Mutation of the Acetylcholine Receptorε-Subunit Promoter in Congenital

Myasthenic SyndromePhilip Nichols, MRCP,* Rebecca Croxen, DPhil,* Angela Vincent, FRCPath,* Richard Rutter, MA,*

Michael Hutchinson, FRCP,† John Newsom-Davis, FRS,* and David Beeson, PhD*

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission.Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most casesresults from mutations within the coding region of the AChR « subunit. However, studies in mice have established thatsynapse-specific expression of AChR is dependent on a sequence contained within the AChR-subunit promoter regions,termed an N-box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic featuresconsistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynap-tic. Single-strand conformational polymorphism analysis identified an abnormal conformer in the AChR «-subunit genepromoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates withrecessive inheritance of a single point mutation, a transition (C3T) in the N-box of the «-subunit promoter. Analysis ofan intercostal biopsy from 1 of the patients showed a dramatic reduction in «-subunit mRNA levels compared with diseaseand normal controls. This is the first evidence in humans that an N-box mutation can lead to disruption of «-subunittranscription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR-deficiency congenitalmyasthenic syndrome.

Nichols P, Croxen R, Vincent A, Rutter R, Hutchinson M, Newsom-Davis J, Beeson D. Mutation of theacetylcholine receptor ε-subunit promoter in congenital myasthenic syndrome. Ann Neurol 1999;45:439–443

Congenital myasthenic syndrome (CMS) is a heteroge-neous group of inherited disorders of neuromusculartransmission.1 In contrast to acquired myasthenia gra-vis, there is no response to immunotherapy and anti-AChR-specific autoantibodies are absent. The inheri-tance pattern is usually autosomal recessive but may beautosomal dominant.2,3 Patients usually present in theneonatal period with ocular, bulbar, or respiratorysymptoms exacerbated by crying, or in the first 2 yearsof life with fluctuating ocular palsies, excessive fatiga-bility, and delayed motor milestones, although oneform (the slow channel syndrome) may present later inlife. The most common form, in our experience, isAChR deficiency in which muscle biopsies frequentlyshow a greatly reduced number of AChRs.4

Mutations within the genes encoding muscle AChRsubunits5 or the collagen tail of acetylcholinesterase6

have been shown to cause CMS. Most AChR-deficiencymutations described occur in the ε-subunit gene codingregion and give rise either to null alleles, or to alleles thatgenerate ε-subunit polypeptides that are assembled into

functional adult AChR only at very low levels.7–10 How-ever, the causal mutation in some AChR-deficiency pa-tients remains unknown.11 An alternative pathogenicmechanism12 could be mutations within the AChR-subunit promoter regions, resulting in a disruption ofAChR-subunit mRNA transcription and reducedAChR-subunit synthesis.

We provide here the first full description of a ho-mozygous mutation within the ε-subunit promoter re-gion resulting in the clinical phenotype of AChR defi-ciency, which we previously reported briefly.13 Themutation is present in 2 siblings, from consanguineousparents, and results in the loss of ε-subunit transcrip-tion and adult AChR production. The mutation is asingle-base transition (C3T), which disrupts a critical6-bp promoter element known as the N-box. This pro-moter element has been shown to be important in thesynapse-specific transcriptional regulation of AChRsubunits in other species,14,15 but this is the first evi-dence of its role in AChR production in humans andits importance in the pathogenesis of a form of CMS.

From the *Neurosciences Group, Institute of Molecular Medicine,John Radcliffe Hospital, Headington, Oxford, UK; and †Depart-ment of Clinical Neurology, St Vincent’s Hospital, Dublin, Ireland.

Received Oct 1, 1998, and in revised form Nov 24. Accepted forpublication Dec 1, 1998.

Address correspondence to Dr Beeson, Neurosciences Group, Insti-tute of Molecular Medicine, John Radcliffe Hospital, Headington,Oxford OX3 9DS, UK.

Copyright © 1999 by the American Neurological Association 439

Materials and MethodsMuscle Biopsy, mRNA, and DNA Patient SamplesIntercostal muscle biopsies were studied for end-plate AChRnumbers (by 125I-a-bungarotoxin binding), morphology, andelectrophysiology, as described by Vincent and colleagues.4

RNA was isolated from intercostal muscle biopsies by usingRNAzol B (AMS Biotechnology, UK), following instructionsprovided by the manufacturer. Oligo(dT) cellulose chroma-tography was used to obtain poly(A)1 RNA. Genomic DNAwas isolated from peripheral blood by using the Nucleon IIDNA extraction kit (Nucleon Biosciences). Approval for theuse of human muscle tissues was received from the CentralOxford Research Ethics Committee.

