- Email: [email protected]
Severe congenital myasthenic syndrome associated with novel biallelic mutation of the CHRND gene
Journal Pre-proof
Severe congenital myasthenic syndrome associated with novel biallelic mutation of the CHRND gene ¨ , Carmen Bonanno , Carmelo Rodolico , Ana Topf Francesca Maria Foti , Wei-Wei Liu , David Beeson , Antonio Toscano , Hanns Lochmuller ¨ PII: DOI: Reference:
S0960-8966(20)30038-9 https://doi.org/10.1016/j.nmd.2020.02.012 NMD 3806
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
16 June 2019 14 January 2020 16 February 2020
¨ , Francesca Maria Foti , Please cite this article as: Carmen Bonanno , Carmelo Rodolico , Ana Topf Wei-Wei Liu , David Beeson , Antonio Toscano , Hanns Lochmuller , Severe congenital myasthenic ¨ syndrome associated with novel biallelic mutation of the CHRND gene, Neuromuscular Disorders (2020), doi: https://doi.org/10.1016/j.nmd.2020.02.012
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
Highlights
Neuromuscular junction proteins are essential for transmission effectiveness Cholinergic receptor nicotinic δ subunit mutations are rare but possibly fatal Congenital myasthenic syndromes neonatal onset can be severe and life-threatening Myasthenic syndromes diagnosis is tricky,extensive genetic approach is required
1
Severe congenital myasthenic syndrome associated with novel biallelic mutation of the CHRND gene
Carmen Bonanno
a
and Carmelo Rodolico a*, Ana Töpf b, Francesca Maria Foti c, Wei-Wei Liu d,
David Beeson d, Antonio Toscano a, Hanns Lochmüller e a
Department of Clinical and Experimental Medicine, Unit of Neurology and Neuromuscular
Diseases, University of Messina, Messina, Italy b
John Walton, Muscular Dystrophy Research Centre, Institute of Translational and Clinical
Research, Newcastle University and Newcastle Hospitals, Newcastle upon Tyne, UK c
Neonatology and Neonatal Intensive Care Unit, "Bianchi-Melacrino-Morelli" Hospital, Reggio
Calabria, Italy d
Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK e
Department of Neuropediatrics and Muscle Disorders, Medical Center – University of Freiburg,
Faculty of Medicine, Freiburg, Germany and Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Catalonia, Spain Children's Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Canada and Division of Neurology, Department of Medicine, The Ottawa Hospital, Ottawa, Canada *These two authors contributed equally to the paper
Corresponding Author: Carmelo Rodolico Department of Clinical and Experimental Medicine, Unit of Neurology and Neuromuscular Disease, University of Messina, Messina, Italy Phone: 00390902213501 Email: [email protected] ABSTRACT
2
Congenital myasthenic syndromes (CMS) are a group of inherited disorders caused by mutations in genes encoding proteins essential for neuromuscular transmission. CMS is characterized by fatigable muscle weakness with onset at birth or in early childhood; rarely, symptoms may present later. The most frequently involved proteins are choline acetyltransferase, the endplate species of acetylcholinesterase and the acetylcholine receptor subunits. Defects in the cholinergic receptor nicotinic delta subunit (CHRND) are a rare cause for CMS but they should be considered in patients with a severe, early onset disease, with respiratory distress. We describe two sisters, clinically and genetically diagnosed with CMS, carrying two heteroallelic variants in the CHRND gene: c.730C>T; p.(Arg244Cys) and c.1304T>C; p.(Leu435Pro). The first variant has already been described yet no clinical relevance has been proved; the second one, is a novel variant documented here for the first time. These two cases expand the clinical spectrum of CMS linked to CHRND mutations.
Key words: lethal phenotype; AChR delta subunit; children; precocious onset.
3
1. INTRODUCTION Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of the neuromuscular transmission. Although clinical manifestations vary by subtype, CMS are usually characterized by fatigable muscle weakness (ocular, bulbar, limb muscles) with onset at birth or in early childhood; rarely, symptoms may present later. The main proteins involved in the pathogenesis of CMS are: choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor subunits (CHRNA, CHRNB, CHRND, CHRNE), rapsyn, plectin, Na(v)1.4, the muscle specific protein kinase (MuSK), agrin, downstream of tyrosine kinase 7 (Dok7), and glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [1]. Depending on the location of the primary defect within the neuromuscular junction (NMJ) congenital myasthenic syndromes can be classified as presynaptic, synaptic, or postsynaptic [2]. To date, the number of genes already identified and related to a specific CMS subtype is exceeding thirty, however, 30-50% of patients with congenital myasthenic syndromes do not carry mutations in known CMS-causing genes [3]. Mutations of the δ AchR subunit (CHRND) are a very rare cause for CMS but they should be considered in patients with a severe, early onset disease with respiratory involvement [4-5]. We describe the clinical phenotype and the neurophysiological pattern associated to two heteroallelic variants in the CHRND gene in two sisters belonging to a non-consanguineous Italian family. Parents are asymptomatic carriers of one variant and do not show any clinical signs of a neuromuscular disorder. Both sisters showed generalized hypotonia at birth, sucking and swallowing difficulty, weak cry, severe eyelid ptosis and ophthalmoparesis. During the first days of life, they experienced acute respiratory distress requiring invasive ventilation. Neostigmine (0.15 mg three times a day, TID) was ineffective and salbutamol (200 mcg/die) was started with an initial partial response only in case two. These two cases expand the clinical spectrum of CMS linked to
4
CHRND mutations, in fact, there are only few reports of heteroallelic, recessive CHRND mutations in children born with CMS. 2. CASE REPORTS 2.1 Clinical details Case 1. A one-month old female was admitted to the department of Neonatology and Neonatal Intensive Care Unit with a severe generalized hypotonia, sucking and swallowing difficulty, weak cry, severe eyelid ptosis and ophthalmoparesis. She was the second child of nonconsanguineous healthy parents, and her brother had no relevant medical family history. She was born full term after a normal pregnancy but reduced fetal movements were reported. At physical examination, her weight, length, and head circumference were normal for her age. Neurologic examination revealed severe generalized hypotonia, severe bilateral eyelid ptosis, ophthalmoparesis, sucking and swallowing difficulty and weak cry. During the first days of life she experienced a severe acute respiratory distress that required invasive ventilation. A treatment with neostigmine 0.15 mg TID was promptly started without substantial benefit except a partial improvement in ptosis. She never weaned from ventilation and died at the age of three months.
