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Human Molecular Genetics, 2003, Vol. 12, No. 23 3055-3066
DOI: 10.1093/hmg/ddg334
© 2003 Oxford University Press

A frameshifting mutation in CHRNE unmasks skipping of the preceding exon

Kinji Ohno*, Margherita Milone, Xin-Ming Shen and Andrew G. Engel

Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN, USA

Received July 8, 2003; Accepted September 29, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 PATIENTS AND METHODS
 REFERENCES
 
A frameshifting 7 bp deletion ({varepsilon}553del7) in exon 7 of CHRNE encoding the acetylcholine receptor {varepsilon} subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that {varepsilon}553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsense-mediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even {varepsilon}553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from {varepsilon}553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or {varepsilon}553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that {varepsilon}EF157V and {varepsilon}E154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 PATIENTS AND METHODS
 REFERENCES
 
Congenital myasthenic syndromes (CMS) are caused by genetic defects of presynaptic, synaptic, or postsynaptic molecules at the motor endplate (EP) (1). Mutations in CHAT encoding the choline acetyltransferase impair acetylcholine (ACh) resynthesis at the motor nerve terminal and cause CMS with episodic apnea (2). Mutations in COLQ encoding the collagenic tail subunit of acetylcholinesterase cause EP acetylcholinesterase deficiency (3,4). Mutations in acetylcholine receptor (AChR) subunit genes alter AChR channel kinetics and/or cause EP AChR deficiency (1,5). Mutations in RAPSN encoding rapsyn, a 43 kDa postsynaptic structural protein that clusters AChR, also cause EP AChR deficiency (6,7).

The adult-type muscle nicotinic AChR is composed of four homologous {alpha}, ß, {delta} and {varepsilon} subunits encoded by CHRNA1, CHRNB1, CHRND and CHRNE, respectively, and has the stoichiometry of {alpha}2ß{delta}{varepsilon}. The fetal-type muscle nicotinic AChR harbors the {gamma} subunit encoded by CHRNG instead of the {varepsilon} subunit, and has the stoichiometry of {alpha}2ß{delta}{gamma}. When the {varepsilon} subunit is defective owing to a null or low-expressor mutation in CHRNE, expression of the fetal-type {gamma}-AChR at the EP partially rescues the phenotype (8–11). Because null mutations in other AChR subunits are probably fatal due to lack of a substituting subunit, low-expressor or null mutations of CHRNE account for most cases of CMS.

Nonsense-mediated mRNA decay (NMD) is a quality-control surveillance mechanism that eliminates mRNA harboring a premature termination codon (PTC) (12–14). In mammalian cells, an exon–exon junction protein complex (EJC) is deposited 20–24 nucleotides upstream of exon–exon junctions (15). EJC recruits some other factors and promotes mRNA export to cytoplasm (16). If translation terminates less than 50–55 nucleotides upstream of the most 3' exon–exon junction, translating ribosomes remove the junction-bound EJC (17). If translation terminates more than 50–55 nucleotides upstream of the last exon–exon junction, the remaining EJC and the associated hUpf1-3 initiate removal of the 5' cap and the exonucleolytic degradation of mRNA (18).

Splicing of pre-mRNA occurs in a macromolecular splicesome composed of five small nuclear ribonucleoproteins (U1, U2, U4, U5 and U6 snRNPs) and non-snRNP proteins (19,20). Cis-acting elements for pre-mRNA splicing include: (i) the 5' splice site with the consensus sequence of MAG/GURAGU (M=A or C, R=A or G) (21,22), where the delimiter represents an exon–intron boundary; (ii) the 3' splice site with the consensus sequence of YAG/G (Y=U or C) (21); (iii) the branch point sequence with the consensus sequence of CURAY, YNYURAY, or YUVAY (V=A, G, or C), where an invariant A (underlined) is located 11–40 nucleotides upstream of the 3' splice site (21,23); (iv) the polypyrimidine tract between the branch point and the 3' splice site, in which U is preferred to C (24,25); and (v) exonic splicing enhancers (ESEs) and exonic splicing silencers (26,27). In splicesome assembly, U1 snRNP binds to the 5' splice site, U2 snRNP to the branch point, U2AF65 to the polypyrimidine tract and to U2 snRNP, U2AF35 to the 3' splice site, and SR proteins to ESEs (20,28).

