Identification of previously unreported mutations in CHRNA1, CHRNE ...

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Feb 16, 2010 - Abstract Congenital myasthenic syndromes are rare genetic disorders compromising neuromuscular transmis- sion. The defects are mainly ...
J Neurol (2010) 257:1119–1123 DOI 10.1007/s00415-010-5472-0

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Identification of previously unreported mutations in CHRNA1, CHRNE and RAPSN genes in three unrelated Italian patients with congenital myasthenic syndromes Raffaella Brugnoni • Lorenzo Maggi • Eleonora Canioni • Isabella Moroni • Chiara Pantaleoni • Stefano D’Arrigo • Daria Riva • Ferdinando Cornelio • Pia Bernasconi • Renato Mantegazza

Received: 19 October 2009 / Revised: 12 January 2010 / Accepted: 15 January 2010 / Published online: 16 February 2010 Ó Springer-Verlag 2010

Abstract Congenital myasthenic syndromes are rare genetic disorders compromising neuromuscular transmission. The defects are mainly mutations in the muscle acetylcholine receptor, or associated proteins rapsyn and Dok7. We analyzed three unrelated Italian patients with typical clinical features of congenital myasthenic syndrome, who all benefitted from cholinesterase inhibitors. We found five mutations: a previously unreported homozygous aG378D mutation in the CHRNA1 gene, a previously unreported heterozygous eY8X mutation associated with a known heterozygous eM292del deletion in the CHRNE gene, and the common heterozygous N88K mutation associated with a previously unreported heterozygous IVS1 ? 2T [ G splice site mutation in the RAPSN gene. All three patients had two mutant alleles; parents or offspring with a single mutated allele were asymptomatic, thus all mutations exerted their effects recessively. The previously unreported

R. Brugnoni Laboratory NBS Biotech, Fondazione Istituto Neurologico Carlo Besta, Milan, Italy L. Maggi  E. Canioni  F. Cornelio  P. Bernasconi  R. Mantegazza (&) Department of Neurology IV, Fondazione Istituto Neurologico Carlo Besta, Via Celoria 11, 20133 Milan, Italy e-mail: [email protected] I. Moroni Division of Child Neurology, Fondazione Istituto Neurologico Carlo Besta, Milan, Italy C. Pantaleoni  S. D’Arrigo  D. Riva Developmental Neurology Unit, Fondazione Istituto Neurologico Carlo Besta, Milan, Italy

mutations are likely to reduce the number of AChRs at the motor endplate, although the aG378D mutation might produce a mild fast channel syndrome. The aG378D mutation was recessive, but recessive CHRNA1 mutations have rarely been reported previously, so studies on the effect of this mutation at the cellular level would be of interest. Keywords Congenital myasthenic syndrome  Acetylcholine receptor  Rapsyn  CHRNA1, CHRNE, and RAPSN genes

Introduction The congenital myasthenic syndromes (CMS) are a heterogeneous group of inherited diseases characterized by impaired neuromuscular transmission, resulting in weakness and fatigability [1]. CMS, usually autosomal recessive, can be associated with abnormalities of acetylcholine release, acetylcholinesterase activity, or ACh receptor (AChR) function or density, and are accordingly classified into presynaptic, synaptic, or postsynaptic forms [1, 2]. Postsynaptic forms are the most common and are usually due to AChR deficiency. The AChR deficiency is generally due to mutations in AChR subunits (usually the CHRNE gene encoding the AChR-e subunit), in the AChR-clustering protein rapsyn, or in the MuSK-interacting cytoplasmic protein Dok-7 [2–6]. The most common RAPSN mutation, particularly among Europeans, is N88K, which produces CMS when present homozygously or when another RAPSN mutation is present on the other allele [4, 5]. Here we describe previously unreported mutations in each of the CHRNA1, CHRNE, and RAPSN genes, identified in three unrelated Italian patients with CMS.