Case 2. A one-month old female; the younger sister of the first case. She came to our observation with the same clinical picture and neurological features as her sister. In fact, she also experienced a severe respiratory insufficiency followed by orotracheal intubation and assisted ventilation four weeks after birth. She started treatment with neostigmine at 0.15 mg TID by nasogastric tube with poor response thus, therapy was changed to salbutamol sulphate 200 mcg (drops administrated through nasogastric tube) three times a day. After three days of treatment, an improvement in swallowing, eyelid ptosis and in particular respiratory function was observed. Twelve days later no invasive ventilation was required and she was extubated. Unfortunately, a week later in coincidence with fever, orotracheal intubation was required again. Salbutamol dosage was increased to 200 mcg four times a day with no benefit. She died at four months of age due to respiratory failure. 5
2.2 Laboratory tests and electromyography studies Serum antibodies against acetylcholine receptor (AChR) were negative and creatine kinase (CK) levels were normal in both sisters. They both underwent electrophysiological studies. Repetitive nerve stimulations (RNS) on tibialis anterior showed a CMAP decremental response of 55%, and the single fiber electromyography (SFEMG) showed an abnormal augmentation of jitter and the presence of conduction blocking, suggesting an abnormal neuromuscular transmission.
2.3 Muscle biopsy Vastus lateralis muscle biopsy was performed only in case 2 after informed consent obtained from the parents. The only evidence found was fibers size variability and the presence of type 2 atrophic fibers in the 9.4 ATPase staining.
2.4 Genetic analysis DNA from case 2 and her parents was subjected to whole exome sequencing (WES) at deCODE genetics, Iceland using Illumina Nextera Rapid Capture exome kit (37 Mb) and sequenced 90 nt paired-end on Illumina HiSeq2000 sequencer. Sequence alignment, variant calling and functional annotation were performed with Burrows Wheel Aligner, Genomes Analysis Tool Kit and Variant Effect Predictor (VEP). Pathogenic or likely pathogenic variants were sought by applying standard filtering criteria for rare diseases, i.e. minor allele frequency (MAF) <1% and moderate to high VEP together with a gene list including all the genes known to be associated with CMS. Two variants in the CHRND gene were identified: a missense change (NC_000002.11:g.233394759C>T; NM_000751.2:c.730C>T; NP_000742.1:p.(Arg244Cys) previously reported in another CMS patient [6] and found in four heterozygous carriers, but no homozygotes, in >250,000 control chromosomes (0.001% [7]) and a novel missense variant (NC_000002.11:g.233398985T>C; 6
NM_000751.2:c.1304T>C; NP_000742.1:p.(Leu435Pro) which is absent in the control population. Aminoacid p.Leu435 is highly conserved in mammals and located in the transmembrane domain, while p.Arg244 falls at the very end of the ligand binding domain of the δ-subunit. In addition, the novel substitution p.(Leu435Pro) change is predicted to be highly damaging by all the in silico tools tested (SIFT, PolyPhen-2 and MutationTaster), and a Combined Annotation-Dependent Depletion score (CADD) of 27.3. 2.5 Functional studies Expression of AChR harbouring the missense mutations p.(Arg244Cys) and p.(Leu435Pro) on the cell surface of HEK293 cells was tested by measuring the binding of radiolabelled alphabungarotoxin (125I α-BuTx) following transfection with cDNA encoding wild type AChR αβεδ subunits and αβεδ [R223C] and [L414P] (numbering for the mature δ-subunit polypeptide without the N-terminal signal sequence). The results were normalised against α-BuTx binding to wild type and represent the mean ± SD of six samples, p<0.0001 (unpaired t-test) (Fig. 1). AChR expression of δL414P was markedly reduced to ~20% compared to the wild type, whereas δR223C was essentially not incorporated into cell surface expression of the AChR.