Mutational AnalysisPOLYMERASE CHAIN REACTION (PCR) AND SINGLE-STRAND CONFORMATIONAL POLYMORPHISM (SSCP) ANAL-YSIS. PCR amplifications for SSCP analysis were performedon genomic DNA for regions covering the AChR ε-subunitand ε-promoter regions. The PCR amplifications for SSCP,which gave rise to unique conformers for the affected pa-tients, were performed by using the following primer pairs:59 CTG CTA CTA GAA TTG TGG TTG CAG G 39 and59 CCT GCT GCG TGG TTC TCA GGG TTA 39 toamplify a 305-bp fragment 59 of the ATG translation startsite of the ε subunit.

SEQUENCE ANALYSIS. Cosmid and PCR-amplified frag-ments of genomic DNA were purified from agarose gels byusing the QIAGEN QIAquick gel extraction kit, subclonedin pGEM-3Z (Promega), and then sequenced by using theSequenase version 2.0 DNA sequencing kit (USB/Amer-sham). MspI restriction endonuclease digestions were per-formed according to the manufacturer’s instructions.

REVERSE TRANSCRIPTION–PCR (RT-PCR) ANALYSIS. First-strand cDNA was prepared from poly(A)1 RNA isolatedfrom intercostal muscle biopsies taken from Patient S.M.,Patient R.A., and an unrelated healthy individual. cDNAwas synthesized from 1 mg poly(A)1 RNA in a reaction vol-ume of 50 ml, using standard conditions and a mixture ofrandom hexanucleotide and oligo(dT)12–18 primers. PCR cy-cling for amplifications of muscle cDNAs used an annealingtemperature of 60°C for the a, b, g, and d primer pairs and50°C for the ε primer pair. PCR reactions were performed in20 ml in buffer containing 50 mM KCl, 10 mM Tris-HCl,pH 9, 2 mM MgCl2, 0.1% Triton X-100, dNTPs each at200 mM, 1 mM each primer, 0.5 U of Taq polymerase(BRL), and 1 ml of the first-strand cDNA reaction. Thesubunit-specific primer pairs used were as follows: a 59 GATGAA GTA AAT CAG ATC GTG AC 39 and 59 TTG TTATAG AGA ACA AGG TCT GG 39; b 59 CTG AAG GAGGAC TGG CAG TTT GTG 39 and 59 GGT ACG TGGCGT CCA GGA AGA TGA CTA G 39; g 59 GAG TTAGGG CTG AGC CAG TTC TGT G 39 and 59 GTC TGGTGA GGG CAG GTA GGG GCG TG 39; d 59 GAA CTCTTC AAT GAG CTG AAG CC 39 and 59 GAT GAAGCG CTT GTC CTG CAC GTT G 39; and ε 59 ACTCTC ACC ACT AGC GTC 39 and 59 GCG GAT GATGAG CGA GTA 39.

ResultsPatientsPatients S.M. and C.M. were brother and sister from alarge pedigree in which the parents were consanguineousbut asymptomatic. The patients had 5 living unaffectedsiblings, but 1 male sibling had died at 3 months of agefrom a respiratory infection complicated by respiratoryfailure and may have been affected but undiagnosed.

S.M. first presented at the age of 6 years with ptosisand mild proximal limb weakness. Clinically he re-sponded to acetylcholinesterase inhibitors but had noresponse to a thymectomy performed at his home cen-tre. Examination at the age of 28 years showed mildptosis and slight reduction in eye abduction bilaterally,proximal fatigable weakness in arms and legs and somemild distal weakness in the upper limbs. Antibodies toAChR were absent. Electromyography showed a 12%(normal, ,8%) decrement in the compound muscleaction potential (abductor digiti minimi) at 3-Hz stim-ulation, and single-fiber electromyographic studiesshowed a marked increase in mean jitter of 123 msec(normal, ,35 msec; extensor digitorum communis;n 5 5). An intercostal muscle biopsy showed smallminiature end-plate potential amplitudes in the pres-ence of eserine (mean 6 SEM, 0.39 6 0.07; controlvalues in eserine, 0.99 6 0.09 mV), a reduction inend-plate AChR numbers (mean 6 SEM, 0.34 6 0.04125I-a-bungarotoxin binding sites 107/end plate; nor-mal values, .1.0), and a marked increase in the lengthof the cholinesterase-stained end-plate regions (mean 6SEM, 16.6 6 5.3 sarcomeres; normal values, 7–8).4

C.M. showed symptoms similar to her brother. The re-maining 5 siblings and their parents were also examined,and none had any features on examination or in theirhistory to suggest that they were affected by CMS.

