Brain, Vol. 124, No. 7, 1362-1372,
July 2001
© 2001 Oxford University Press
End-plate 
- and 
-subunit mRNA levels in AChR deficiency syndrome due to -subunit null mutations
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1 Neurosciences Group, Institute of Molecular Medicine, The John Radcliffe Hospital, Headington, Oxford and 2 Department of Neurobiology, The Medical School, University of Newcastle, Newcastle upon Tyne, UK
Correspondence to:
Dr D. Beeson, Neurosciences Group, Institute of Molecular Medicine, The John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK E-mail: dbeeson{at}hammer.imm.ox.ac.uk
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Abstract |
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Acetylcholine receptor (AChR) deficiency is the most common of the congenital myasthenic syndromes (CMS). Typically, the number of AChRs, measured by
-bungarotoxin binding, is reduced to 10–30% of normal levels, the miniature end-plate potentials are correspondingly reduced, and there are morphological changes at the motor end-plates. The majority of these syndromes are due to either missense or frameshift mutations within the gene encoding the adult-specific 
-subunit. These are often null mutations, but some mutant -subunits can be incorporated, at low levels, into functional AChRs in transfected cell lines. It is not clear, therefore, whether upregulation of the mutant -subunit mRNA could generate sufficient AChR to support neuromuscular transmission, albeit at a reduced level. Conversely, it might be that the mutant -subunit transcripts are subject to mRNA surveillance and `nonsense-mediated' loss, leading to reduced -subunit mRNA expression. In either case, it is thought that neuromuscular transmission may be provided partly or entirely by incorporation of the foetal-specific 
-subunit into end-plate AChR. -Subunit mRNA is expressed at low levels in normal human muscle, but might be upregulated in CMS. The study of mRNA levels for AChR subunits should improve our understanding of genotype–phenotype relationships in CMS. Here we have defined homozygous -subunit mutations in four unrelated families with AChR deficiency and studied the steady-state levels of mRNA for AChR subunits at the motor end-plates by in situ hybridization. Although we demonstrated that each mutation would lead to almost complete absence of surface adult AChR expression, we detected similar robust expression of - and -subunit mRNAs at end-plates of patient and control muscles, suggesting that mRNA transcripts for the -subunit are neither upregulated nor degraded preferentially. Interestingly, we were unable to detect any increase in -subunit mRNA expression at CMS end-plates. Transgenic mice lacking the -subunit die 2–3 months after birth, suggesting that 2ß
2 pentamers cannot sustain neuromuscular transmission. Therefore, we tentatively conclude that the persistent low level expression of the -subunit, which is present in normal human muscles as well as in AChR deficiency syndromes, is sufficient to enable patients with -subunit null alleles to survive. AChR; congenital myasthenic syndrome; in situ hybridization; mRNA
AChR = acetylcholine receptor; -BuTx = -bungarotoxin; CMS = congenital myasthenic syndrome; M = transmembrane domain; NMJ = neuromuscular junction; PCR = polymerase chain reaction; RT–PCR = reverse transcriptase–PCR; SSCP = single-strand conformation polymorphism
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
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The congenital myasthenic syndromes (CMS) are a heterogeneous group of inherited disorders of neuromuscular transmission, which, in common with the immune-mediated myasthenias, are characterized by fatiguable muscle weakness. They are caused by a variety of genetic defects. The commonest lead to acetylcholine receptor (AChR) deficiency, in which a severe reduction in the number of AChRs at the motor end-plate can be demonstrated in binding studies using labelled
-bungarotoxin (-BuTx) (Vincent et al., 1981
).
The muscle AChR pentamer exists as two isoforms, an adult form comprising 2ß-subunits and a foetal form compris- ing 2ß-subunits (Mishina et al., 1986). The subunits are homologous, each consisting of a long N-terminal extracellular domain followed by three transmembrane domains (M1–M3), an intracellular cytoplasmic domain, a final transmembrane domain (M4) and an extracellular C-terminus. Each is encoded by a separate gene of between 10 and 12 exons.