3. DISCUSSION
The present report describes the clinical features of two sisters clinically and genetically diagnosed with CMS associated to variants in the CHRND gene. These two cases aim to describe a severe and lethal phenotype related exclusively to a CHRND variant, without any associated syndromic features. Furthermore, no facial dysmorphisms, usually reported as a typical feature of CMS, were manifested. In the literature, little is known about the peculiarities of the CMS phenotypes related to CHRND mutations [8]. To date a few syndromes associated to CHRND mutations are described both for autosomal recessive and dominant inheritance pattern [9]. Mutations in the CHRND gene may be associated to a multiple pterygium syndrome lethal (MUPSL), slow-channel CMS 7
(SCCMS), fast-channel CMS (FCCMS), and AChR deficiency [1].
In multiple pterygium
syndrome is characterized by a webbing of the skin (pterygium) at the joints and foetal akinesia before birth. The lethal multiple pterygium syndrome is fatal before birth or very soon after birth [10]. Slow-channel syndrome causes prolonged synaptic currents and action potentials. It is very rare, caused by dominant mutations in ligand-binding or pore domains of the AChR and patients worsen if treated with pyridostigmine [11,12]. Fast-channel syndrome is caused by a recessive mutation in AChR subunit allele, accompanied by a null or low-expressing mutation or, rarely, by another fast-channel mutation on the other allele. Patients with this syndrome respond well to pyridostigmine [13]. When recessive missense, nonsense, or splice site and promoter region mutations in the CHRND gene cause AChR deficiency, a CMS can result. Brownlow et al. [4] described a child who presented at birth with joint contractures after a pregnancy characterized by greatly reduced fetal movements, and who, thanks to a mutational screening, was subsequently found to have an autosomal recessive CMS due to two compound heterozygous mutations within the AChR δ subunit gene. Functional work showed that p.756del2 (c.820_820+1delAG according to current nomenclature) was a null allele, whilst p.Glu59Lys was predicted to result in fast decay of endplate currents due to shorter than normal channel activations. A 20-year-old woman who had moderately severe to severe myasthenic symptoms since birth, no anti-AChR antibodies, a decremental response of the compound muscle action potential on repetitive stimulation and a poor response to pyridostigmine was found to carry compound
heterozygous
variants
in
the
AChR
δ
subunit
gene
(p.Leu42Pro
and
p.Ile58Lys/p.Val93Leu). Furthermore, she had a similarly affected sibling who died at age 11 months [14]. Other examples of CHRND mutations associated to a lethal phenotype were reported by Michalk et al. [15]. The authors found compound heterozygous mutations (p.Arg443* and p.Phe74Leu) in the CHRND gene in multiple sibs of a German family and a homozygous nonsense (p.Trp57*) variant in a Turkish consanguineous family, both resulting in a lethal multiple pterygium 8
syndrome. A benign phenotype in a 43-year-old Chinese male has been reported by Feng et al. He has a relatively mild condition and a good response to pyridostigmine. He was diagnosed with 2 new mutations [c.59G>A (exon2) and c.423G>C (exon5)] in the CHRND gene [16]. Aharoni et al. evaluated the genetical and clinical features of a forty-five CMS patients population and CHRND mutations were encountered in 2 patients belonging to the same family, showing a c.389A>T homozygous variant. For these two cases clinical features are not outlined [17]. In
our
reported
cases
WES
analysis
revealed
(NC_000002.11:g.233398985T>C; NM_000751.2:c.1304T>C)
a
novel
CHRND
variant
in both sisters inherited in
compound heterozygosity with a previously reported variant (NC_000002.11:g.233394759C>T; NM_000751.2:c.730C>T). Segregation analysis identified the c.730C>T variant as maternal, and the c.1304T>C as paternal; the pedigree indicated an autosomal recessive (AR) inheritance pattern (Fig.2). On the basis of the phenotype–genotype correlation already known (OMIM #100720), mutations in the CHRND gene are suggested to be pathogenic for CMS. Our patients had a severe condition that responded poorly to pyridostigmine and even salbutamol. We can speculate that neostigmine was ineffective due to the deleterious effect of the mutations on AChR expression and that salbutamol, in the second case, resulted partially effective because of a stabilizing effect of the β-adrenergic agonists on the neuromuscular junction structure. No other cases with such a precocious onset, fast and lethal course are reported in the literature except for a few associated with arthrogryposis multiplex congenita [4]. The identified variants fall in highly conserved aminoacid residues of the δ AChR subunit, suggesting a crucial role of these residues for the proper functioning of the receptor. Variant p.Arg244Cys essentially ablates surface expression of the AChR and variant p.Leu235Pro resulted in severely reduced levels of cell surface expression of the AChR, consistent with the compound heterozygous variants of the δ AChR gene (CHRND) in our patients causing a severe phenotype of CMS.