Isolation of the AChR «-Subunit Promoter RegionSequence encoding the promoter region of the humanAChR ε-subunit gene was derived from a chromosome17 library cosmid clone containing the AChRε-subunit gene.16 Hybridization using a 32P-labeledoligonucleotide (59 CAC GCA GCA GGA TGG CAAGGG 39), that contains the translation start site for thehuman ε subunit,17 identified a 4.3-kb BamHI cosmidfragment from which 1,574 bp 59 to the ATG trans-lation start site were sequenced (EMBL accession num-ber Z84811). Figure 1A details part of the humanε-subunit promoter sequence aligned with mouse,showing a high degree of sequence identity close to thetranslation start site and conservation in the position ofE- and N-box promoter elements between species. Theε-promoter sequence is less well conserved betweenspecies further upstream (data not shown).

Promoter Region Mutation AnalysisSSCP ANALYSIS. DNA was obtained from the parentsand all the unaffected siblings as well as the 2 patients.

440 Annals of Neurology Vol 45 No 4 April 1999

PCR amplifications for the region containing the mus-cle AChR ε-subunit promoter sequence immediately 59to the translation start site were performed. SSCP anal-ysis of the amplified products showed an abnormalconformer for the 2 affected siblings that was not evi-dent in control individuals or when screening 60 pa-tients with other CMS (data not shown). Screening thecoding regions of the AChR ε-subunit gene from the 2affected siblings did not detect any additional abnor-mal conformers.

SEQUENCE ANALYSIS. Sequence analysis of the AChRε-subunit gene promoter was performed on cloned PCRproducts amplified from genomic DNA derived from theaffected siblings. The sequence demonstrated a homozy-gous single nucleotide transition ε-156C3T within theN-box region of the ε-subunit promoter (see Fig 1B) forthe 2 patients, disrupting an MspI restriction site (Fig 2A).MspI digests of PCR amplifications of genomic DNAfrom the 2 patients and the unaffected family members

confirmed that the mutation cosegregates with diseasethrough the family (see Fig 2B). As predicted for autoso-mal recessive inheritance, both parents were heterozygousfor the mutation as were 2 of the unaffected siblings, theothers being homozygous wild type.

AChR-Subunit Expression in Skeletal MusclePCR amplifications were performed on cDNA derivedfrom intercostal muscle from Patient S.M., and from anormal individual. In addition, as a positive control forε-subunit mRNA stability, PCR amplification was per-formed on similarly prepared cDNA derived from an-other AChR-deficiency patient. This patient (R.A.)shows recessive inheritance of an AC dinucleotide in-sertion at nucleotide position 627 of the ε-subunitgene (ε627ins2) that predicts truncation of the ε sub-unit within the N-terminal extracellular domain. Am-plification of cDNA encoding the ε subunit for S.M. isdramatically reduced when compared with the AChR-deficiency Patient R.A. or the unaffected control indi-vidual. Indeed, signal from the S.M. sample could notbe detected by ethidium bromide staining under ultravi-

Fig 1. (A) Alignment of the human and mouse acetylcholinereceptor (AChR) «-subunit gene promoter sequences demon-strating the conservation of genomic sequence and position ofE-box and N-box promoter elements. Nucleotide numbering isgiven with respect to the start of the mature «-subunitpolypeptide sequence. (B) Genomic DNA sequence analysis(antisense strand) of wild-type and patient (S.M.) AChR«-subunit promoter regions showing a G3A transition (sensestrand C 131 T) within the N-box.

Fig 2. (A) Schematic representation of the human acetylcho-line receptor (AChR) «-subunit gene promoter region, aroundthe transcription start site, showing the position of E-box andN-box elements and the abolition of the MspI site by the«-156C3T mutation. (B) AChR «-subunit gene promoterpolymerase chain reaction products from affected siblings andimmediate family members digested with MspI. Wild-typesequence yields cleaved products of 209 bp and 96 bp, withmutant sequence remaining uncleaved at 305 bp. Both par-ents and 2 unaffected siblings are heterozygous for the«-156C3T mutation. The affected patients are homozygous.■ and F 5 affected individuals; n and N 5 asymptomaticcarriers as defined by the molecular analysis.

Nichols et al: AChR Promoter Mutation 441

olet light (Fig 3), or by Southern hybridization and pro-longed exposure to autoradiography (data not shown). Bycontrast, equally robust amplification was seen for a-, b-,g-, and d-subunit cDNA for each sample. g-SubunitcDNA could be readily amplified from both the healthymuscle sample and the AChR-deficiency samples (see Fig3). These results strongly suggest that the N-box mutationprofoundly impairs mRNA transcription from theε-subunit gene in Patient S.M.

