Brain, Vol. 125, No. 5, 1005-1013,
May 2002
© 2002 Guarantors of Brain
A newly identified chromosomal microdeletion and an N-box mutation of the AChR
gene cause a congenital myasthenic syndrome
1 Genzentrum and Friedrich-Baur-Institut, Ludwig-Maximilians-University Munich, Germany and 2 Department of Pharmacology/Neurobiology, Biozentrum, University of Basel, Switzerland
Correspondence to: H. Lochmüller, Genzentrum München, Feodor-Lynen-Strasse 25, 81377 München, Germany E-mail: hanns{at}lmb.uni-muenchen.de
*These authors contributed equally to this work
Received August 2, 2001. Revised November 14, 2001. Accepted December 10, 2001.
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Congenital myasthenic syndromes (CMSs) are frequently caused by mutations of the coding region of the acetylcholine receptor epsilon subunit (AChR
) gene leading to a reduced expression of the acetylcholine receptor (AChR) at the postsynaptic membrane. Two recent observations have linked two different N-box mutations of the human AChR promoter to a clinical CMS phenotype. N-boxes are regulatory sequence elements of mammalian promoters that confer synapse-specific expression of several genes, including the AChR subunit genes. Here, we report on a novel point mutation (-154G
A) in the N-box of the AChR promoter in a German CMS pedigree. Semiquantitative analysis of AChR mRNA levels in the patient’s muscle indicated significantly impaired AChR expression. We provide additional evidence of a pathogenic role for this mutation using the mutated promoter (-154GA) driving a heterologous gene (luciferase) in rat skeletal muscle. We show that agrin-induced gene expression is significantly reduced by the N-box mutant (mt) compared with the wild-type (wt) promoter. Refined haplotype analysis and direct sequencing revealed maternal inheritance of the mutant AChR promoter (-154GA) together with paternal inheritance of a chromosomal microdeletion (
1290 bp) encompassing the promoter and the first two exons of the AChR gene in the index patient. In conclusion, we provide genetic and functional evidence that a mutation of the AChR subunit promoter (-154GA) causes CMS due to the reduction of gene expression in skeletal muscle. Moreover, this is the first report of a chromosomal microdeletion affecting an AChR gene. This type of mutation may be missed in standard screening techniques of CMS patients. Keywords: acetylcholine receptor; congenital myasthenic syndrome; epsilon subunit; gene deletion; promoter mutation
Abbreviations: AChR = acetylcholine receptor; AChR = epsilon subunit of the acetylcholine receptor; bp = base pair; CMS = congenital myasthenic syndrome; kb = kilobase pair; mt = mutant; RACE = rapid amplification of cDNA ends; RT–PCR = reverse transcription PCR; SNP = single nucleotide polymorphism; wt = wild type
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Introduction |
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Congenital myasthenic syndromes (CMSs) are a heterogeneous group of disorders, where neuromuscular transmission is impaired by inherited defects (Engel, 1994
; Beeson et al., 1998; Vincent et al., 2000; Engel, 2001). The underlying molecular defects identified to date include mutations in a gene encoding the collagenic tail subunit of acetylcholinesterase (ColQ) (Donger et al., 1998; Ohno et al., 1998) (synaptic defect), in a gene encoding choline acetyltransferase (Ohno et al., 2001) (presynaptic defect) and mutations in acetylcholine receptor (AChR) subunit genes (Engel et al., 1999) (postsynaptic defects).
The latter are most frequently affecting the gene encoding the epsilon subunit of AChR (AChR). Some missense mutations of the AChR gene lead to kinetic abnormalities of the channel. However, most AChR mutations reported to date are missense, nonsense, splice site mutations or single base pair (bp) deletions that result in a loss of functional AChR at the postsynaptic membrane. Rearrangements and chromosomal deletions of AChR genes have not yet been reported. Severe deficiency of the adult AChR at the neuromuscular junction leads to the clinical phenotype of CMS, and is usually inherited in autosomal-recessive traits (Engel et al., 1999; Vincent et al., 2000; Croxen et al., 2001).