9
The diagnosis of CMS is usually based on clinical data such as onset of symptoms (at birth or in early childhood), fatigable weakness, and a positive family history associated to an abnormal response on repetitive nerve stimulation. However, in neonates making a differential diagnosis from other neuromuscular disorders is often challenging. Furthermore, drugs with mechanisms countering specific neuromuscular defects have been found to be effective in appropriate CMS subtypes, but harmful in other CMS subtypes with molecular defects inverse to the drug action [1]. An early genetic diagnosis is important for treatment, prognosis and possible future prenatal diagnosis. Our case report adds to the number of CMS affected individuals identified with rare mutations of the acetylcholine receptor delta subunit gene. We highlight the importance of an extensive genetic approach, using an expanded panel of genes implicated in neuromuscular transmission disorders. Funding Study funded by European Commission’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 2012-305121 (NEUROMICS). DB if funded by MRC Programme Grant MR/M006824 Declaration of interests: none
10
REFERENCES [1] Engel, A. G., Shen, X. M., Selcen, D., Sine, S. M. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol 2015; 420–34. https://doi.org/10.1016/S1474-4422(14)70201-7.
[2] Barisic N, Chaouch A, Muller JS, Lochmüller H. Genetic heterogeneity and pathophysiological mechanisms in congenital myasthenic syndromes. Eur J Paediatr Neurol 2011; 15:189e96. https://doi.org/10.1016/j.ejpn.2011.03.006.
[3] Nicole S, Azuma Y, Bauché S, Eymard B, Lochmüller H, Slater C. Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions. J Neuromuscul Dis 2017; 4:269-84. https://doi.org/10.3233/JND-170257.
[4] Brownlow S, Webster R, Croxen R, Brydson M, Neville B, Lin JP, et al. Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita. J Clin Invest 2001;108:125–30, https://doi.org/10.1172/JCI12935.
[5] Muller J, Mihaylova V, Abicht A, Lochmuller H. Congenital myasthenic syndromes: Spotlight on genetic defects of neuromuscular junction. Expert Rev Mol Med 2007; 9:1e20, https://doi.org/10.1017/S1462399407000427.
[6] https://www.ncbi.nlm.nih.gov/clinvar/variation/585671/, last access 08 June 2019
[7] https://gnomad.broadinstitute.org/gene/ENSG00000135902, last access 08 June 2019 11
[8] Abicht A, Dusl M, Gallenmuller C, Guergueltcheva V, Schara U, Della Marina A, et al. Congenital myasthenic syndromes: achievements and limitations of phenotype-guided geneafter-gene sequencing in diagnostic practice: a study of 680 patients. Hum Mutat 2012;33:1474–84, https://doi.org/10.1002/humu.22130.
[9] Ohno K, Engel AG. Congenital myasthenic syndromes: gene mutations. Neuromuscul Disord 2004;14:117-22. https://doi.org/10.1016/S0960-8966(02)00171-2.
[10] Van RN, Wilkin P, Englert Y, et al. The lethal multiple pterygium syndromes. Am J Med Genet 2010;17:803–7 https://doi.org/10.1002/ajmg.1320170412.
[11] Gomez CM, Maselli RA, Vohra BP, Navedo M, Stiles JR, Charnet P et al. Novel delta subunit mutation in slow-channel syndrome causes severe weakness by novel mechanisms. Ann Neurol. 2002 Jan; 51(1):102-12 https://doi.org/10.1002/ana.10077
[12] Shen XM, Milone M, Wang HL, Banwell B, Selcen D, Sine SM et al. Slow-channel myasthenia due to novel mutation in M2 domain of AChR delta subunit. Ann Clin Transl Neurol. 2019 Oct;6(10):2066-78. https://doi.org/10.1002/acn3.50902.
[13] Engel AG. The therapy of congenital myasthenic syndromes [published correction appears in Neurotherapeutics. 2019 Jan;16(1):244]. Neurotherapeutics. 2007;4(2):252–57. https://10.1016/j.nurt.2007.01.001
12
[14] Shen XM, Fukuda T, Ohno K, Sine SM, Engel AG. Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating. J Clin Invest 2008; 118:1867–76. https://doi.org/10.1172/JCI34527.
[15] Michalk A, Stricker S, Becker J, Rupps, R, Pantzar, T, Miertus, J, et al. Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders. Am. J. Hum. Genet 2008; 82: 464-76. https://doi.org/10.1016/j.ajhg.2007.11.006.
[16] Feng H, Zhou H. New compound heterozygous variants of the cholinergic receptor nicotinic delta subunit gene in a Chinese male with congenital myasthenic syndrome: A case report. Medicine (Baltimore). 2017;96(51):e8981. doi:10.1097/MD.0000000000008981
[17] Aharoni S, Sadeh M, Shapira Y, Edvardson, S., Daana, M., Dor-Wollman, T.,et al. Congenital myasthenic syndrome in Israel: Genetic and clinical characterization. Neuromuscul Disord. 2017;27(2):136–40. https://doi:10.1016/j.nmd.2016.11.014
13
Fig. 1 - AChR surface expression test, 125I α-BuTx binding to AChR expressed on the surface of HEK293 cells following transfection with cDNA encoding wild type AChR αβεδ subunits and αβεδ [L414P] and [R223C]. The results are normalised against α-BuTx binding to wild type and represent the mean ± SD of six samples, p<0.0001.
