- Email: [email protected]
Clinical variability of CMS-EA (congenital myasthenic syndrome with episodic apnea) due to identical CHAT mutations in two infants
European Journal of Paediatric Neurology (2005) 9, 7–12
www.elsevier.com/locate/ejpn
ORIGINAL ARTICLE
Clinical variability of CMS-EA (congenital myasthenic syndrome with episodic apnea) due to identical CHAT mutations in two infants ¨llerb, E. Paucic-Kirincicc, M. Gazdikc, K. Lah-Tomulicc, N. Barisica, J.S. Mu ¨llerb, A. Abichtb,* A. Pertlb, J. Serticd, N. Zurake, H. Lochmu a
Department of Pediatrics, University Medical School, Zagreb, Croatia Department of Neurology, and Gene Center, Friedrich-Baur-Institute, ¨nchen, Germany Ludwig-Maximilians-University, Mu c Department of Pediatrics, Children’s Hospital Kantrida, Rijeka, Croatia d Genetic Laboratory, Clinical Medical Center Zagreb, Zagreb, Croatia e Department of Neurology, University Medical School, Zagreb, Croatia b
Received 20 September 2004; accepted 26 October 2004
KEYWORDS Congenital myasthenic syndrome; Episodic apnea; CHAT mutation; Choline acetyltransferase (ChAT)
Summary Congenital myasthenic syndromes (CMS) result from mutations in various synapse-associated genes. Mutations in the choline acetyltransferase (CHAT) gene cause a presynaptic CMS associated with episodic apnea (CMS-EA). We present two unrelated Croatian children affected by CMS-EA. Beside other clinical findings characteristic for CMS, both patients manifested intermittent apneas since early infancy. Whereas the course of disease is mild in the female patient (patient 2), the male patient (patient 1) experienced recurrent and severe episodes of apnea despite adequate treatment with AChE-inhibitors and shows a global developmental delay with delayed myelination and signs of hypoxic–ischemic injury in brain imaging. Interestingly, sequencing of the CHAT gene revealed identical, compound heterozygous mutations S694C and T354M in both children. These findings are in line with a remarkable clinical heterogeneity observed in patients with CHAT mutations and emphasize the potential role of apneic crises for the development of secondary hypoxic brain damage and psychomotor retardation. Q 2004 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Address: Genzentrum Mu ¨nchen, Feodor-Lynen-Str. 25, 81377 Mu ¨nchen, Germany. Tel.: C49 89 2180 76887; fax: C49 89 2180 76999. E-mail address: [email protected] (A. Abicht).
Congenital myasthenic syndromes (CMS) are a group of rare disorders caused by inherited impairment of the safety margin of neural transmission on
1090-3798/$ - see front matter Q 2004 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2004.10.008
8 presynaptic, synaptic, and postsynaptic level.1,2 Currently, underlying genetic defects have been identified in several genes encoding synapse-associated proteins. The genetic alterations most frequently observed are missense or nonsense point mutations of the acetylcholine receptor (AChR) epsilon subunit gene (CHRNE), and of the rapsyn gene (RAPSN), both leading to postsynaptic defects.2 Less frequently found are mutations of other AChR subunit genes, mutations of the gene encoding the collagenic tail of acetylcholinesterase (ColQ) (synaptic defects),3–5 and mutations of the choline acetyltransferase gene (CHAT) (presynaptic defect).6 Recent studies have shown that ChAT and the vesicular acetylcholine transporter (VAChT) are involved in acetylcholine synthesis, formation of cholinergic vesicles and normal function of the cholinergic nervous system. Both genes CHAT and VACHT are located on chromosome 10q11, VACHT being located within the first intron of CHAT.7 In the CHAT gene, Ohno et al.6 reported 10 different recessive loss-of-function mutations in five CMS-EA kinships causing reduced expression of ChAT and/or reduced catalytic efficiency. Four additional CHAT mutations have recently been described.8,9 Clinically, CMS usually present in early infancy with symptoms similar to other inherited and acquired neuromuscular and metabolic disorders. Generalized muscle hypotonia and weakness, poor suck and cry, feeding difficulties, and developmental delay may be the first signs of CMS. Ocular symptoms including ptosis and ophtalmoparesis are common findings, other symptoms like scoliosis and arthrogryposis are less frequent. Two types of CMS— caused by either CHAT, or RAPSN mutations—are frequently associated with sudden respiratory insufficiency. For CMS with underlying CHAT mutations, this led to the term CMS with episodic apnea (CMS-EA).6 Sudden episodes of respiratory distress and bulbar weakness may be provoked by infections, fever, and conditions of stress. Apnea may require rapid initiation of ventilatory support or cause sudden death.10 Episodes of respiratory weakness may be prevented or mitigated by anticholinesterase medication. Between crisis, ptosis and fatigable muscle weakness of variable degree may be the only symptom of CMS-EA.6,8–10 A decremental EMG response on standard repetitive stimulation testing may be absent, but may be provoked by exercise or a 10Hz pre-stimulation for 5 min. Typically, this is followed by slow recovery of the amplitude of the compound muscle action potential (CMAP) over several minutes.6,10 Some phenotypic heterogeneity regarding onset,
N. Barisic et al. frequency, and severity of crises has been described, even within kinships.6,8–10 We report on two Croatian infants with CMS-EA carrying identical CHAT mutations. The patients revealed a markedly different, clinical presentation. One patient showed a relatively benign clinical course, while the other patient exhibited recurrent respiratory failure, refractory to treatment, significant developmental delay and hypoxic brain damage.
Patients and methods Patients Two patients out of two independent families were examined and followed by the authors. Both families are residing in Croatia. Consanguinity of the parents was not reported for either family.