Alternatively, mutation of regulatory elements may also cause reduced or inadequate gene expression. Therefore, the promoter region of the human AChR-subunit gene was investigated thoroughly (Nichols et al., 1999; Ohno et al., 1999). This region contains several sequence elements such as E-boxes and one N-box that are crucial for the developmental and tissue-specific regulation of gene expression (Fig. 1A). N-boxes have been identified to direct the synapse-specific expression of mammalian genes encoding AChR subunits (Koike et al., 1995; Duclert et al., 1996) and other proteins such as utrophin (Gramolini et al., 1999; Khurana et al., 1999) and acetylcholinesterase (Chan et al., 1999). The N-box of the AChR gene is highly conserved among different species, including man (Ohno et al., 1999). Indeed, two recent reports indicated that mutations of this promoter element may be responsible for CMS (Fig. 1A). In both reports (Nichols et al., 1999; Ohno et al., 1999), the mutant allele was shown to cosegregate with the congenital myasthenic phenotype. Analysis of an intercostal muscle biopsy of one patient revealed a reduction in the number of end-plate AChRs and a reduction in -subunit mRNA levels (Nichols et al., 1999).
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Here, we report on a new mutation (
-154GA) of the AChR N-box motif in a German CMS family. We present direct evidence that this mutation results in severely impaired gene expression. We also, to our knowledge, provide the first report of a chromosomal microdeletion affecting an AChR gene. |
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Patients
A 36-year-old German CMS patient has been followed up in our department for the last 15 years. Starting in early childhood, he presented with myasthenic symptoms including ptosis, limited eye movements, and mild facial and limb weakness. On several occasions, tests for anti-AChR antibodies were negative. Currently, he presents with marked bilateral ptosis, severe bilateral reduction in eye abduction, and fatigable weakness more prominent in proximal than in distal limb muscles. Repetitive stimulation of a proximal motor nerve (N. accessorius) revealed a decremental response of the compound muscle action potential by 30%, indicative of disturbed neuromuscular transmission. The patient responded well to acetylcholinesterase inhibitors; currently he receives pyridostigmine bromide (10 mg/kg/day). An immunosuppressive treatment with azathioprine (100 mg/day) for 6 years was not successful and was therefore discontinued. Both parents and all six siblings (three male, three female) of the patient are clinically unaffected.
DNA samples, muscle biopsy and mRNA samples
Venous blood samples were obtained from the patient, his parents and four siblings. Genomic DNA was isolated from 10 ml of peripheral blood using a blood and tissue culture DNA extraction kit following the manufacturer’s recommendations (Qiagen, Hilden, Germany). A muscle biopsy of the patient was obtained from the left biceps. Control muscle tissue was obtained from orthopaedic surgery for a non-muscle-related reason. RNA was isolated from muscle tissue using standard methods (Chomczynski and Sacchi, 1987).
Sequence analysis and restriction digest
PCR primers were designed to amplify the promoter region, all 12 exons and flanking intronic regions of the AChR-subunit gene (Abicht et al., 1999) (DDBJ/EMBL/GenBank accession no. AF105999). PCR-amplified fragments were purified by the QIAquick PCR purification kit (Qiagen) and sequenced with an Applied Biosystems model 377 DNA sequencer and fluorescein-labelled dideoxy terminators (Perkin-Elmer, Foster City, Calif., USA). Sequence numbering is given with respect to the start of the mature -subunit polypeptide sequence (nucleotide position +1) (Beeson et al., 1993). Screening for the mutation -154GA was performed by restriction digest of PCR products from the patient, his family members, 42 unrelated patients with CMS, and 50 normal controls. A 623 bp fragment containing the promoter region of the AChR -subunit was amplified by PCR using primers 5'-gaatctctgtaccgcagggcta-3' and 5'-agcccctgtccgtaccgagaa-3'. The -154GA mutation results in the loss of a MspI site. The mutant allele remains undigested, whereas the wild-type allele yields two fragments (465 and 158 bp). Restriction enzyme digestion was carried out at 37°C for 2 h by adding 15 U of MspI in 20 µl of reaction mixture. Restriction fragments were size fractionated on a 2% agarose gel containing ethidium bromide.