14
Fig. 2 - Pedigree showing an autosomal recessive (AR) inheritance pattern.
15
Severe congenital myasthenic syndrome associated with novel biallelic mutation of the CHRND gene ¨ , Carmen Bonanno , Carmelo Rodolico , Ana Topf Francesca Maria Foti , Wei-Wei Liu , David Beeson , Antonio Toscano , Hanns Lochmuller ¨ PII: DOI: Reference:
S0960-8966(20)30038-9 https://doi.org/10.1016/j.nmd.2020.02.012 NMD 3806
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
16 June 2019 14 January 2020 16 February 2020
¨ , Francesca Maria Foti , Please cite this article as: Carmen Bonanno , Carmelo Rodolico , Ana Topf Wei-Wei Liu , David Beeson , Antonio Toscano , Hanns Lochmuller , Severe congenital myasthenic ¨ syndrome associated with novel biallelic mutation of the CHRND gene, Neuromuscular Disorders (2020), doi: https://doi.org/10.1016/j.nmd.2020.02.012
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
Highlights
Neuromuscular junction proteins are essential for transmission effectiveness Cholinergic receptor nicotinic δ subunit mutations are rare but possibly fatal Congenital myasthenic syndromes neonatal onset can be severe and life-threatening Myasthenic syndromes diagnosis is tricky,extensive genetic approach is required
1
Severe congenital myasthenic syndrome associated with novel biallelic mutation of the CHRND gene
Carmen Bonanno
a
and Carmelo Rodolico a*, Ana Töpf b, Francesca Maria Foti c, Wei-Wei Liu d,
David Beeson d, Antonio Toscano a, Hanns Lochmüller e a
Department of Clinical and Experimental Medicine, Unit of Neurology and Neuromuscular
Diseases, University of Messina, Messina, Italy b
John Walton, Muscular Dystrophy Research Centre, Institute of Translational and Clinical
Research, Newcastle University and Newcastle Hospitals, Newcastle upon Tyne, UK c
Neonatology and Neonatal Intensive Care Unit, "Bianchi-Melacrino-Morelli" Hospital, Reggio
Calabria, Italy d
Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK e
Department of Neuropediatrics and Muscle Disorders, Medical Center – University of Freiburg,
Faculty of Medicine, Freiburg, Germany and Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Catalonia, Spain Children's Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Canada and Division of Neurology, Department of Medicine, The Ottawa Hospital, Ottawa, Canada *These two authors contributed equally to the paper
Corresponding Author: Carmelo Rodolico Department of Clinical and Experimental Medicine, Unit of Neurology and Neuromuscular Disease, University of Messina, Messina, Italy Phone: 00390902213501 Email: [email protected] ABSTRACT
2
Congenital myasthenic syndromes (CMS) are a group of inherited disorders caused by mutations in genes encoding proteins essential for neuromuscular transmission. CMS is characterized by fatigable muscle weakness with onset at birth or in early childhood; rarely, symptoms may present later. The most frequently involved proteins are choline acetyltransferase, the endplate species of acetylcholinesterase and the acetylcholine receptor subunits. Defects in the cholinergic receptor nicotinic delta subunit (CHRND) are a rare cause for CMS but they should be considered in patients with a severe, early onset disease, with respiratory distress. We describe two sisters, clinically and genetically diagnosed with CMS, carrying two heteroallelic variants in the CHRND gene: c.730C>T; p.(Arg244Cys) and c.1304T>C; p.(Leu435Pro). The first variant has already been described yet no clinical relevance has been proved; the second one, is a novel variant documented here for the first time. These two cases expand the clinical spectrum of CMS linked to CHRND mutations.
Key words: lethal phenotype; AChR delta subunit; children; precocious onset.
3
1. INTRODUCTION Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of the neuromuscular transmission. Although clinical manifestations vary by subtype, CMS are usually characterized by fatigable muscle weakness (ocular, bulbar, limb muscles) with onset at birth or in early childhood; rarely, symptoms may present later. The main proteins involved in the pathogenesis of CMS are: choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor subunits (CHRNA, CHRNB, CHRND, CHRNE), rapsyn, plectin, Na(v)1.4, the muscle specific protein kinase (MuSK), agrin, downstream of tyrosine kinase 7 (Dok7), and glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [1]. Depending on the location of the primary defect within the neuromuscular junction (NMJ) congenital myasthenic syndromes can be classified as presynaptic, synaptic, or postsynaptic [2]. To date, the number of genes already identified and related to a specific CMS subtype is exceeding thirty, however, 30-50% of patients with congenital myasthenic syndromes do not carry mutations in known CMS-causing genes [3]. Mutations of the δ AchR subunit (CHRND) are a very rare cause for CMS but they should be considered in patients with a severe, early onset disease with respiratory involvement [4-5]. We describe the clinical phenotype and the neurophysiological pattern associated to two heteroallelic variants in the CHRND gene in two sisters belonging to a non-consanguineous Italian family. Parents are asymptomatic carriers of one variant and do not show any clinical signs of a neuromuscular disorder. Both sisters showed generalized hypotonia at birth, sucking and swallowing difficulty, weak cry, severe eyelid ptosis and ophthalmoparesis. During the first days of life, they experienced acute respiratory distress requiring invasive ventilation. Neostigmine (0.15 mg three times a day, TID) was ineffective and salbutamol (200 mcg/die) was started with an initial partial response only in case two. These two cases expand the clinical spectrum of CMS linked to
4
CHRND mutations, in fact, there are only few reports of heteroallelic, recessive CHRND mutations in children born with CMS. 2. CASE REPORTS 2.1 Clinical details Case 1. A one-month old female was admitted to the department of Neonatology and Neonatal Intensive Care Unit with a severe generalized hypotonia, sucking and swallowing difficulty, weak cry, severe eyelid ptosis and ophthalmoparesis. She was the second child of nonconsanguineous healthy parents, and her brother had no relevant medical family history. She was born full term after a normal pregnancy but reduced fetal movements were reported. At physical examination, her weight, length, and head circumference were normal for her age. Neurologic examination revealed severe generalized hypotonia, severe bilateral eyelid ptosis, ophthalmoparesis, sucking and swallowing difficulty and weak cry. During the first days of life she experienced a severe acute respiratory distress that required invasive ventilation. A treatment with neostigmine 0.15 mg TID was promptly started without substantial benefit except a partial improvement in ptosis. She never weaned from ventilation and died at the age of three months.