DNA samples Venous blood samples were obtained from the patients, their parents, grandparents, and siblings, where available. Genomic DNA was isolated using a blood and tissue culture DNA extraction kit according to the manufacturer’s recommendations (Promega, Mannheim, Germany).
Sequence analysis, restriction digest PCR primers were designed based on the published genomic structure (GenBank accession numbers AF 305893–AF 305906) of the CHAT gene. We amplified all 18 exons and flanking intronic regions of the CHAT gene as described before.8 PCR-amplified fragments were purified from agarose gel using the NucleoSpin extraction kit (Macherey and Nagel, Du ¨ren, Germany) and sequenced with an Applied Biosystems model 3100 Avant DNA sequencer and fluorescence-labelled dideoxy terminators (Perkin– Elmer, Norwalk, CT, USA). The patients’ relatives and 50 European normal controls (20 of Croatian origin) were screened for the novel mutation of the CHAT gene (T354M) by restriction analysis: a PCR was performed using the primers 5 0 -cggcccactcgctcctcccgt-3 0 and 5 0 -ggatctgttcactcagttgagaaaga-3 0 to amplify a 107 bp fragment containing the mutation T354M in exon 10. The mutation T354M abolishes a TaiI restriction site. The mutant allele remains undigested, whereas the wild-type allele yields two fragments (83 and 24 bp). Restriction enzyme digestion was carried out at 65 8C for 4 h by adding 10 units of TaiI in 30 ml of reaction mixture.
Clinical variability of CMS-EA due to CHAT mutations Restriction fragments were size-fractionated on a 4% agarose gel containing ethidium bromide. Testing for the mutation S694C in exon 18 was performed by PCR using the primers 5 0 -taattcagtcaaacccccaggtgg-3 0 and 5 0 -ggaccctgaggacagggagctgtgga-3 0 to amplify a 487 bp fragment. A BfaI digest yields 208, 146, 76, 30 and 27 bp fragments for the wild-type allele. The mutation S694C abolishes a BfaI restriction site, therefore resulting in fragments of 238, 146, 76 and 27 bp length. Restriction enzyme digestion was carried out at 37 8C for 4 h by adding 10 units of BfaI in 30 ml of reaction mixture. Restriction fragments were sizefractionated on a 4% agarose gel containing ethidium bromide.
Results Clinical features We present two unrelated patients from two nonconsanguineous Croatian kinships: a boy (patient 1) aged 19 months, and a girl (patient 2) aged 26 months at the time of the last examination. Both patients suffer from CMS-EA with crisis provoked by intercurrent infections, exercise, and excitement. Whereas in patient 2 episodic apneas started at the age of 9 months and are well controlled since onset of AChE-inhibitor therapy, patient 1 showed an earlier onset, developmental delay and brain damage despite AChE-inhibitor therapy. Patient 1 The mother of patient 1 noted reduced fetal movements during pregnancy. Her son was born at 38 weeks, gestation by Cesarean section. Apgar score was 9/10, but generalized hypotonia was noticed in the newborn. At the second day of life, the boy developed signs of mild respiratory distress. A first apneic attack with generalized pronounced hypotonia, floppy jaw, severe ptosis, and irregular weak and shallow respiration was noticed at the age of 1 month. After the patient had experienced two additional episodes of apnea he was admitted to the hospital at the age of 2 months. The frequency of EA increased to almost daily occurrence at the age of 3 months. EA was frequently associated with perioral cyanosis and loss of consciousness, and occasionally with tonic–clonic seizures. EA often occurred in the prone position, about 5 min after meals, and were also provoked by respiratory infections and excitement. In general, apnea could be terminated either by tactile stimulation or by mouth to mouth resuscitation. At the age of 7 and 17 months, the patient manifested severe apnea
9 requiring artificial ventilation for 6 and 10.5 weeks, respectively. Consequently, the patient developed several complications of prolonged artificial ventilation including infections, pneumothorax and pneumomediastinum. Acetylcholinesterase inhibitors (pyridostigmine bromide) were introduced at the age of 7 months at a dosage of 4 mg/kg/day. However, there was no permanent, positive response to treatment. Temporarily, the frequency of apnoic episodes decreased to 1–2 episodes per month, but increased after a few weeks to 2–3 episodes per week. On the last examination at age 19 months, the motor and speech development of patient 1 was delayed. He did not walk unassisted. He manifested generalized and fatigable muscle weakness mostly in the upper limb girdle, ptosis, hyperextensible joints, inspiratory stridor, pronounced ptosis and brisk tendon reflexes. In a 10 Hz repetitive stimulation of the median nerve no decremental response was found in rested muscle. MRI scans at the age of 9 months showed delayed myelination, slight cortical atrophy in the frontal and temporal region, and widening of the ventricles. MRI scans at the age of 18 months showed white matter hyperintensities in the occipital region, a mild delay in myelination of the centrum semiovale, and atrophy of the corpus callosum. Usually, EEG recordings were normal, occasionally focal discharges were detected. Patient 2 Patient 2, a girl, was born after an uneventful pregnancy at 37 weeks gestation. At birth she manifested generalized hypotonia and respiratory insufficiency (Apgar score 4/3) and was intubated and ventilated immediately after birth. She recovered a few hours afterwards and was extubated and discharged from hospital after 7 days. At 7 months, she developed mild generalized muscle weakness and intermittent ptosis, most evident in the evening. At 9 months, during a respiratory infection, she had several episodes of respiratory distress resulting in respiratory failure accompanied by muscle hypotonia and loss of consciousness. She was intubated and ventilated for 2 days. Subsequently, treatment with acetylcholinesterase inhibitors (pyridostigmine bromid 7 mg/kg/day) was started. No additional episodes of apnea occurred. At the age of 19 months, the dosage was slowly tapered to 3 mg/kg/day of pyridostigmine bromid. At the last examination at the age of 26 months, the patient showed normal psychomotor and speech development. There were signs of generalized fatigable muscle weakness and mild ptosis in the late afternoon.