In AChR deficiency, which is a recessive disorder, mutations are concentrated at the AChR -subunit gene locus (reviewed in Engel et al., 1999; Beeson and Newsom-Davis, 2000), although a rare instance of heteroallelic mutations at the ß-subunit gene locus has been reported (Quiram et al., 1999). The AChR deficiency mutations of the -subunit are located along the length of the 12 exons of the gene and in the promoter region, although many are in the region encoding the cytoplasmic loop between M3 and M4. Different mechanisms may underlie the AChR deficiency state. Mutations that truncate the -subunit prior to the M3–M4 cytoplasmic loop are almost certainly null alleles, whereas some missense mutations and some truncations near the C-terminus can result in low expression of functional adult AChR. The recent identification of mutations within the -subunit gene promoter region (Nichols et al., 1999; Ohno et al., 1999) strongly suggests that the AChR deficiency, in these cases, is caused by reduced levels of -subunit mRNA. Nonsense and frameshift mutations have been shown to activate nonsense-mediated mRNA decay pathways that markedly reduce levels of mutated RNA (reviewed in Beelman and Parker, 1995; Czaplinski et al., 1999; Mitchell and Tollervey, 2000). Therefore, for some other CMS cases, it is possible that the rapid turnover and thus reduced level of mutated mRNA is responsible for the reduced expression of end-plate AChR.
Strikingly, mice that show recessive inheritance of -subunit null alleles die 2–3 months after birth (Witzemann et al., 1996; Missias et al., 1997), whereas humans with similar truncated -subunits may be only mildly affected (Engel et al., 1999). It has been proposed that incorporation of the (foetal) subunit into end-plate AChR pentamers enables these CMS patients to survive (Engel et al., 1996; Ohno et al., 1997).
In a previous study, we investigated end-plate AChR numbers (by [125I]-BuTx binding and immunocytochemistry), morphology and electrophysiology in the muscle biopsies from four unrelated patients with AChR deficiency (Slater et al., 1997). Here, in each of the four cases, we have identified the AChR subunit mutations, three of which are novel. We have characterized the functional effects of these mutations by measuring -BuTx binding to mutant AChR expressed in HEK293 cells and investigated the compensatory mechanisms that allow survival of humans with -subunit null alleles, by in situ hybridization to AChR -, - and -subunit mRNAs using muscle previously biopsied from these patients.
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Material and methods |
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mRNA and DNA samples
RNA was isolated from muscle biopsies using RNAzol B (AMS Biotechnology, Abingdon, UK) following instructions provided by the manufacturer. Genomic DNA was isolated from peripheral blood using the Nucleon II DNA extraction kit (Nucleon Biosciences, Coatbridge, Scotland, UK). First-strand cDNA was synthesized from total RNA using oligo(dT)15 primer. Approval for the use of human muscle materials was obtained from the local Research Ethics Committees in both Newcastle and Oxford.
Mutational analysis: single-strand conformation polymorphism and DNA sequence analysis
Amplicons containing the 12 coding exons and intron– exon boundary sequences of the -subunit gene were generated using flanking oligonucleotide primers and the polymerase chain reaction (PCR) on genomic DNA. Single-strand conformation polymorphism (SSCP) analysis was performed as previously described (Croxen et al., 1997). cDNA and genomic fragments were sequenced using the T7 Sequenase Version 2.0 DNA sequencing kit (USB/Amersham Pharmacia, Little Chalfont, UK) or the ABI 377 automated DNA sequencer. DNA sequence changes were confirmed by PCR, and restriction endonuclease digestions carried out according to the manufacturer's instructions.
Expression constructs encoding mutant -subunits
Respective mutant -subunit cDNAs were generated in pcDNA3.1 (Invitrogen, Groningen, The Netherlands). 1293insG (Patient 1) was generated using a forward primer modified to contain the additional G nucleotide: 5'-GCCACCGGCGAGGAAGTGTCCGACTGGGTGCGCA-TGGGGGAATGC-3'. A complementary reverse primer located within the 3'-untranslated region was used to generate an amplicon that was first subcloned into pGEM5Z and then subcloned into the wild-type -subunit cDNA using SgrAI and ApaI restriction sites. -Subunit cDNA lacking exon 9 (Patient 2) was generated by amplification of patient cDNA with primers 5'-CTGAGGATACTGTCACCATCA-3' and 5'-CTCGCCGGTGGCCTCCTGATCTCT-3', subcloning and ligating the mutant fragment into the wild-type sequence at the ClaI and SfiI restriction sites. For 1208ins19 (Patient 3), a mutagenic primer containing the 19 bp (base pairs) repeat, 5'-GCCTGGGCGCCCCGCCGCCCCCGAGGTCC-GCTGCTGCCCCCGAGGTCCGCTGCTGTGTGGATGCC-GTGAA-3' was introduced into the wild-type sequence using the GeneEditorTM in vitro site-directed mutagenesis system (Promega, Southampton, UK). P331L (Patient 4) was generated with oligonucleotide 5'-CTGGAGCTGCTGCTG- CGCCTCCTGGGCT-3' using the SculptorTM in vitro mutagenesis system (Amersham Pharmacia Biotech). Mutant -subunit cDNAs were sequenced to check for the presence of the mutation and the absence of additional sequence changes.