Genotype and polymorphism analysis
Five polymorphic microsatellite markers on chromosome 17p13 flanking the AChR-subunit gene (D17S1175, D17S926, D17S849, D17S1828, D17S1810) were chosen based on information obtained from Genome Database (GDB) and Généthon online services. Markers were amplified by PCR using fluorescence-labelled primers, and the size of PCR products was determined on sequencing gels using the GeneScan and GenoTyper software according to the manufacturer’s instructions (Perkin Elmer Biosystems, Norwalk, Conn., USA). To characterize intrafamiliar genotype variation further, the intronic polymorphism IVS3+54CA was analysed in all family members by direct sequencing of PCR products obtained with primers 5'-tgccctggacaagacctcacac-3' (intron 3) and 5'-aacaataatcgtccgggcctcg-3' (intron 5). Furthermore, in the 3'-untranslated region of the AChR gene, UniSTS 83674 (GenBank UniSTS database RH76075) was analysed in all family members using the recommended primers.
Mapping of the transcriptional initiation site in humans
First-strand cDNA synthesis and quantification of specific RNA transcripts was performed using the SMARTTM Rapid Amplification cDNA Ends (RACE) Amplification Kit (Clontech, Palo Alto, Calif., USA) according to the manufacturer’s instructions. The reverse transcription reaction was performed under standard conditions in a reaction volume of 10 µl, using 1 µg of total RNA from muscle biopsies, 200 U SuperscriptTM RNase H– (Gibco-BRL, Karlsruhe, Germany) and 1 µl each of the 5'-RACE cDNA synthesis primer CDS and the SMART II oligo (Clontech).
For mapping of the human transcriptional initiation site of the AChR gene, RACE experiments using an AChR exon 5-specific antisense primer 5'-cgtcacggagccgccctcgta-3' and the Universal Primer Mix (Clontech) were conducted on control muscle cDNA. This was followed by nested PCR with the AChR exon 2-specific antisense primer 5'-cagtgagatgagattcgtcag-3' and Nested Universal Primer (Clontech). In total, eight RACE products of eight independently performed reactions were analysed.
Semi-quantitative reverse transcription–PCR (RT–PCR) analysis
First-strand cDNA synthesis was carried out as described above for both patient and control samples. For quantification of the AChR
-subunit gene transcripts (DDBJ/EMBL/GenBank accession no. XM 010902), PCR was performed using 1 µl of the reverse transcription reaction mixture and the gene-specific primers 5'-ccgaggtgaaaagtgccatcg-3' (exon 8) and 5'-tgagtcgacctgcaaacacgg-3' (exon 9). PCR cycling was carried out with 91°C for 30 s, 50°C for 30 s and 72°C for 1 min. The efficiency of quantification was monitored after cycles 30, 32, 36, 39, 42 and 46. All PCR reactions were performed in an Eppendorf Personal Cycler (Hamburg, Germany). For quantification of specific mRNA transcripts of the AChR gene, 2 µl of the reverse transcribed products were amplified using Universal Primer Mix (Clontech) and the aforementioned AChR exon 5-specific antisense primer according to the following protocol: 5 cycles at 94°C for 30 s, 55°C for 30 s and 68°C for 5 min, and 40 cycles at 94°C for 1 min, 58°C for 1 min and 68°C for 3 min. Subsequently, nested PCR (25 cycles at 94°C for 5 s, 50°C for 30 s, 72°C for 1 min) was performed in duplicate using Nested Universal Primer (Clontech) and the above-mentioned AChR exon 2-specific antisense primer with 1.5 and 2.5 µl of the first PCR product, respectively.