10 Electroencephalography and EMG studies were normal. Repetitive stimulation at 10 Hz for 5 min resulted in a mild decremental response (10–20%) in facial and distal muscles. There was no decremental response in resting muscle without prior 10 Hz stimulation. A 4 year-old brother and both parents of the female patient are asymptomatic.
Molecular genetic analysis Direct sequencing of the CHAT gene on chromosome 10q11.2 in both patients revealed identical
N. Barisic et al. compound heterozygous mutations: Mutation 2081COG (S694C) in exon 18 and mutation 1061COT (T354M) in exon 10. No additional mutation was found in any of the other translated exons of the CHAT gene. Analysis of exons 10 and 18 in the parents of patients 1 and 2 and the healthy brother of patient 2 (Fig. 1) indicated that both fathers are heterozygous carriers of the mutation T354M while both mothers are heterozygous carriers of the mutation S694C. In the mother of patient 1 all exons of CHAT were sequenced to exclude a second mutation. The healthy brother of
Figure 1 Restriction enzyme analysis in families 1 and 2: to detect the CHAT mutation T354M (1061COT), a PCR fragment of 107 bp of exon 10 was amplified. Since the mutation abolishes a TaiI site, the mutated allele remains undigested, whereas the wildtype allele yields two fragments (83 and 24 bp). To detect the CHAT mutation S694C (2081COG), a PCR fragment of 487 bp containing exon 18 was amplified. The mutation abolishes one of the BfaI sites, yielding a 238 bp fragment instead of 208 and 30 bp in the wildtype allele. The size of the other fragments remains unchanged. In both families, the patients (II:1 and III:1, respectively) carry both mutations heterozygously. In family 1, the patient’s father (I:2) is carrier of T354M, whereas the mother (I:1) is heterozygous carrier of the S694C mutation. In family 2, the patient’s father and grandmother (II:1 and I:2) carry T354M heterozygously, whereas the mother (II:2) carries S694C. The healthy brother and the grandfather of patient 2 do not carry any of the mutations.
Clinical variability of CMS-EA due to CHAT mutations patient 2 does not carry any mutation. Both mutations are missense mutations leading to an amino acid exchange. The mutation S694C has been described, previously.9 The mutation T354M was not detected in 50 healthy controls.
Discussion We present two unrelated infants with CMS-EA, both carrying the same compound heterozygous missense mutations of the CHAT gene. One of the missense mutations detected in our patients, the mutation 1061COT (T354M) in exon 10, is a novel mutation that has not been described before. We hypothesize that CHAT (T354M) is pathogenic because the mutation was not detected in 100 control chromosomes, the mutation segregates with the disease phenotype in both families in an autosomal recessive trait, and the mutation affects an amino acid that is highly conserved at this position in mammals.11 Based on the rat ChAT crystal structure,11 the amino acid substitution T354M is distant from active sites and binding sites of the enzyme. The second missense mutation, 2081COG (S694C) in exon 18 of the CHAT gene, was identified heterozygously in both patients. Compound heterozygous CHAT (S694C) has been described previously in a patient with CMS-EA.9 In this report, CHAT (S694C) resulted in a phenotype characterized by mild proximal muscle weakness associated with life threatening apnea. The parent carrying heterozygous CHAT (S694C), but no second CHAT mutation, was reported clinically unaffected.9 Based on the rat ChAT crystal structure, S694C may alter the choline binding site. 9,11 However, functional studies have not been carried out for both mutations, and the exact molecular consequences of the mutations remain speculative for the time being. Interestingly, the mother of patient 1 heterozygous for the mutation CHAT (S694C) reported on subjective complaints (day-time dependent ptosis and fatigue), but regrettably was not available for objective examination (clinically or electrophysiologically), whereas the mother of patient 2 who carries the same mutation was clinically unaffected. Sequencing of all exons of the CHAT gene did not reveal any additional mutation in the mother of patient 1. Similarly, heterozygous parents have been reported to be unaffected.6,9 Heterozygous ChAT knockout mice having a 50–60% reduction of ChAT activity appear clinically normal.12 However, there
11 is indication that a decrease of ChAT activity below 50% of the normal may provoke functional impairment of neuromuscular transmission.6 Similarly, a decremental EMG response was reported in a clinically unaffected parent heterozygous for a recessive COLQ mutation.5 Despite identical genotypes for CHAT, patients 1 and 2 exhibit markedly different clinical phenotypes of CMS-EA. Patient 2 presented with mild myasthenic symptoms, a normal psychomotor development and a benign course of disease. Following one episode of respiratory distress at the age of 9 months, specific therapy with AChEinhibitors (pyridostigmine bromide) was started, and no further EA occurred. Patient 1 showed global developmental delay, episodes of respiratory distress manifested earlier (at the age of 1 month) and occurred more frequently, and cumulated in severe apnea with prolonged respiratory failure despite adequate AChE treatment. In addition, the patient experienced tonic–clonic seizures during EA, repeatedly. Brain MRI scans performed in patient 1 are compatible with hypoxic–ischemic brain lesion associated with signs of delayed myelination. Cholinergic neurons are distributed widely throughout the central and peripheral nervous system, where they are involved in motor function, autonomic nervous system, neuronal growth and developmental plasticity.11,13,14 A decrease in ChAT activity has been associated with a number of developmental and neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, Schizophrenia, Rett syndrome, and Sudden Infant Death Syndrome (SIDS).15 