Expression studies
Respective mutant -subunit cDNAs, in combination with wild-type , ß and , were transfected in HEK293 cells grown on 6-well tissue culture plates and transfected using polyethylenimine. [125I]-BuTx binding was measured 2 days after transfection. Surface binding was measured by overlaying the cells in PBS (phosphate-buffered saline) containing 10 nM [125I]-BuTx and 1 mg/ml bovine serum albumin for 1 h. Cells were washed four times with PBS, removed from the plate with 60 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5 mM phenylmethylsulphonyl fluoride and 1.25% Triton X-100, and the amount of bound [125I]-BuTx determined. Binding of [125I]-BuTx to -subunit-containing surface AChR was established using -subunit-specific polyclonal antisera as described previously (Croxen et al., 1999). The antiserum raised against amino acids 327–413 of the -subunit (Beeson et al., 1996a) recognized the mutant subunits from patients 1, 3 and 4 but not from patient 2. An -subunit-specific antiserum derived from a patient with myasthenia gravis (Beeson et al., 1996b) was used to immunoprecipitate mutant -subunits from patient 2.
In situ hybridization
In vitro transcription from full-length cDNA clones (Beeson et al., 1993) was used to generate 35S-labelled sense and antisense riboprobes for the -, - and -subunits of the human muscle AChR. The plasmids were linearized with HindIII (, and antisense), XbaI ( sense), XhoI ( sense) or EcoRI ( sense). In vitro transcription was performed in the presence of SP6 or T7 polymerases, and the resulting RNA was subjected to limited alkaline hydrolysis designed to produce probe fragments of ~250 bases which were separated from unincorporated nucleotides using NAP-5 columns (Amersham Pharmacia Biotech.) The optimal probe concentration was determined from preliminary experiments. The - and -subunit probes were used at a concentration of 40 000 c.p.m./µl and the probe at 5000 c.p.m./µl. The sense probe was used as a negative control for the specificity of the antisense probe.
Transverse cryosections of biopsied muscle were fixed in paraformaldehyde and reacted to demonstrate the presence of neuromuscular junctions using a modified indigogenic esterase method as previously described (Young et al., 1998). After thorough washing, the sections were refixed and processed for in situ hybridization. In brief, sections were treated with proteinase K and acetic anhydride to facilitate probe penetration, dehydrated through graded alcohols and air-dried before hybridization with 35S-labelled sense or antisense probe. After a series of washes of increasing stringency, the sections were dipped in Kodak NTB 2 emulsion. They were exposed for 7 days at 4°C before developing and counterstaining with acidified toluidine blue. The sites of probe hybridization were visualized as accumulations of silver grains. No such labelling was seen in sections hybridized with the sense probe or in no-probe controls.
The sections were examined using bright and dark field optics on a Leica DMRA microscope. Neuromuscular junctions (NMJs) were identified in bright field images by their high cholinesterase activity. Fields containing NMJs were then recorded in dark field illumination using a SPOT-2 CCD camera (Diagnostic Instruments Inc., Michigan, USA). As a measure of the relative amounts of probe binding at the NMJs, the intensity of light scattered by the silver grains within a circle of 12 µm radius, centred on the NMJ, was determined (Vater et al., 1998) using Scion Image software. Except in one instance, light scatter from at least six NMJs was analysed and, for many, >20 NMJs were analysed.
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Results |
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Clinical features
The clinical features of the patients studied here have been described previously (Slater et al., 1997
) and are summarized in Table 1 which is modified from this report. In brief, generalized fatiguable muscle weakness was present in all patients. Weakness of ocular and facial muscles was prominent, with onset of symptoms in infancy. On muscle biopsy, each showed an extended area of end-plate acetylcholinesterase staining, reduced postsynaptic membrane folds and reduced end-plate [125I]-BuTx binding.