Reporter constructs
PCR fragments of the 5'-untranslated region of the AChR gene, including upstream regulatory elements, promoter and transcriptional initiation site (position –622 to position –90), were generated using the genomic DNA of the patient and of a normal control. To facilitate subcloning, restriction sites for MluI and HindIII were added using primers 5'-gcgacgcgtgaatctctgtaccgcagggcta-3' and 5'-gcgaagcttagctctggcaggcttggag-3'. Promoter fragments were subcloned into pGL3-Basic Vector, a commercially available vector containing an SV40 enhancer located downstream of a luciferase gene (Promega, Madison, Wisc., USA). This system has been used previously to test promoter function (Si et al., 1997). The nucleotide sequence of the subcloned fragments was confirmed by DNA sequencing. The wild-type (wt) and the mutant (mt) constructs differ only at position –154. Plasmid DNA was purified using an endotoxin-free kit (Qiagen) according to the manufacturer’s instructions, and DNA was resuspended in sterile PBS (phosphate-buffered saline) to a final concentration of 2–4 µg/µl.
In vivo reporter gene assays
The mt or wt AChR reporter constructs were mixed at a ratio of 18: 1 with the pRL-TK vector (Promega) encoding Renilla luciferase under the control of the herpes simplex virus thymidine kinase promoter. The AChR reporter/pRL-TK mixture was diluted to 100 ng/µl and a cDNA encoding full-length chicken neural agrin cAgrin7A4B8 (Denzer et al., 1995) was added at a dilution of 10 ng/µl. For controls, empty pcDNA1 vector (Invitrogen, Carlsbad, Calif., USA) was used. The resulting plasmid mixtures were injected into 
40 single soleus muscle fibres of young adult rats (body weight 120 g) as described elsewhere (Jones et al., 1997; Meier et al., 1997; Briguet and Ruegg, 2000). Two weeks after injection, the muscle was removed and frozen in liquid nitrogen. The proximal part of the soleus muscle (injected region) was homogenized in 500 µl ‘passive lysis buffer’ (Promega). Firefly and Renilla luciferase activities of 20 µl lysate were measured using the dual-luciferase reporter assay (Promega). Firefly luciferase activity was normalized to the Renilla luciferase activity derived from the pRL-TK vector. For statistical analysis, an unpaired t-test was used with significance defined as P < 0.1.
Southern blot analysis
To test the hypothesis of a chromosomal rearrangement/deletion of the paternal AChR allele, Southern blot analysis was carried out. Genomic DNA (10 µg each) from the patient, his father and a control person were digested with MspI, separated by gel electrophoresis on agarose gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and hybridized with [-32P]dATP- and [-32P]dCTP-radiolabelled probes. Probes were generated by PCR and restriction digest as follows: a 740 bp fragment was amplified from control genomic DNA using primers 5'-gaatctctgtaccgcagggcta-3' (AChR promoter, 270 bp upstream of the N-box) and 5'-cagtgagatgagattcgtcag-3' (AChR exon 2, 470 bp downstream of the N-box), and digested by MspI. The wt N-box contains a MspI restriction site (see above). Two fragments (270 and 470 bp, respectively) were separated on a 2% agarose gel, purified and used as probes. Membranes were hybridized overnight at 65°C in an ExpressHybTM Hybridization Solution (Clontech) and washed for 30 min each at 65°C in 2x SSC, 0.1x SSC and 0.01x SSC, respectively. Mt N-box alleles were detected as hybridization signals of 1260 bp with both probes, while wt alleles resulted in hybridization signals of 790 bp with the upstream probe and 470 bp with the downstream probe.