All CHAT mutations observed in CMS-EA so far localize to the coding region shared by all isoforms of ChAT that are expressed in the nervous system.16 However, none of these patients revealed evidence for a significant central or autonomic nervous system involvement in addition to the neuromuscular transmission defect. Explanations for this selectivity may be differences among neuromuscular synapses and synapses of the CNS regarding presynaptic levels of ChAT, choline, or acetyl-CoA; rates of choline uptake; or rates of ACh release under conditions of increased neuronal impulse flow.2 We may speculate, that the delay in CNS development and neuronal myelination observed in patient 1 are directly caused by the deficiency of ChAT in the brain. The expression of ChAT and vAChT in cholinergic CNS neurons are co-regulated by ciliary neurotrophic factor, cAMP, cholinergic differentiation factor/leukemia inhibitory factor and retinoids. 15,17 Alteration in
12 neurotransmitter release impair the normal pattern of neurotransmitter expression and growth related proteins in developing motoneurons in the rat.18 Therefore, impaired cholinergic transmission may influence CNS maturation and capacity for neuroprotection. However, it appears more likely that the observed damage in the brain of patient 1 reflects a hypoxic– ischemic encephalopathy secondary to repeated, severe respiratory failure. Infants with SIDS showed decreased activity of ChAT in the striatum, hypothalamus, nucleus arcuatus, dorsal motor nucleus of vagus and in the hypoglossal nucleus.15,19 Impaired maturation of brainstem structures may play a role in the pathogenesis of SIDS.19 Coincidence of CHAT mutations, and a family history for SIDS has been reported by others.10 Our case illustrates the importance of effective management of EA to prevent secondary hypoxia and brain damage in CMS-EA patients. Although treatment with pyridostigmine bromide was not equally effective in both patients prophylactic anticholinesterase treatment is recommended to prevent or mitigate respiratory failure, even for patients asymptomatic between crisis.2 In a respiratory crisis, patients should be treated with AChE-inhibitors.10 Parents of CMS-EA infants should be instructed to apply an inflatable rescue bag (ambu) and a fitted mask. It has been suggested to avoid the use of aminoglycoside antibiotics in patients with CMS-EA.10 Similarly, anoxic convulsions need to be treated, but application of diazepam and phenobarbitone may worsen the respiratory depression.10
Acknowledgements We thank the patients and their families for participating in this study. We wish to thank Milivoj Novak, MD and Miran Cvitkovic, MD for treating patient 1 in the intensive care unit. We thank Luka Brcic, MD and Ivan Lehman, MD, for technical assistance in preparing the manuscript, Marko Rados, PhD for analyzing MR scans, and Leo Pazanin, MD for muscle histology. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to HL and AA, and by a Collaborative Linkage Grant from the NATO Science Foundation (LST.CLG.980296) to HL and NB. JSM receives a scholarship from the Boehringer Ingelheim Fonds.
N. Barisic et al.
References 1. Engel AG. Myasthenic syndromes. In: Engel AG, editor. Myology, F.-A.C.e. New York: McGraw-Hill; 1994. p. 1798– 835. 2. Engel AG, Ohno K, Sine SM. Congenital myasthenic syndromes: progress over the past decade. Muscle Nerve 2003; 27:4–25. 3. Donger C, Krejci E, Serradell AP, et al. Mutation in the human acetylcholinesterase-associated collagen gene, COLQ, is responsible for congenital myasthenic syndrome with endplate acetylcholinesterase deficiency (Type Ic). Am J Hum Genet 1998;63:967–75. 4. Ohno K, Brengman J, Tsujino A, Engel AG. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme. Proc Natl Acad Sci USA 1998;95:9654–9. 5. Ohno K, Engel AG, Brengman JM, et al. The spectrum of mutations causing end-plate acetylcholinesterase deficiency. Ann Neurol 2000;47:162–70. 6. Ohno K, Tsujino A, Brengman JM, et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc Natl Acad Sci USA 2001;98:2017–22. 7. Eiden LE. The cholinergic gene locus. J Neurochem 1998;70: 2227–40. 8. Schmidt C, Abicht A, Krampfl K, et al. Congenital myasthenic syndrome due to a novel missense mutation in the gene encoding choline acetyltransferase. Neuromuscul Disord 2003;13:245–51. 9. Maselli RA, Chen D, Mo D, et al. Choline acetyltransferase mutations in myasthenic syndrome due to deficient acetylcholine resynthesis. Muscle Nerve 2003;27:180–7. 10. Byring RF, Pihko H, Tsujino A, et al. Congenital myasthenic syndrome associated with episodic apnea and sudden infant death. Neuromuscul Disord 2002;12:548–53. 11. Cai Y, Cronin CN, Engel AG, et al. Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J 2004;23:2047–58. 12. Brandon EP, Lin W, D’Amour KA, et al. Aberrant patterning of neuromuscular synapses in choline acetyltransferasedeficient mice. J Neurosci 2003;23:539–49. 13. Lauder JM, Schambra UB. Morphogenetic roles of acetylcholine. Environ Health Perspect 1999;107(Suppl 1):65–9. 14. Karczmar AG. Brief presentation of the story and present status of studies of the vertebrate cholinergic system. Neuropsychopharmacology 1993;9:181–99. 15. Oda Y. Choline acetyltransferase: the structure, distribution and pathologic changes in the central nervous system. Pathol Int 1999;49:921–37. 16. Engel AG, Ohno K, Sine SM. Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat Rev Neurosci 2003;4:339–52. 17. Berse B, Blusztajn JK. Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor alpha, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line. J Biol Chem 1995;270:22101–4. 18. Sharp PS, Dekkers J, Dick JR, Greensmith L. Manipulating transmitter release at the neuromuscular junction of neonatal rats alters the expression of ChAT and GAP-43 in motoneurons. Brain Res Dev Brain Res 2003;146:29–38. 19. Kinney HC, Filiano JJ, Harper RM. The neuropathology of the sudden infant death syndrome. A review. J Neuropathol Exp Neurol 1992;51:115–26.