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Mutational analysis
Patient 1
Screening of the AChR
-subunit gene by SSCP analysis revealed an abnormal conformer for exon 12 of the -subunit gene. DNA sequencing showed that the patient is homozygous for a single nucleotide insertion, 1293insG. Confirmation of this mutation and of its segregation within the family was established by restriction endonuclease digestion using an oligonucleotide primer that created a BslI site in the presence of 1293insG. The mutation predicts truncation of the -subunit after amino acid residue 431 and the addition of three missense amino acids (Figs 1A and B, and 5)
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Patient 2
SSCP analysis revealed an abnormal conformer for exon 9 of the AChR
-subunit gene. DNA sequencing identified a homozygous transversion of the first nucleotide of intron 9, IVS9+1G
T (Fig. 2A). This mutation was confirmed by restriction endonuclease digestion with AflII (Fig. 2B). Both parents were seen to be heterozygous for this mutation, as were the patient's two sisters. To demonstrate the effect of this mutation on splicing of the -subunit RNA transcript, we extracted RNA from the patient muscle biopsy and performed reverse transcriptase–PCR (RT–PCR) using oligonucleotides that amplify between sequences in exons 2 and 11 (Fig. 2C). Sequencing of the amplicon from the propositus demonstrates that the mRNA transcript skips precisely from exon 8 to exon 10, and the 115 bp that correspond to exon 9 have been lost (Fig. 2D). Loss of exon 9 will result in truncation of the -subunit after amino acid residue 286 and the addition of 39 missense amino acids (Fig. 5
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Patient 3
An abnormal conformer was identified by SSCP analysis in exon 11 of the AChR
-subunit gene (Fig. 3A). PCR amplification of exon 11 showed a homozygous insertion within this exon, and DNA sequence analysis identified the mutation as 1208ins19, in which there is a duplication of the 19 nucleotides 1190–1208 (Fig. 3B). This mutation predicts truncation of the -subunit after amino acid 403 and the addition of 37 missense amino acids (Fig. 5).
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Patient 4
SSCP analysis showed an abnormal conformer in exon 9. DNA sequence analysis identified a homozygous transition,
C992T (Fig. 4). The mutation was confirmed by restriction endonuclease digestions which showed both parents and an unaffected sister to be heterozygous, whereas the propositus and his affected brother are both homozygous (Fig. 4B). The missense mutation, P331L, causes substitution of a proline residue located on the cytoplasmic side of M3 that is conserved both between species and between subunits (Fig. 4).
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Expression studies
To investigate the functional properties of the mutant
-subunits, we expressed them in mammalian cells and studied their ability to bind [125I]-BuTx. In vitro mutagenesis was used to generate expression vectors for -subunit cDNAs containing the four mutations. These mutant cDNAs, in combination with wild-type -, ß- and -subunit cDNAs, were transfected into HEK293 cells and surface [125I]-BuTx binding determined 48 h later. Some surface [125I]-BuTx binding was detected for all transfections using mutant -subunit cDNAs, and for the ß cDNA combination (Fig. 6A). Since this might all have come from the ß combination present in each case, we used an -subunit-specific antiserum to immunoprecipitate surface [125I]-BuTx–AChR and thus give a measure of the amounts of mutant -subunits incorporated into the surface AChRs. We have demonstrated previously that this serum can detect similar truncated mutant -subunits (Croxen et al., 1999). Only wild-type AChR was detected by this immunoprecipitation procedure (Fig. 6B). Thus the mutant -subunits are not incorporated into surface AChR pentamers and the surface binding must be due to complexes of ß subunits. These -omitted complexes show very limited function (Ohno et al., 1998).
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To determine if the mutant
-subunits can associate with the -subunit, we determined the level of -BuTx binding generated by co-transfection of mutant or wild-type -subunit cDNA with -subunit cDNA (Fig. 6C). Compared with wild-type , alone and mutants gave reduced -BuTx binding. This may indicate a failure of mutant -subunits to assemble with the -subunit, the accelerated breakdown of mutant dimers or instability of -mutant mRNA and thus loss of mutant -subunit expression.
In situ hybridization
We used in situ hybridization to investigate steady-state levels of mRNA encoding the AChR -, - and -subunits in biopsy sections from control and patient muscle. Material from Patient 2 was not suitable for study. End-plate regions were identified by cholinesterase staining, as described in Material and methods. Following hybridization with the -specific antisense probe, clusters of grains were associated with junctional nuclei, and occasionally with other nuclei in the same fibre profile, in control subjects and in the three patients studied (Fig. 7). In addition, there was a generalized increase in grain density over the cytoplasm of these muscle fibres. No clusters of grains or increased cytoplasmic labelling were found in profiles that did not contain NMJs.