Determination of the deletion breakpoints
PCR amplification was carried out on the genomic DNA of the patient and of a control using the primer 5'-gtcggcacagtcagtaaagagg-3' (AChR intron 11). PCR conditions were as follows: 5 cycles at 94°C for 6 min, 52°C for 1 min, 68°C for 15 min, and 35 cycles at 94°C for 1 min, 55°C for 1 min, 68°C for 10 min. Subsequently, nested PCR was performed under the same conditions using the primers 5'-ccctggatcccgaccttagcat-3' [1.6 kilobases (kb) upstream of AChR at a BamHI restriction site] and 5'-tgccctggacaagacctcacac-3' (AChR intron 3). In addition to the expected band of 2.2 kb in length (wt allele), a strong 950 bp was detected in the patient (deletion allele). Direct sequencing of the 950 bp band in the antisense direction revealed the proximal and distal deletion breakpoints. The same method has been used to detect the deletion allele in the patient’s relatives.
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Results |
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Transcription start site of the human AChR
gene in human skeletal muscleRecently, we and others have analysed the human and rodent AChR
promoter (Ohno et al., 1999; Stucka et al., 2000). In the present study, we mapped the transcription start site of the human AChR gene by direct sequencing of RACE products. Heterogeneity was observed in eight independent experiments using skeletal muscle from different healthy individuals and different amounts of RNA, indicating that multiple transcription start sites may exist. However, in five out of eight experiments the main transcription start site was assigned to position –88 (data not shown).
Mutation analysis in the German CMS pedigree
Sequence analysis of the promoter region of the AChR gene revealed a GA transition at nucleotide position –154 (-154GA) in the index patient II:1 (Fig. 2) of our German CMS pedigree. The mutation affects the fourth position of the conserved 6 bp core of the N-box sequence (Fig. 1A). To exclude additional mutations we sequenced the entire coding region of the AChR gene of the patient. No other mutation was found. The mutation -154GA was detected in neither 42 unrelated CMS-patients nor 50 healthy controls. In index patient II:1, the wt AChR promoter sequence was detectable neither by direct sequencing nor by restriction digest of PCR products, suggesting a homozygous mutation, i.e. two identically mutated N-box alleles (Fig. 2). Restriction analysis (Fig. 2) revealed that unaffected family members are either carrying the N-box mutation heterozygously (I:1 and II:2) or do not carry the mutation (I:2, II:3, II:4 and II:5). This is in accordance with an autosomal recessive trait. Surprisingly, the father (I:2) of the index patient did not carry a mutated N-box allele heterozygously. We hypothesized that one of the following three mechanisms may be responsible for this unexpected finding: (i) non-paternity; (ii) germline N-box mutation of the paternal AChR allele; or (iii) chromosomal deletion/rearrangement of the paternal AChR allele.
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Refined haplotype analysis of the AChR
locus in the German CMS pedigreeBy haplotype analysis using five polymorphic microsatellite markers on chromosome 17p13 (Figs 1B and 3) flanking the AChR
gene (D17S1175, D17S926, D17S849, D17S1828, D17S1810), non-paternity of the patient’s father was found to be highly unlikely since the patient (II:1) and his father (I:2) share an identical allele for all markers tested.
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Haplotype analysis confirmed that the index patient inherited the allele with the N-box mutation (
-154GA) from his mother (I:1), as did his unaffected sister II:2 (Fig. 3). To exclude a chromosomal rearrangement of the patient’s AChR locus, Southern blot analysis was performed. Only fragments with the mutated N-box allele were detected, suggestive of a chromosomal deletion but not suggestive of a chromosomal rearrangement (data not shown). Therefore, we hypothesized that the patient’s paternal allele harbours a chromosomal deletion of the entire, or at least parts of the AChR gene, resulting in a ‘hemizygosity’ for the N-box mutation in the patient (Figs 1 and 3).
The putative deletion was mapped between markers D17S1175 (5' of the AChR gene, distance 5 kb) and D17S1828 (3' of the AChR gene), which were found heterozygously in the index patient (II:1) and/or the patient’s father (I:2) (Figs 1B and 3). A single nucleotide polymorphism (SNP) in the 3'-untranslated sequence of the AChR gene (RH76075; NT_010823 variation 1341821: GT) and a polymorphism in intron 3 of the AChR gene (IVS3+54CA) cosegregate with the N-box mutation on the maternal allele, but are not found in the father. The patient (II:1) was found to be heterozygous for both polymorphisms. Therefore, the paternal allele is deleted neither in the 3'-untranslated sequence of the AChR gene, nor the coding part of the AChR gene downstream of intron 3.