www.elsevier.com/locate/ejpn
ORIGINAL ARTICLE
Clinical variability of CMS-EA (congenital myasthenic syndrome with episodic apnea) due to identical CHAT mutations in two infants ¨llerb, E. Paucic-Kirincicc, M. Gazdikc, K. Lah-Tomulicc, N. Barisica, J.S. Mu ¨llerb, A. Abichtb,* A. Pertlb, J. Serticd, N. Zurake, H. Lochmu a
Department of Pediatrics, University Medical School, Zagreb, Croatia Department of Neurology, and Gene Center, Friedrich-Baur-Institute, ¨nchen, Germany Ludwig-Maximilians-University, Mu c Department of Pediatrics, Children’s Hospital Kantrida, Rijeka, Croatia d Genetic Laboratory, Clinical Medical Center Zagreb, Zagreb, Croatia e Department of Neurology, University Medical School, Zagreb, Croatia b
Received 20 September 2004; accepted 26 October 2004
KEYWORDS Congenital myasthenic syndrome; Episodic apnea; CHAT mutation; Choline acetyltransferase (ChAT)
Summary Congenital myasthenic syndromes (CMS) result from mutations in various synapse-associated genes. Mutations in the choline acetyltransferase (CHAT) gene cause a presynaptic CMS associated with episodic apnea (CMS-EA). We present two unrelated Croatian children affected by CMS-EA. Beside other clinical findings characteristic for CMS, both patients manifested intermittent apneas since early infancy. Whereas the course of disease is mild in the female patient (patient 2), the male patient (patient 1) experienced recurrent and severe episodes of apnea despite adequate treatment with AChE-inhibitors and shows a global developmental delay with delayed myelination and signs of hypoxic–ischemic injury in brain imaging. Interestingly, sequencing of the CHAT gene revealed identical, compound heterozygous mutations S694C and T354M in both children. These findings are in line with a remarkable clinical heterogeneity observed in patients with CHAT mutations and emphasize the potential role of apneic crises for the development of secondary hypoxic brain damage and psychomotor retardation. Q 2004 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Address: Genzentrum Mu ¨nchen, Feodor-Lynen-Str. 25, 81377 Mu ¨nchen, Germany. Tel.: C49 89 2180 76887; fax: C49 89 2180 76999. E-mail address: [email protected] (A. Abicht).
Congenital myasthenic syndromes (CMS) are a group of rare disorders caused by inherited impairment of the safety margin of neural transmission on
1090-3798/$ - see front matter Q 2004 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2004.10.008
8 presynaptic, synaptic, and postsynaptic level.1,2 Currently, underlying genetic defects have been identified in several genes encoding synapse-associated proteins. The genetic alterations most frequently observed are missense or nonsense point mutations of the acetylcholine receptor (AChR) epsilon subunit gene (CHRNE), and of the rapsyn gene (RAPSN), both leading to postsynaptic defects.2 Less frequently found are mutations of other AChR subunit genes, mutations of the gene encoding the collagenic tail of acetylcholinesterase (ColQ) (synaptic defects),3–5 and mutations of the choline acetyltransferase gene (CHAT) (presynaptic defect).6 Recent studies have shown that ChAT and the vesicular acetylcholine transporter (VAChT) are involved in acetylcholine synthesis, formation of cholinergic vesicles and normal function of the cholinergic nervous system. Both genes CHAT and VACHT are located on chromosome 10q11, VACHT being located within the first intron of CHAT.7 In the CHAT gene, Ohno et al.6 reported 10 different recessive loss-of-function mutations in five CMS-EA kinships causing reduced expression of ChAT and/or reduced catalytic efficiency. Four additional CHAT mutations have recently been described.8,9 Clinically, CMS usually present in early infancy with symptoms similar to other inherited and acquired neuromuscular and metabolic disorders. Generalized muscle hypotonia and weakness, poor suck and cry, feeding difficulties, and developmental delay may be the first signs of CMS. Ocular symptoms including ptosis and ophtalmoparesis are common findings, other symptoms like scoliosis and arthrogryposis are less frequent. Two types of CMS— caused by either CHAT, or RAPSN mutations—are frequently associated with sudden respiratory insufficiency. For CMS with underlying CHAT mutations, this led to the term CMS with episodic apnea (CMS-EA).6 Sudden episodes of respiratory distress and bulbar weakness may be provoked by infections, fever, and conditions of stress. Apnea may require rapid initiation of ventilatory support or cause sudden death.10 Episodes of respiratory weakness may be prevented or mitigated by anticholinesterase medication. Between crisis, ptosis and fatigable muscle weakness of variable degree may be the only symptom of CMS-EA.6,8–10 A decremental EMG response on standard repetitive stimulation testing may be absent, but may be provoked by exercise or a 10Hz pre-stimulation for 5 min. Typically, this is followed by slow recovery of the amplitude of the compound muscle action potential (CMAP) over several minutes.6,10 Some phenotypic heterogeneity regarding onset,
N. Barisic et al. frequency, and severity of crises has been described, even within kinships.6,8–10 We report on two Croatian infants with CMS-EA carrying identical CHAT mutations. The patients revealed a markedly different, clinical presentation. One patient showed a relatively benign clinical course, while the other patient exhibited recurrent respiratory failure, refractory to treatment, significant developmental delay and hypoxic brain damage.
Patients and methods Patients Two patients out of two independent families were examined and followed by the authors. Both families are residing in Croatia. Consanguinity of the parents was not reported for either family.
DNA samples Venous blood samples were obtained from the patients, their parents, grandparents, and siblings, where available. Genomic DNA was isolated using a blood and tissue culture DNA extraction kit according to the manufacturer’s recommendations (Promega, Mannheim, Germany).