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After hybridization with the
-specific antisense probe, clusters of grains were localized to junctional nuclei in control and CMS muscle. For the probe, there was no generalized cytoplasmic labelling in any of the samples. Following hybridization with the -specific antisense probe, there were dense grain clusters at regions with esterase activity in 19-week foetal muscle. No such accumulations were seen in sections from adult control muscle or in sections from any of the three patients studied. Quantitative analysis of the in situ hybridization signals indicated no obvious differences between controls and CMS patients in the steady-state levels of mRNA encoding the AChR - and -subunits. Moreover, there was no detectable increase in levels of -subunit mRNA at the end-plates of the CMS patients. |
Discussion |
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We have identified the mutations that underlie AChR deficiency syndrome in four unrelated patients. Each patient carries a different homozygous mutation. Three mutations have not been reported previously: a splice site mutation,
IVS9+1GT, a 19 bp duplication, 1208ins19, and a missense mutation P331L. The fourth (Patient 1) had a single nucleotide insertion, 1293insG (Engel et al., 1996; Sieb et al., 2000). Expression studies in HEK293 cells demonstrated that each mutation results in severely reduced surface expression of 2ß AChR pentamers. We then used in situ hybridization to investigate steady-state levels of AChR subunit mRNAs at the motor end-plates from three of the four CMS patients. Contrary to expectation, the presence of a mutation within the -subunit-coding sequence neither had a detectable effect on the levels of -subunit mRNA, nor was there evidence for compensatory upregulation of -subunit mRNA.
One problem in quantifying levels of AChR mRNA in mature muscle is that transcription of adult AChR mRNAs is restricted almost exclusively to subsynaptic and perisynaptic nuclei which make up only a few percent of the total number of nuclei within a muscle fibre. Therefore, for accurate RT–PCR quantification of AChR mRNA levels at NMJs, it is necessary to know the number of end-plates within a biopsy sample before RNA preparation. To overcome the technical difficulties that this causes, we chose the technique of in situ hybridization to detect subunit-specific mRNA directly in the vicinity of NMJs within control and patient biopsies. Although in situ hybridization is not as sensitive a method as RT–PCR, we obtained robust signals from end-plate regions (Fig. 7) with signal strengths that would enable any obvious upregulation or downregulation of mRNA to be detected easily. However, in situ hybridization might not detect subtle changes in the steady-state expression levels.
The four mutations that we have identified affect their respective mRNA transcript/-subunit polypeptide through different mechanisms: 1293insG causes a frameshift immediately prior to M4 that generates a stop codon after three additional missense amino acids; IVS9+1GT alters RNA splicing causing skipping of exon 9, loss of M3 and a frameshift that generates 39 missense amino acids followed by a stop codon; 1208ins19 is a duplication of 19 nucleotides resulting in a frameshift that generates 37 missense amino acids followed by a stop codon; and P331L alters a highly conserved proline residue on the cytoplasmic side of M3. The predicted consequence of the RNA splice-site change of the first nucleotide of an intron is loss of the preceding exon. For IVS9+1GT, we used RT–PCR and DNA sequencing to confirm that in vivo exon 9 is excised from the mature mRNA transcript. We, and others (Ohno et al., 1998; Croxen et al., 2000), previously have identified the mutation 1206ins19, and here we report another mutation, 1208ins19. These two mutations, that each lead to CMS, suggest that the structure of the -subunit gene in this region makes it susceptible to a 19 bp duplication.
At least two mechanisms could account for reduced surface AChR expression. Aberrant mRNA transcripts may be recognized by RNA surveillance mechanisms and degraded rapidly through the nonsense-mediated decay pathways. RNA surveillance mechanisms have evolved to ensure that termination of translation occurs at the appropriate codon within the transcript. They are thought to depend upon the local ribonucleoprotein environment around the termination codon, and their interaction with sequences downstream from the stop signal may activate nonsense-mediated decay. Three of the four mutations we identified could, potentially, generate mRNA transcripts that are subject to nonsense-mediated mRNA decay pathways and thus cause a severe reduction in levels of mutant mRNA. The -subunit mRNA transcripts we used for in situ hybridizations contained frameshifts, a missense substitution or the wild-type sequence, but we observed no great difference in transcript levels. mRNA surveillance mechanisms are thought to be most effective in identifying nonsense codons early within the mRNA transcript, whereas the earliest frameshifts that we identify here occur after exon 8 of the -subunit gene. However, we obtained robust RT–PCR amplification from other CMS patient -subunit mRNAs that contain nonsense or frameshift mutations close to the 5' end of the transcripts (R. Croxen and D. Beeson, unpublished observations). These results suggest that a reduction in -subunit mRNA levels is unlikely to account for the abnormal phenotype in the majority of AChR deficiency syndromes.