Identification of the deletion breakpoints in the AChR gene
We wished to determine the deletion breakpoints of the paternal AChR allele by direct sequencing. Therefore, the above-mentioned polymorphisms were used for allele-specific PCR in order to map the 3' deletion breakpoint to exon 2 of the AChR gene (data not shown). The deletion was confirmed by PCR and direct sequencing of the paternal allele. The deletion extends over 1290 bp [position 1.335.845 to 1.337.134 on contig NT_010823 (DDBJ/EMBL/GenBank accession no.)], thus removing the promoter region, exon 1 and the 5'-part of exon 2 of the AChR gene (Fig. 4). The 3'-breakpoint is located within exon 2 of the AChR gene, while the 5'-breakpoint is located within a sequence element highly homologous to Alu. Ten base pairs in between the two breakpoints do not show homology to any sequence at this locus. The deleted paternal allele is also present in the patient’s healthy brother (II:3), but not in combination with the maternal allele carrying the mutated N-box (Fig. 3).
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Semi-quantitative RT–PCR analysis
Semi-quantitative RT–PCR analysis of
- and -subunit mRNA isolated from a muscle biopsy of the patient and an unaffected control showed robust and approximately equal amounts of -subunit cDNA in both muscles. By contrast, significantly reduced and barely detectable AChR expression was seen in the patient’s muscle compared with that of the control (Fig. 5). In the patient, direct sequencing of barely detectable fragments of a length consistent with wt transcripts was carried out after an additional reamplification step. Interestingly, besides correctly spliced, full-length AChR transcripts, aberrantly spliced products were detected containing exon 2 of AChR spliced to parts of the coding region of the GP1BA (glycoprotein Ib, platelet, alpha polypeptide) gene, located 50 kb centromeric of the AChR gene locus (data not shown).
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In vivo expression studies of AChR
promoter constructsIn order to determine whether the mutation
-154GA of the N-box element would affect gene expression in vivo, reporter gene constructs driven by the human wt or mt AChR promoter fragment were injected into rat soleus muscle. No difference in expression was observed between wt and mt promoter constructs in the absence of agrin (Fig. 6, mt/ –agrin). While agrin increased expression of the wt promoter construct >4-fold (wt/+agrin), agrin had no effect on the expression of the mutant AChR promoter (mt/+agrin).
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Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
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In this study, we report on a chromosomal deletion of the AChR
gene and a newly identified mutation (-154GA) of the AChR N-box motif in a German CMS family. So far, numerous AChR subunit gene mutations have been identified. Most of them are recessive loss-of-function mutations (Engel et al., 1999; Croxen et al., 2001). In our index patient, the hemizygous N-box mutation is uncovered by the heteroallelic AChR deletion. We show that the hemizygous N-box mutation results in severe reduction of AChR expression in the patient’s skeletal muscle (Fig. 4), which is in agreement with a previously reported homozygous N-box mutation (Fig. 1) (Nichols et al., 1999).
At present, the underlying genetic defects leading to CMS cannot be elucidated in all CMS families. As most of the studies have focused on the coding regions of the AChR and other CMS-related genes, further analysis of regulatory sequences may reveal additional pathogenic mutations. In this study, we characterized the transcriptional start site of the human AChR gene by RACE analysis and found a major transcription start site at position –88. Compared with transcription initiation sites of the rat or mouse AChR gene (Sanes et al., 1991; Durr et al., 1994), the human transcripts initiate 40 and 60 bp downstream, respectively. Mutations close to the transcription start site have not been detected in CMS patients so far, but may also impair AChR gene expression.