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Patients and methods We studied three patients referred to the Besta Neurological Institute, and six healthy relatives. All genetic analyses were performed at the Laboratory NBS Biotech. None of the patients had a family history of CMS and all were negative for anti-AChR and anti-MuSK antibodies. Patient 1, an 18-year-old male, was born from nonconsanguineous parents. He presented at birth with bilateral pes tortus, respiratory distress, weak crying, and swallowing deficiency. Subsequently bilateral ptosis, ophthalmoparesis, and hoarse voice developed, but psychomotor development was normal. At age three, myasthenia gravis (MG) was diagnosed following a positive Tensilon test. At age nine marked decrease in CMAPs after repetitive facial nerve stimulation was found. Pyridostigmine (30 mg four times daily) resulted in progressive improvement in muscle strength. At latest examination, upper and lower limb muscle strength, was normal but ptosis, ophthalmoparesis and dysphonia persisted. Patient 2, a 56-year-old woman, has had bilateral ptosis and severe ophthalmoparesis without diplopia since age two. She acquired walking late and never ran. At age five she had nasal speech, weak neck extensors and weak proximal limb muscles. At age 20 repetitive spinal accessory nerve stimulation suggested MG; pyridostigmine (60 mg four times daily) produced clear though partial benefit. At 49 years she was started on diaminopyridine (5 mg three times daily), with further improvement. At latest follow-up she had bilateral ophthalmoplegia and eyelid ptosis, limb weakness and fatigability. Patient 3 is a 20-year woman. At birth she had bilateral ptosis, horizontal gaze palsy and feeding difficulties. At 14 months she developed respiratory distress requiring mechanical ventilation. Repetitive spinal accessory nerve stimulation indicated MG, and pyridostigmine (15 mg four times daily) was started with good response. At 5 years she developed dysphagia and dysphonia during a febrile episode. At present she has bilateral eyelid ptosis and still takes pyridostigmine (60 mg four times daily). Genetic analysis Written informed consent was obtained from all patients and relatives. Genomic DNA was extracted from peripheral blood lymphocytes. All exons and adjacent intronic regions of the CHRNA1 and CHRNE genes were sequenced. Exon 2 of the RAPSN gene was screened for the N88K mutation and if the patient was heterozygous for this mutation the whole gene was analyzed. PCR amplification employed specific intronic primers, followed by bidirectional sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster

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City, CA) on an ABI PRISM 3100 Analyzer (Applied Biosystems). Sequences were analyzed by SeqScape v2.1.1 software (Applied Biosystems) and compared with those in GenBank (accession number: NT_005403.16 for CHRNA1; NT_010718.15 for CHRNE; NT_009237.17 for RAPSN). Following PCR of the promoter region of CHRNE, we also analyzed the 6 bp promoter element (N-box) by digestion with MspI (Roche Diagnostics) [7]. Previously unreported mutations were compared with sequences in 100 unrelated healthy sex- and age-matched Italians as controls.

Results In patient 1, a previously unreported homozygous missense mutation (c.1133G [ A) in exon 8 of CHRNA1 was found (Fig. 1a), resulting in change from glycine to aspartate (neutral to acidic amino acid) at position 378 (aG378D) of the protein. This mutation is located in a curved a-helix cytoplasmic subdomain of the AChR a subunit (Fig. 1d) [8]. Both asymptomatic parents were heterozygous for the mutation (Figs. 1b and 1c). Prenatal testing of the patient’s brother was negative for the mutation. Two heterozygous mutations in CHRNE were found in patient 2. One is a previously unreported c.24T [ G substitution in exon 2, which changes tyrosine to a stop codon at position 8 (eY8X) of the N-terminus of the e subunit (Figs. 2a and 2d). The second was a known inframe three base-pair deletion (e874–876del3 in exon 9) resulting in deletion of methionine at 292 (M292del) in the e subunit M3 domain (Figs. 2b and 2d) [9]. The eY8X mutation was present heterozygously in the patient’s asymptomatic daughter (Fig. 2c). The half-brother had no mutations. DNA of the proband’s parents was unavailable as both had died; they presented no evidence of neuromuscular disease in life. Patient 3 was heterozygous for the N88K mutation of RAPSN (Fig. 3a). Entire gene sequencing revealed a previously unreported splice mutation IVS1 ? 2T [ G in the 50 donor splice site of intron 1 (Fig. 3b), present (as is the N88K mutation) in the tetratricopeptide repeat (TPR) region of the protein (Fig. 3d). The mother was heterozygous for N88K and the father was heterozygous for IVS1 ? 2T [ G (Fig. 3c). Muscle from the patient and his father was unavailable for analysis of the splice mutation transcript.