Sequence analysis, restriction digest PCR primers were designed based on the published genomic structure (GenBank accession numbers AF 305893–AF 305906) of the CHAT gene. We amplified all 18 exons and flanking intronic regions of the CHAT gene as described before.8 PCR-amplified fragments were purified from agarose gel using the NucleoSpin extraction kit (Macherey and Nagel, Du ¨ren, Germany) and sequenced with an Applied Biosystems model 3100 Avant DNA sequencer and fluorescence-labelled dideoxy terminators (Perkin– Elmer, Norwalk, CT, USA). The patients’ relatives and 50 European normal controls (20 of Croatian origin) were screened for the novel mutation of the CHAT gene (T354M) by restriction analysis: a PCR was performed using the primers 5 0 -cggcccactcgctcctcccgt-3 0 and 5 0 -ggatctgttcactcagttgagaaaga-3 0 to amplify a 107 bp fragment containing the mutation T354M in exon 10. The mutation T354M abolishes a TaiI restriction site. The mutant allele remains undigested, whereas the wild-type allele yields two fragments (83 and 24 bp). Restriction enzyme digestion was carried out at 65 8C for 4 h by adding 10 units of TaiI in 30 ml of reaction mixture.
Clinical variability of CMS-EA due to CHAT mutations Restriction fragments were size-fractionated on a 4% agarose gel containing ethidium bromide. Testing for the mutation S694C in exon 18 was performed by PCR using the primers 5 0 -taattcagtcaaacccccaggtgg-3 0 and 5 0 -ggaccctgaggacagggagctgtgga-3 0 to amplify a 487 bp fragment. A BfaI digest yields 208, 146, 76, 30 and 27 bp fragments for the wild-type allele. The mutation S694C abolishes a BfaI restriction site, therefore resulting in fragments of 238, 146, 76 and 27 bp length. Restriction enzyme digestion was carried out at 37 8C for 4 h by adding 10 units of BfaI in 30 ml of reaction mixture. Restriction fragments were sizefractionated on a 4% agarose gel containing ethidium bromide.
Results Clinical features We present two unrelated patients from two nonconsanguineous Croatian kinships: a boy (patient 1) aged 19 months, and a girl (patient 2) aged 26 months at the time of the last examination. Both patients suffer from CMS-EA with crisis provoked by intercurrent infections, exercise, and excitement. Whereas in patient 2 episodic apneas started at the age of 9 months and are well controlled since onset of AChE-inhibitor therapy, patient 1 showed an earlier onset, developmental delay and brain damage despite AChE-inhibitor therapy. Patient 1 The mother of patient 1 noted reduced fetal movements during pregnancy. Her son was born at 38 weeks, gestation by Cesarean section. Apgar score was 9/10, but generalized hypotonia was noticed in the newborn. At the second day of life, the boy developed signs of mild respiratory distress. A first apneic attack with generalized pronounced hypotonia, floppy jaw, severe ptosis, and irregular weak and shallow respiration was noticed at the age of 1 month. After the patient had experienced two additional episodes of apnea he was admitted to the hospital at the age of 2 months. The frequency of EA increased to almost daily occurrence at the age of 3 months. EA was frequently associated with perioral cyanosis and loss of consciousness, and occasionally with tonic–clonic seizures. EA often occurred in the prone position, about 5 min after meals, and were also provoked by respiratory infections and excitement. In general, apnea could be terminated either by tactile stimulation or by mouth to mouth resuscitation. At the age of 7 and 17 months, the patient manifested severe apnea
9 requiring artificial ventilation for 6 and 10.5 weeks, respectively. Consequently, the patient developed several complications of prolonged artificial ventilation including infections, pneumothorax and pneumomediastinum. Acetylcholinesterase inhibitors (pyridostigmine bromide) were introduced at the age of 7 months at a dosage of 4 mg/kg/day. However, there was no permanent, positive response to treatment. Temporarily, the frequency of apnoic episodes decreased to 1–2 episodes per month, but increased after a few weeks to 2–3 episodes per week. On the last examination at age 19 months, the motor and speech development of patient 1 was delayed. He did not walk unassisted. He manifested generalized and fatigable muscle weakness mostly in the upper limb girdle, ptosis, hyperextensible joints, inspiratory stridor, pronounced ptosis and brisk tendon reflexes. In a 10 Hz repetitive stimulation of the median nerve no decremental response was found in rested muscle. MRI scans at the age of 9 months showed delayed myelination, slight cortical atrophy in the frontal and temporal region, and widening of the ventricles. MRI scans at the age of 18 months showed white matter hyperintensities in the occipital region, a mild delay in myelination of the centrum semiovale, and atrophy of the corpus callosum. Usually, EEG recordings were normal, occasionally focal discharges were detected. Patient 2 Patient 2, a girl, was born after an uneventful pregnancy at 37 weeks gestation. At birth she manifested generalized hypotonia and respiratory insufficiency (Apgar score 4/3) and was intubated and ventilated immediately after birth. She recovered a few hours afterwards and was extubated and discharged from hospital after 7 days. At 7 months, she developed mild generalized muscle weakness and intermittent ptosis, most evident in the evening. At 9 months, during a respiratory infection, she had several episodes of respiratory distress resulting in respiratory failure accompanied by muscle hypotonia and loss of consciousness. She was intubated and ventilated for 2 days. Subsequently, treatment with acetylcholinesterase inhibitors (pyridostigmine bromid 7 mg/kg/day) was started. No additional episodes of apnea occurred. At the age of 19 months, the dosage was slowly tapered to 3 mg/kg/day of pyridostigmine bromid. At the last examination at the age of 26 months, the patient showed normal psychomotor and speech development. There were signs of generalized fatigable muscle weakness and mild ptosis in the late afternoon.