Case 2. A one-month old female; the younger sister of the first case. She came to our observation with the same clinical picture and neurological features as her sister. In fact, she also experienced a severe respiratory insufficiency followed by orotracheal intubation and assisted ventilation four weeks after birth. She started treatment with neostigmine at 0.15 mg TID by nasogastric tube with poor response thus, therapy was changed to salbutamol sulphate 200 mcg (drops administrated through nasogastric tube) three times a day. After three days of treatment, an improvement in swallowing, eyelid ptosis and in particular respiratory function was observed. Twelve days later no invasive ventilation was required and she was extubated. Unfortunately, a week later in coincidence with fever, orotracheal intubation was required again. Salbutamol dosage was increased to 200 mcg four times a day with no benefit. She died at four months of age due to respiratory failure. 5
2.2 Laboratory tests and electromyography studies Serum antibodies against acetylcholine receptor (AChR) were negative and creatine kinase (CK) levels were normal in both sisters. They both underwent electrophysiological studies. Repetitive nerve stimulations (RNS) on tibialis anterior showed a CMAP decremental response of 55%, and the single fiber electromyography (SFEMG) showed an abnormal augmentation of jitter and the presence of conduction blocking, suggesting an abnormal neuromuscular transmission.
2.3 Muscle biopsy Vastus lateralis muscle biopsy was performed only in case 2 after informed consent obtained from the parents. The only evidence found was fibers size variability and the presence of type 2 atrophic fibers in the 9.4 ATPase staining.
2.4 Genetic analysis DNA from case 2 and her parents was subjected to whole exome sequencing (WES) at deCODE genetics, Iceland using Illumina Nextera Rapid Capture exome kit (37 Mb) and sequenced 90 nt paired-end on Illumina HiSeq2000 sequencer. Sequence alignment, variant calling and functional annotation were performed with Burrows Wheel Aligner, Genomes Analysis Tool Kit and Variant Effect Predictor (VEP). Pathogenic or likely pathogenic variants were sought by applying standard filtering criteria for rare diseases, i.e. minor allele frequency (MAF) <1% and moderate to high VEP together with a gene list including all the genes known to be associated with CMS. Two variants in the CHRND gene were identified: a missense change (NC_000002.11:g.233394759C>T; NM_000751.2:c.730C>T; NP_000742.1:p.(Arg244Cys) previously reported in another CMS patient [6] and found in four heterozygous carriers, but no homozygotes, in >250,000 control chromosomes (0.001% [7]) and a novel missense variant (NC_000002.11:g.233398985T>C; 6
NM_000751.2:c.1304T>C; NP_000742.1:p.(Leu435Pro) which is absent in the control population. Aminoacid p.Leu435 is highly conserved in mammals and located in the transmembrane domain, while p.Arg244 falls at the very end of the ligand binding domain of the δ-subunit. In addition, the novel substitution p.(Leu435Pro) change is predicted to be highly damaging by all the in silico tools tested (SIFT, PolyPhen-2 and MutationTaster), and a Combined Annotation-Dependent Depletion score (CADD) of 27.3. 2.5 Functional studies Expression of AChR harbouring the missense mutations p.(Arg244Cys) and p.(Leu435Pro) on the cell surface of HEK293 cells was tested by measuring the binding of radiolabelled alphabungarotoxin (125I α-BuTx) following transfection with cDNA encoding wild type AChR αβεδ subunits and αβεδ [R223C] and [L414P] (numbering for the mature δ-subunit polypeptide without the N-terminal signal sequence). The results were normalised against α-BuTx binding to wild type and represent the mean ± SD of six samples, p<0.0001 (unpaired t-test) (Fig. 1). AChR expression of δL414P was markedly reduced to ~20% compared to the wild type, whereas δR223C was essentially not incorporated into cell surface expression of the AChR.
