-87.
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[14]
Exome Aggregation Consortium (ExAC), Cambridge, MA (URL: http://exac.broadinstitute.org) [March 30 2015].
[15]
Osaki Y, Nishino I, Murakami N, et al. Mitochondrial abnormalities in selenium-deficient myopathy. Muscle Nerve 1998;21:637-9.
[16]
Jeffree RL, Wills EJ, Harper C. An unusual myopathy: speckled muscle fibers due to enlarged mitochondria. Muscle Nerve 2007;36:118-22.
FIGURE 1. Muscle sections of Case 1. (a-c) modified Gomori-trichrome (mGT, 400x) staining showing myopathic changes. (d-e) COX staining showing focal absence in muscle fibers (400x). (f) Electron microscopy (EM) showing zones of relative mitochondrial depletion (Bar = 2μm Mag 6000x). A highresolution version of this slide for use with the Virtual Microscope is available as eSlide: VM02070
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