10 Electroencephalography and EMG studies were normal. Repetitive stimulation at 10 Hz for 5 min resulted in a mild decremental response (10–20%) in facial and distal muscles. There was no decremental response in resting muscle without prior 10 Hz stimulation. A 4 year-old brother and both parents of the female patient are asymptomatic.
Molecular genetic analysis Direct sequencing of the CHAT gene on chromosome 10q11.2 in both patients revealed identical
N. Barisic et al. compound heterozygous mutations: Mutation 2081COG (S694C) in exon 18 and mutation 1061COT (T354M) in exon 10. No additional mutation was found in any of the other translated exons of the CHAT gene. Analysis of exons 10 and 18 in the parents of patients 1 and 2 and the healthy brother of patient 2 (Fig. 1) indicated that both fathers are heterozygous carriers of the mutation T354M while both mothers are heterozygous carriers of the mutation S694C. In the mother of patient 1 all exons of CHAT were sequenced to exclude a second mutation. The healthy brother of
Figure 1 Restriction enzyme analysis in families 1 and 2: to detect the CHAT mutation T354M (1061COT), a PCR fragment of 107 bp of exon 10 was amplified. Since the mutation abolishes a TaiI site, the mutated allele remains undigested, whereas the wildtype allele yields two fragments (83 and 24 bp). To detect the CHAT mutation S694C (2081COG), a PCR fragment of 487 bp containing exon 18 was amplified. The mutation abolishes one of the BfaI sites, yielding a 238 bp fragment instead of 208 and 30 bp in the wildtype allele. The size of the other fragments remains unchanged. In both families, the patients (II:1 and III:1, respectively) carry both mutations heterozygously. In family 1, the patient’s father (I:2) is carrier of T354M, whereas the mother (I:1) is heterozygous carrier of the S694C mutation. In family 2, the patient’s father and grandmother (II:1 and I:2) carry T354M heterozygously, whereas the mother (II:2) carries S694C. The healthy brother and the grandfather of patient 2 do not carry any of the mutations.
Clinical variability of CMS-EA due to CHAT mutations patient 2 does not carry any mutation. Both mutations are missense mutations leading to an amino acid exchange. The mutation S694C has been described, previously.9 The mutation T354M was not detected in 50 healthy controls.
Discussion We present two unrelated infants with CMS-EA, both carrying the same compound heterozygous missense mutations of the CHAT gene. One of the missense mutations detected in our patients, the mutation 1061COT (T354M) in exon 10, is a novel mutation that has not been described before. We hypothesize that CHAT (T354M) is pathogenic because the mutation was not detected in 100 control chromosomes, the mutation segregates with the disease phenotype in both families in an autosomal recessive trait, and the mutation affects an amino acid that is highly conserved at this position in mammals.11 Based on the rat ChAT crystal structure,11 the amino acid substitution T354M is distant from active sites and binding sites of the enzyme. The second missense mutation, 2081COG (S694C) in exon 18 of the CHAT gene, was identified heterozygously in both patients. Compound heterozygous CHAT (S694C) has been described previously in a patient with CMS-EA.9 In this report, CHAT (S694C) resulted in a phenotype characterized by mild proximal muscle weakness associated with life threatening apnea. The parent carrying heterozygous CHAT (S694C), but no second CHAT mutation, was reported clinically unaffected.9 Based on the rat ChAT crystal structure, S694C may alter the choline binding site. 9,11 However, functional studies have not been carried out for both mutations, and the exact molecular consequences of the mutations remain speculative for the time being. Interestingly, the mother of patient 1 heterozygous for the mutation CHAT (S694C) reported on subjective complaints (day-time dependent ptosis and fatigue), but regrettably was not available for objective examination (clinically or electrophysiologically), whereas the mother of patient 2 who carries the same mutation was clinically unaffected. Sequencing of all exons of the CHAT gene did not reveal any additional mutation in the mother of patient 1. Similarly, heterozygous parents have been reported to be unaffected.6,9 Heterozygous ChAT knockout mice having a 50–60% reduction of ChAT activity appear clinically normal.12 However, there
11 is indication that a decrease of ChAT activity below 50% of the normal may provoke functional impairment of neuromuscular transmission.6 Similarly, a decremental EMG response was reported in a clinically unaffected parent heterozygous for a recessive COLQ mutation.5 Despite identical genotypes for CHAT, patients 1 and 2 exhibit markedly different clinical phenotypes of CMS-EA. Patient 2 presented with mild myasthenic symptoms, a normal psychomotor development and a benign course of disease. Following one episode of respiratory distress at the age of 9 months, specific therapy with AChEinhibitors (pyridostigmine bromide) was started, and no further EA occurred. Patient 1 showed global developmental delay, episodes of respiratory distress manifested earlier (at the age of 1 month) and occurred more frequently, and cumulated in severe apnea with prolonged respiratory failure despite adequate AChE treatment. In addition, the patient experienced tonic–clonic seizures during EA, repeatedly. Brain MRI scans performed in patient 1 are compatible with hypoxic–ischemic brain lesion associated with signs of delayed myelination. Cholinergic neurons are distributed widely throughout the central and peripheral nervous system, where they are involved in motor function, autonomic nervous system, neuronal growth and developmental plasticity.11,13,14 A decrease in ChAT activity has been associated with a number of developmental and neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, Schizophrenia, Rett syndrome, and Sudden Infant Death Syndrome (SIDS).15 