3. DISCUSSION
The present report describes the clinical features of two sisters clinically and genetically diagnosed with CMS associated to variants in the CHRND gene. These two cases aim to describe a severe and lethal phenotype related exclusively to a CHRND variant, without any associated syndromic features. Furthermore, no facial dysmorphisms, usually reported as a typical feature of CMS, were manifested. In the literature, little is known about the peculiarities of the CMS phenotypes related to CHRND mutations [8]. To date a few syndromes associated to CHRND mutations are described both for autosomal recessive and dominant inheritance pattern [9]. Mutations in the CHRND gene may be associated to a multiple pterygium syndrome lethal (MUPSL), slow-channel CMS 7
(SCCMS), fast-channel CMS (FCCMS), and AChR deficiency [1].
In multiple pterygium
syndrome is characterized by a webbing of the skin (pterygium) at the joints and foetal akinesia before birth. The lethal multiple pterygium syndrome is fatal before birth or very soon after birth [10]. Slow-channel syndrome causes prolonged synaptic currents and action potentials. It is very rare, caused by dominant mutations in ligand-binding or pore domains of the AChR and patients worsen if treated with pyridostigmine [11,12]. Fast-channel syndrome is caused by a recessive mutation in AChR subunit allele, accompanied by a null or low-expressing mutation or, rarely, by another fast-channel mutation on the other allele. Patients with this syndrome respond well to pyridostigmine [13]. When recessive missense, nonsense, or splice site and promoter region mutations in the CHRND gene cause AChR deficiency, a CMS can result. Brownlow et al. [4] described a child who presented at birth with joint contractures after a pregnancy characterized by greatly reduced fetal movements, and who, thanks to a mutational screening, was subsequently found to have an autosomal recessive CMS due to two compound heterozygous mutations within the AChR δ subunit gene. Functional work showed that p.756del2 (c.820_820+1delAG according to current nomenclature) was a null allele, whilst p.Glu59Lys was predicted to result in fast decay of endplate currents due to shorter than normal channel activations. A 20-year-old woman who had moderately severe to severe myasthenic symptoms since birth, no anti-AChR antibodies, a decremental response of the compound muscle action potential on repetitive stimulation and a poor response to pyridostigmine was found to carry compound
heterozygous
variants
in
the
AChR
δ
subunit
gene
(p.Leu42Pro
and
p.Ile58Lys/p.Val93Leu). Furthermore, she had a similarly affected sibling who died at age 11 months [14]. Other examples of CHRND mutations associated to a lethal phenotype were reported by Michalk et al. [15]. The authors found compound heterozygous mutations (p.Arg443* and p.Phe74Leu) in the CHRND gene in multiple sibs of a German family and a homozygous nonsense (p.Trp57*) variant in a Turkish consanguineous family, both resulting in a lethal multiple pterygium 8
syndrome. A benign phenotype in a 43-year-old Chinese male has been reported by Feng et al. He has a relatively mild condition and a good response to pyridostigmine. He was diagnosed with 2 new mutations [c.59G>A (exon2) and c.423G>C (exon5)] in the CHRND gene [16]. Aharoni et al. evaluated the genetical and clinical features of a forty-five CMS patients population and CHRND mutations were encountered in 2 patients belonging to the same family, showing a c.389A>T homozygous variant. For these two cases clinical features are not outlined [17]. In
our
reported
cases
WES
analysis
revealed
(NC_000002.11:g.233398985T>C; NM_000751.2:c.1304T>C)
a
novel
CHRND
variant
in both sisters inherited in
compound heterozygosity with a previously reported variant (NC_000002.11:g.233394759C>T; NM_000751.2:c.730C>T). Segregation analysis identified the c.730C>T variant as maternal, and the c.1304T>C as paternal; the pedigree indicated an autosomal recessive (AR) inheritance pattern (Fig.2). On the basis of the phenotype–genotype correlation already known (OMIM #100720), mutations in the CHRND gene are suggested to be pathogenic for CMS. Our patients had a severe condition that responded poorly to pyridostigmine and even salbutamol. We can speculate that neostigmine was ineffective due to the deleterious effect of the mutations on AChR expression and that salbutamol, in the second case, resulted partially effective because of a stabilizing effect of the β-adrenergic agonists on the neuromuscular junction structure. No other cases with such a precocious onset, fast and lethal course are reported in the literature except for a few associated with arthrogryposis multiplex congenita [4]. The identified variants fall in highly conserved aminoacid residues of the δ AChR subunit, suggesting a crucial role of these residues for the proper functioning of the receptor. Variant p.Arg244Cys essentially ablates surface expression of the AChR and variant p.Leu235Pro resulted in severely reduced levels of cell surface expression of the AChR, consistent with the compound heterozygous variants of the δ AChR gene (CHRND) in our patients causing a severe phenotype of CMS.