Discussionε-156C3T is the first mutation identified in theε-subunit promoter region13 that leads to a loss ofmRNA transcription sufficient to cause a deficiency ofadult AChR at the neuromuscular junction. SeveralAChR gene mutations, which produce the clinical phe-notype of AChR-deficiency CMS, have previously beendescribed. All lie within the transcribed region of theAChR ε-subunit gene.7–10 The homozygous N-boxmutation ε-156C3T, which we describe here, dem-onstrates that disruption of elements that controlAChR gene transcription may also cause CMS.

Transcriptional regulation of the muscle AChR has

been studied in some detail in animals, and promoterelements that are highly conserved between specieshave been located close to the translation start site ofall the AChR-subunit genes.19 E-boxes (-CANNTG-;see Fig 1A) that interact with the MyoD family oftranscription factors are thought to play a critical rolein muscle-specific expression.20–23 Another promotertarget sequence, termed the N-box (-TTCCGG-), hasbeen proposed as the site for regulation of synapse-specific expression of the AChR subunits.14,15,24,25

ARIA (neuregulin) secreted from the motor nerve ter-minal is thought to act via postsynaptic surface (erbB)receptors to increase intracellular Ras/mitogen-activatedprotein kinase activity. It is proposed that Ras/mitogen-activated protein kinase–responsive transcrip-tion factors such as GABP (GA-binding protein) thenact at the level of transcription via the N-box.26 Whenthe N-box in the mouse ε-subunit promoter was mu-tated there was a significant reduction in synapse-specific expression from reporter genes. It is notewor-thy that the most dramatic reduction (;70%) wasproduced by a mutation identical to that found in ourpatients. Moreover, the C3T transition that is presentin our patients produced the greatest reduction(;90%) in GABP binding to the N-box sequence.27

The in vivo consequences of the N-box mutation inhumans was demonstrated by loss of expression of theAChR ε-subunit mRNA on RT-PCR of cDNA fromour patient’s intercostal muscle biopsy, indicating thatthe AChR deficiency at the end plate, noted by re-duced 125I-a-bungarotoxin binding, occurs secondaryto a defect in ε-subunit mRNA transcription.

It is not known how great a reduction in theε-subunit mRNA transcription is needed to cause amyasthenic syndrome. However, the loss of ε-subunitmRNA expression in Patient S.M., which is concor-dant with the myasthenic clinical phenotype, appearsto be more dramatic than the reduction in reportergene expression observed for the same mutation in theanimal studies. The use of a minimal promoter regionin the experimental studies may not fully reflect tran-scriptional control in vivo, or alternatively, in humans,the ε-subunit gene N-box element may play a greaterrole in the control of transcription than is evident fromanimal studies.

PCR amplifications, in accordance with our previousresults,28 show that g-subunit mRNA can be readilydetected in human muscle from both control individ-uals and AChR-deficiency CMS patients by RT-PCR.Both patch-clamp and immunocytochemical evidencehas been obtained showing the presence of fetal AChRat the end plates of CMS patients with null mutationsof the ε-subunit gene.7,8 It is therefore probable thatthe expression of g subunit, albeit at low levels, enablesneuromuscular transmission to occur through fetalAChRs in our patients.

Fig 3. Reverse transcription–polymerase chain reaction analysisof a-, b-, g-, d-, and «-subunit mRNA isolated from inter-costal muscle biopsies of Patient S.M., an additional acetylcho-line receptor–deficiency Patient R.A. («627ins2), and an un-affected control. Each primer pair amplifies across at least oneintron/exon boundary. The a primer pair amplifies across thealternatively spliced P3A exon.18 Products were run on 2%agarose gels and visualized under ultraviolet light after stain-ing with ethidium bromide. M 5 marker lane.

442 Annals of Neurology Vol 45 No 4 April 1999

This family demonstrates that an AChR-subunit pro-moter mutation can underlie the clinical phenotype ofAChR-deficiency CMS. Just as the AChR-subunit mu-tations described earlier have given insight into the func-tional characteristics of the AChR,29–33 this promotermutation has confirmed in vivo and in vitro animalstudies suggesting the importance of the N-box in thesynapse-specific transcriptional regulation of the muscleAChR. Identification of further CMS mutations 59 to theAChR-subunit coding regions may highlight additionalelements essential in the control of AChR synthesis.

This study was supported by the Medical Research Council andMyasthenia Gravis Association/Muscular Dystrophy Group of GreatBritain. We thank Prof K. Mills, Department of Clinical Neurol-ogy, Radcliffe Infirmary, Oxford, for the electrophysiology studies.

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