All CHAT mutations observed in CMS-EA so far localize to the coding region shared by all isoforms of ChAT that are expressed in the nervous system.16 However, none of these patients revealed evidence for a significant central or autonomic nervous system involvement in addition to the neuromuscular transmission defect. Explanations for this selectivity may be differences among neuromuscular synapses and synapses of the CNS regarding presynaptic levels of ChAT, choline, or acetyl-CoA; rates of choline uptake; or rates of ACh release under conditions of increased neuronal impulse flow.2 We may speculate, that the delay in CNS development and neuronal myelination observed in patient 1 are directly caused by the deficiency of ChAT in the brain. The expression of ChAT and vAChT in cholinergic CNS neurons are co-regulated by ciliary neurotrophic factor, cAMP, cholinergic differentiation factor/leukemia inhibitory factor and retinoids. 15,17 Alteration in
12 neurotransmitter release impair the normal pattern of neurotransmitter expression and growth related proteins in developing motoneurons in the rat.18 Therefore, impaired cholinergic transmission may influence CNS maturation and capacity for neuroprotection. However, it appears more likely that the observed damage in the brain of patient 1 reflects a hypoxic– ischemic encephalopathy secondary to repeated, severe respiratory failure. Infants with SIDS showed decreased activity of ChAT in the striatum, hypothalamus, nucleus arcuatus, dorsal motor nucleus of vagus and in the hypoglossal nucleus.15,19 Impaired maturation of brainstem structures may play a role in the pathogenesis of SIDS.19 Coincidence of CHAT mutations, and a family history for SIDS has been reported by others.10 Our case illustrates the importance of effective management of EA to prevent secondary hypoxia and brain damage in CMS-EA patients. Although treatment with pyridostigmine bromide was not equally effective in both patients prophylactic anticholinesterase treatment is recommended to prevent or mitigate respiratory failure, even for patients asymptomatic between crisis.2 In a respiratory crisis, patients should be treated with AChE-inhibitors.10 Parents of CMS-EA infants should be instructed to apply an inflatable rescue bag (ambu) and a fitted mask. It has been suggested to avoid the use of aminoglycoside antibiotics in patients with CMS-EA.10 Similarly, anoxic convulsions need to be treated, but application of diazepam and phenobarbitone may worsen the respiratory depression.10
Acknowledgements We thank the patients and their families for participating in this study. We wish to thank Milivoj Novak, MD and Miran Cvitkovic, MD for treating patient 1 in the intensive care unit. We thank Luka Brcic, MD and Ivan Lehman, MD, for technical assistance in preparing the manuscript, Marko Rados, PhD for analyzing MR scans, and Leo Pazanin, MD for muscle histology. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to HL and AA, and by a Collaborative Linkage Grant from the NATO Science Foundation (LST.CLG.980296) to HL and NB. JSM receives a scholarship from the Boehringer Ingelheim Fonds.
N. Barisic et al.
References 1. Engel AG. Myasthenic syndromes. In: Engel AG, editor. Myology, F.-A.C.e. New York: McGraw-Hill; 1994. p. 1798– 835. 2. Engel AG, Ohno K, Sine SM. Congenital myasthenic syndromes: progress over the past decade. Muscle Nerve 2003; 27:4–25. 3. Donger C, Krejci E, Serradell AP, et al. Mutation in the human acetylcholinesterase-associated collagen gene, COLQ, is responsible for congenital myasthenic syndrome with endplate acetylcholinesterase deficiency (Type Ic). Am J Hum Genet 1998;63:967–75. 4. Ohno K, Brengman J, Tsujino A, Engel AG. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme. Proc Natl Acad Sci USA 1998;95:9654–9. 5. Ohno K, Engel AG, Brengman JM, et al. The spectrum of mutations causing end-plate acetylcholinesterase deficiency. Ann Neurol 2000;47:162–70. 6. Ohno K, Tsujino A, Brengman JM, et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc Natl Acad Sci USA 2001;98:2017–22. 7. Eiden LE. The cholinergic gene locus. J Neurochem 1998;70: 2227–40. 8. Schmidt C, Abicht A, Krampfl K, et al. Congenital myasthenic syndrome due to a novel missense mutation in the gene encoding choline acetyltransferase. Neuromuscul Disord 2003;13:245–51. 9. Maselli RA, Chen D, Mo D, et al. Choline acetyltransferase mutations in myasthenic syndrome due to deficient acetylcholine resynthesis. Muscle Nerve 2003;27:180–7. 10. Byring RF, Pihko H, Tsujino A, et al. Congenital myasthenic syndrome associated with episodic apnea and sudden infant death. Neuromuscul Disord 2002;12:548–53. 11. Cai Y, Cronin CN, Engel AG, et al. Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J 2004;23:2047–58. 12. Brandon EP, Lin W, D’Amour KA, et al. Aberrant patterning of neuromuscular synapses in choline acetyltransferasedeficient mice. J Neurosci 2003;23:539–49. 13. Lauder JM, Schambra UB. Morphogenetic roles of acetylcholine. Environ Health Perspect 1999;107(Suppl 1):65–9. 14. Karczmar AG. Brief presentation of the story and present status of studies of the vertebrate cholinergic system. Neuropsychopharmacology 1993;9:181–99. 15. Oda Y. Choline acetyltransferase: the structure, distribution and pathologic changes in the central nervous system. Pathol Int 1999;49:921–37. 16. Engel AG, Ohno K, Sine SM. Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat Rev Neurosci 2003;4:339–52. 17. Berse B, Blusztajn JK. Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor alpha, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line. J Biol Chem 1995;270:22101–4. 18. Sharp PS, Dekkers J, Dick JR, Greensmith L. Manipulating transmitter release at the neuromuscular junction of neonatal rats alters the expression of ChAT and GAP-43 in motoneurons. Brain Res Dev Brain Res 2003;146:29–38. 19. Kinney HC, Filiano JJ, Harper RM. The neuropathology of the sudden infant death syndrome. A review. J Neuropathol Exp Neurol 1992;51:115–26.