9
The diagnosis of CMS is usually based on clinical data such as onset of symptoms (at birth or in early childhood), fatigable weakness, and a positive family history associated to an abnormal response on repetitive nerve stimulation. However, in neonates making a differential diagnosis from other neuromuscular disorders is often challenging. Furthermore, drugs with mechanisms countering specific neuromuscular defects have been found to be effective in appropriate CMS subtypes, but harmful in other CMS subtypes with molecular defects inverse to the drug action [1]. An early genetic diagnosis is important for treatment, prognosis and possible future prenatal diagnosis. Our case report adds to the number of CMS affected individuals identified with rare mutations of the acetylcholine receptor delta subunit gene. We highlight the importance of an extensive genetic approach, using an expanded panel of genes implicated in neuromuscular transmission disorders. Funding Study funded by European Commission’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 2012-305121 (NEUROMICS). DB if funded by MRC Programme Grant MR/M006824 Declaration of interests: none
10
REFERENCES [1] Engel, A. G., Shen, X. M., Selcen, D., Sine, S. M. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol 2015; 420–34. https://doi.org/10.1016/S1474-4422(14)70201-7.
[2] Barisic N, Chaouch A, Muller JS, Lochmüller H. Genetic heterogeneity and pathophysiological mechanisms in congenital myasthenic syndromes. Eur J Paediatr Neurol 2011; 15:189e96. https://doi.org/10.1016/j.ejpn.2011.03.006.
[3] Nicole S, Azuma Y, Bauché S, Eymard B, Lochmüller H, Slater C. Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions. J Neuromuscul Dis 2017; 4:269-84. https://doi.org/10.3233/JND-170257.
[4] Brownlow S, Webster R, Croxen R, Brydson M, Neville B, Lin JP, et al. Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita. J Clin Invest 2001;108:125–30, https://doi.org/10.1172/JCI12935.
[5] Muller J, Mihaylova V, Abicht A, Lochmuller H. Congenital myasthenic syndromes: Spotlight on genetic defects of neuromuscular junction. Expert Rev Mol Med 2007; 9:1e20, https://doi.org/10.1017/S1462399407000427.
[6] https://www.ncbi.nlm.nih.gov/clinvar/variation/585671/, last access 08 June 2019
[7] https://gnomad.broadinstitute.org/gene/ENSG00000135902, last access 08 June 2019 11
[8] Abicht A, Dusl M, Gallenmuller C, Guergueltcheva V, Schara U, Della Marina A, et al. Congenital myasthenic syndromes: achievements and limitations of phenotype-guided geneafter-gene sequencing in diagnostic practice: a study of 680 patients. Hum Mutat 2012;33:1474–84, https://doi.org/10.1002/humu.22130.
[9] Ohno K, Engel AG. Congenital myasthenic syndromes: gene mutations. Neuromuscul Disord 2004;14:117-22. https://doi.org/10.1016/S0960-8966(02)00171-2.
[10] Van RN, Wilkin P, Englert Y, et al. The lethal multiple pterygium syndromes. Am J Med Genet 2010;17:803–7 https://doi.org/10.1002/ajmg.1320170412.
[11] Gomez CM, Maselli RA, Vohra BP, Navedo M, Stiles JR, Charnet P et al. Novel delta subunit mutation in slow-channel syndrome causes severe weakness by novel mechanisms. Ann Neurol. 2002 Jan; 51(1):102-12 https://doi.org/10.1002/ana.10077
[12] Shen XM, Milone M, Wang HL, Banwell B, Selcen D, Sine SM et al. Slow-channel myasthenia due to novel mutation in M2 domain of AChR delta subunit. Ann Clin Transl Neurol. 2019 Oct;6(10):2066-78. https://doi.org/10.1002/acn3.50902.
[13] Engel AG. The therapy of congenital myasthenic syndromes [published correction appears in Neurotherapeutics. 2019 Jan;16(1):244]. Neurotherapeutics. 2007;4(2):252–57. https://10.1016/j.nurt.2007.01.001
12
[14] Shen XM, Fukuda T, Ohno K, Sine SM, Engel AG. Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating. J Clin Invest 2008; 118:1867–76. https://doi.org/10.1172/JCI34527.
[15] Michalk A, Stricker S, Becker J, Rupps, R, Pantzar, T, Miertus, J, et al. Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders. Am. J. Hum. Genet 2008; 82: 464-76. https://doi.org/10.1016/j.ajhg.2007.11.006.
[16] Feng H, Zhou H. New compound heterozygous variants of the cholinergic receptor nicotinic delta subunit gene in a Chinese male with congenital myasthenic syndrome: A case report. Medicine (Baltimore). 2017;96(51):e8981. doi:10.1097/MD.0000000000008981
[17] Aharoni S, Sadeh M, Shapira Y, Edvardson, S., Daana, M., Dor-Wollman, T.,et al. Congenital myasthenic syndrome in Israel: Genetic and clinical characterization. Neuromuscul Disord. 2017;27(2):136–40. https://doi:10.1016/j.nmd.2016.11.014
13
Fig. 1 - AChR surface expression test, 125I α-BuTx binding to AChR expressed on the surface of HEK293 cells following transfection with cDNA encoding wild type AChR αβεδ subunits and αβεδ [L414P] and [R223C]. The results are normalised against α-BuTx binding to wild type and represent the mean ± SD of six samples, p<0.0001.
14
Fig. 2 - Pedigree showing an autosomal recessive (AR) inheritance pattern.
15