Alternatively, mutation of the -subunit polypeptide may cause misfolding, retention within the endoplasmic reticulum and rapid degradation of the polypeptide within the lumen of the endoplasmic reticulum. Expression studies confirmed that AChR pentamers containing these -subunit mutant polypeptides do not reach the cell surface of HEK293 cells. This result for 1293insG is consistent with previous studies in which no channel activity was observed in patch-clamp recordings of similarly transfected HEK293 cells (Engel et al., 1996). Thus it appears that this mechanism is likely to be operating in these CMS patients.
Potentially cryptic splice sites might give rise to novel functional -subunit variants, and we previously have detected -subunit mRNA transcripts in both CMS patients and control individuals that retain the intron between exons 11 and 12 (Croxen et al., 1999). To date, we have not detected the use of cryptic splice sites in the many CMS muscle samples that we have analysed (R. Croxen and D. Beeson, unpublished observations). However, there is evidence that neuromuscular transmission in CMS patients with -subunit null alleles may be mediated through foetal AChR (2ß) (Engel et al., 1996; Ohno et al., 1997). Denervation or the blocking of electrical stimulus to the muscle is known to induce substantial upregulation of the transcription of mRNAs encoding foetal AChR. Therefore, in CMS patients with -subunit null alleles, there may be compensatory upregulation of the foetal () subunit to substitute for the -subunit. However, our in situ hybridization experiments do not support this as they do not detect any obvious upregulation of -subunit mRNA transcripts at the subsynaptic nuclei of patient muscle. Low levels of -subunit mRNA can be detected in normal adult human muscle (MacLennan et al., 1997). We therefore suggest that persistent low level -subunit mRNA transcription from subsynaptic and perisynaptic nuclei, which is present in both healthy individuals and CMS patients, enables sufficient incorporation of the -subunit into AChR pentamers for patient survival. Alternatively, or in addition, some transmission might be mediated through 2ß2 pentamers. Interestingly, as has been reported for mouse muscle (Merlie and Sanes, 1985; Sanes et al., 1991), our in situ hybridizations show that the -subunit mRNA transcription is almost exclusively end-plate specific, whereas some -subunit transcription is associated with extrasynaptic nuclei.
In both AChR deficiency syndromes and myasthenia gravis, the number of end-plate AChRs is reduced. Our results for - and -subunit mRNA expression using in situ hybridization on CMS muscle biopsies contrast with studies of AChR gene expression in patients with myasthenia gravis using quantitative PCR, that showed upregulation of adult AChR subunit mRNA in severe cases (Guyon et al., 1994, 1998). This difference may reflect methodology or be a consequence of the different underlying disease mechanisms.
These studies show that the steady-state levels of -subunit mRNA are not dramatically altered—at least for three patients with AChR deficiency. The results also argue against the proposal that upregulation of foetal AChR provides a compensatory mechanism. Rather, the data suggest that compensatory foetal AChR is provided by the normal residual -subunit mRNA transcription from subsynaptic and extrasynaptic nuclei. Sieb and colleagues recently reported a severely handicapped CMS patient harbouring the 1293insG mutation and suggested that this severe phenotype might be due to homozygosity of the mutation (Sieb et al., 2000). In contrast, our patient 1, although homozygous for this mutation, had a less severe phenotype. Similarly, for other mutations within the AChR -subunit gene, we have noted that different families with the same homozygous mutations can show remarkable differences in disease severity (D. Beeson and J. Newsom-Davis, unpublished observations). Thus, it appears that factors other than the particular -subunit mutation are likely to influence phenotype, and changes in AChR expression would be one possibility. However, the lack of effect on steady-state levels of mRNA encoding the -, - and -subunits that we show here argues against changes in AChR mRNA expression as a major factor in determining phenotypic variation in CMS.
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Notes |
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* Present address: CeNeS Ltd, Cambridge CB4 9ZR, UK
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Acknowledgements |
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We would like to thank John Newsom-Davis for assessing patients and for critical reading of this manuscript. This work was supported by the Myasthenia Gravis Association/Muscular Dystrophy Campaign and the Medical Research Council.
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References |
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Received January 15, 2001. Revised February 23, 2001. Accepted March 5, 2001.
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