Discussion We have found five CMS-associated mutations in three unrelated Italian patients. Three are previously unreported:

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Fig. 1 a Sequence analysis of exon 8 of the CHRNA1 gene shows a previously unreported homozygous missense mutation c.1133G [ A, resulting in the amino acid substitution aG378D. b Sequence analysis of exon 8 in each of the patient’s parents shows each has heterozygous aG378D mutation. c Pedigree: patient 1 is homozygous for the mutation, the asymptomatic parents have one copy each of the mutated gene. Prenatal testing in the patient’s sibling was negative for the mutation. d Schematic representation of the a subunit of the AChR illustrating the position of the aG378D mutation

Fig. 2 a Sequence analysis of exon 2 of the CHRNE gene shows a previously unreported heterozygous mutation c.24T [ G, that changes a TAT tyrosine codon to a TAG stop codon in the e subunit (eY8X). b Sequence analysis of exon 9 shows a train of double peaks indicating a heterozygous deletion 874-876delATG in the CHRNE gene, and predicting deletion of methionine eM292del in the e subunit. c Family tree depicts patient 2 (filled circle) with two mutated alleles, and her daughter with the eY8X mutation (half-circle). The half-brother had no mutations while the patient’s husband was not analyzed. d Schematic representation of the e subunit of the AChR illustrating the positions of the eY8X and eM292del mutations

aG378D in CHRNA1, eY8X in CHRNE, and IVS1 ? 2T [ G in RAPSN. The known mutations were eM292del in CHRNE and N88K in RAPSN. All three

patients had two mutant alleles, and parents and offspring with a single mutated allele were asymptomatic, thus all mutations are recessive.

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Fig. 3 a Sequence analysis of exon 2 shows the common heterozygous mutation c.264C [ A in the RAPSN gene, causing the N88K amino acid substitution. b Sequence analysis of exon 1 shows a previously unreported heterozygous splice mutation IVS1 ? 2T [ G. c Pedigree shows patient 3 with both mutations (closed symbol), the mother with the M88J mutation and father with the splice IVS1 ? 2T [ G mutation. d Schematic representation of the rapsyn protein showing locations of the N88K and IVS1 ? 2T [ G mutations

The missense aG378D mutation in patient 1 occurs at the beginning of the MA domain (a curved a-helix subdomain on the cytoplasmic side) [8] of the AChR a protein (Fig. 1). Unwin [8] investigated residues of the a subunit of the Torpedo marmorata AChR which interact with other subunits, and found that glutamate at 377, isoleucine at 376 and valine at 379 were crucial for interaction with neighboring subunits. Glycine at 378 was found not to be essential, but since it is highly conserved, its substitution for aspartate (a bulky acidic residue) is likely to alter a subunit structure either impairing interaction with other subunits (causing AChR deficiency) or altering channel kinetics to produce a mild fast channel syndrome indistinguishable clinically from AChR deficiency. Patient 2 had previously unreported (eY8X) and known (eM292del) CHRNE mutations (Fig. 2) [9]. The stop codon eY8X occurs after seven amino acids, predicting a truncated non-functional e subunit, not incorporated into the AChR and causing AChR deficiency. The previously-described eY15X mutation also predicts a non-functional protein, and in fact was present heterozygously in two CMS sisters in association with another heterozygous null mutation (e70insG) near the N-terminus [10]. The eM292del mutation is not present in the patient 2’s asymptomatic daughter, while HEK 293 cells with the same mutation do not express AChR at the cell surface [9]. Thus eM292del is probably the main determinant of the CMS phenotype in patient 2. Patient 3, who was negative for CHRNA1 and CHRNE mutations, was found to have a heterozygous N88K mutation. Complete RAPSN sequencing then revealed a

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previously unreported heterozygous splice mutation IVS1 ? 2T [ G (Fig. 3). Both mutations are located on the TPR region of rapsyn which is important for self-association [4, 5]; and while the N88K mutation affects AChR clustering [9], the splice mutation is likely to affect exon splicing, resulting in skipping of exon 1 or retention of intron 1, and hence leading to truncated rapsyn polypeptide and AChR deficiency [11]. A muscle biopsy of patient 3 was not available to determine how this mutation effects rapsyn RNA splicing. The clinical features of our CMS patients are similar to those reported in elsewhere [1, 2, 4, 5] and all benefitted from cholinesterase inhibitors. The three previously unreported mutations are more likely to cause AChR deficiency than changes in the functional properties of the ion channels (although this remains a possibility for patient 1), so it would be useful to confirm this conjecture by further investigations. Finally, since recessive CHRNA1 mutations are rare [2, 3], it would be of interest to examine the molecular effects of the recessive aG378D mutation in this gene. Acknowledgments English.

The authors thank Don Ward for help with the

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