In addition, we report for the first time a chromosomal deletion of the AChR gene encompassing the entire promoter region and the first two exons. This deletion may have arisen through a non-homologous recombination of the paternal allele (Roth and Wilson, 1988). Whereas single bp mutations of AChR subunit genes are readily detected, chromosomal deletions or rearrangements may be missed by conventional PCR analysis employed to screen CMS patients. The frequency of chromosomal deletions of AChR subunit genes is currently unknown. In contrast, chromosomal deletions of the dystrophin gene account for up to 60% of all Duchenne muscular dystrophy cases (Oudet et al., 1992; Mendell et al., 2001). Since the AChR gene is relatively small and does not contain repeated sequences prone to recombinational events, it may be speculated that large deletions occur at a lower frequency if compared with the dystrophin gene.
The promoter mutation -154GA changes the fourth nucleotide of the 6 bp core of the N-box element. Recently, two other mutations in the AChR promoter region have been identified in CMS families (-155GA and -156CT) (Nichols et al., 1999; Ohno et al., 1999), disrupting the conserved N-box motif at positions 2 and 3, respectively (Fig. 1). In both studies as well as in our report, the mt allele cosegregates with the congenital myasthenic phenotype in an autosomal recessive fashion. Homozygous carriers of the mutation show myasthenic symptoms, whereas heterozygous carriers remain unaffected. Similarly, only homozygous or compound heterozygous carriers of null mutations of the coding region of the AChR gene cause a severe deficiency of the adult AChR at the neuromuscular junction that leads to the clinical phenotype of CMS. Therefore, it may be speculated that only a drastic reduction in AChR promoter activity may lead to myasthenic symptoms. Indeed, analysis of an intercostal muscle biopsy of one patient (-156CT) revealed a reduction in the number of end-plate AChRs and a severe reduction in -subunit mRNA levels (Nichols et al., 1999). Similarly, in our patient functional AChR mRNA is merely detectable (Fig. 4).
The AChR promoter is highly conserved among different species and critical for synapse-specific gene expression. From mutagenesis studies in the mouse it is known that mutations of the N-box element and other promoter mutations directly affect gene expression at the endplate (Duclert et al., 1996; Fromm and Burden, 1998; Gramolini et al., 1998; Schaeffer et al., 1998). The effect of the specific point mutation in the N-box motif identified in our patient altering CCGGAA to CCGAAA has been analysed in two studies using mouse promoter reporter gene constructs (Duclert et al., 1996; Schaeffer et al., 1998). These studies revealed reduced reporter gene expression at the endplate due to the N-box mutation. However, direct proof that impaired gene expression results from the N-box mutation of the human AChR promoter has not yet been provided. Moreover, expression levels may not be extrapolated directly from the in vitro to the in vivo situation, since in vivo expression of AChR molecules is mediated via complex pathways (Schaeffer et al., 1998) involving electrical activity and/or nerve-derived trophic factors that may be absent in vitro. Recent evidence strongly suggests that agrin mediates several mechanisms at the neuromuscular junction, including AChR clustering and AChR subunit gene transcription (Ruegg and Bixby, 1998; Briguet and Ruegg, 2000). In the current study, in vivo experimentation revealed that the wt AChR promoter fragment is responsive to neural agrin-mediated activation of gene expression. In contrast, this was not seen using the mt promoter.
In conclusion, we report for the first time on a chromosomal deletion of the AChR gene linked to a CMS phenotype. In addition, we provide genetic and functional evidence that a newly identified mutation of the AChR gene promoter (-154GA) causes a congenital myasthenic syndrome due to the reduction in gene expression in skeletal muscle.
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Acknowledgements |
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We wish to thank the patient and his family for their kind participation in this study. We thank Nancy Larochelle and Dr Reinhardt Rüdel for helpful discussions, and Ursula Klutzny for expert technical assistance. This work was supported by grants from the Deutsche Forschungs gemeinschaft (DFG) and the Fritz Thyssen Stiftung to H.L. and A.A., and by grants from the Swiss National Science Foundation, the Kanton of Basel-Stadt and the Swiss Foundation for Research on Muscle Diseases to M.A.R.
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