Mutations in any of the cis-acting elements above may cause aberrant splicing (29,30), and splicing mutations constitute at least 10% of human disease-causing mutations (31). Common consequences of splicing mutations include exon skipping, activation of a cryptic splice site, creation of a new splice site and intron retention, with ratios of 51, 32, 11 and 6%, respectively (32). In addition to the commonly observed aberrant transcripts, splicing mutations also result in aberrant transcripts affecting multiple contiguous exons (33–45) or remote exons (46–49). Skipping of multiple contiguous exons is probably accounted for by ordered removal of introns and consequent clustering of neighboring exons (36,37). The mechanism that underlies skipping of remote exons has not been studied to date, and the present study focuses on elucidating this mechanism.

Here we report that a frameshifting mutation in CHRNE exon 7 ({varepsilon}553del7) observed in seven CMS patients remarkably increases an aberrantly spliced transcript that skips the preceding 101 bp exon 6 in skeletal muscle and in transfected COS cells. Inhibition of protein synthesis and NMD by anisomycin and optimization of splicing signals for exon 6 reveal that inherently weak splicing signals for exon 6 cause skipping of exon 6 even in wild-type CHNRE, but the exon 6-skipped transcript is degraded by NMD, whereas, in the presence of {varepsilon}553del7, NMD degrades the normally spliced transcript rather than the exon 6-skipped transcript. Presence of weak splicing signals for CHRNE exon 6 also prompted us to search in other CMS patients for ESE-disrupting mutations in exon 6, and we found that two CMS mutations, {varepsilon}EF157V and {varepsilon}E154X, cause aberrant splicing of exon 6.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 PATIENTS AND METHODS
 REFERENCES
 
Endplate studies in patients 1, 2, 3 and 8
The number of {alpha}-bgt binding sites per EP was reduced to 10% or less of normal in patients (Pts) 1, 2, 3 and 8. The amplitude of the miniature EP potentials and currents was decreased to 30% or less of normal, indicating a diminished synaptic response to ACh. Single channel patch clamp analysis of patient EPs revealed that the dominant component of the burst open durations was 1.6- to 3.3-fold longer than normal, and that the conductance of channel events was reduced to 43–48 pS, consistent with expression of the fetal {gamma}-AChR instead of the adult-type {varepsilon}-AChR.

Ultrastructural studies showed that many EP regions were smaller than normal but the structural integrity of the pre- and post-synaptic regions was preserved. The density and distribution of AChR on the postsynaptic membranes, visualized with peroxidase-labeled {alpha}-bgt, were greatly attenuated.

Mutation analysis
Direct sequencing of AChR subunit genes revealed that all patients carry two mutant CHRNE alleles. Pts 1–7 harbor a 7 bp deletion of TGGGCCA at nucleotides 553–559 ({varepsilon}553del7), which occupies positions 12–18 of exon 7. Pts 1, 6 and 7 are heterozygous, and Pts 2–5 are homozygous for {varepsilon}553del7 (Table 1). {varepsilon}553del7 predicts a frameshift, but turned out to have an unexpected splicing effect on the preceding exon 6, as will be shown below. Pt 1 also carries a 3' splice site mutation in intron 9 ({varepsilon}IVS9-1G->C). Pt 6 harbors a 20 bp duplication at nucleotides 1002–1021 ({varepsilon}1021ins20). Pt 7 carries duplication of a G nucleotide at 1098 ({varepsilon}1098insG).


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  		Table 1. CHRNE mutations in nine patients
 
Pt 8 is homozygous for a nonsense mutation at codon 154 ({varepsilon}E154X). Pt 9 carries an inframe 3 bp deletion at nucleotides 470–472 ({varepsilon}470delAGT) that predicts to mutate glutamate and phenylalanine at codons 157 and 158 to valine ({varepsilon}EF157V). Pt 9 also harbors a 3' splice site mutation in intron 4 ({varepsilon}IVS4-2A->C).

We previously reported {varepsilon}553del7 in another patient with EP AChR deficiency, but we did not analyze pre-mRNA splicing (case 3 in 9). The other mutations have not been previously reported.

Family analysis
Family analysis revealed that unaffected members carry no or a single mutant allele, whereas affected siblings harbor two mutant alleles, indicating that each mutation is recessive (Fig. 1A). As no DNA was available from the father of Pt 1, we examined the allelic distributions of {varepsilon}553del7 and {varepsilon}IVS9-1G->C using allele-specific RT–PCR. This revealed that the two mutations are heteroallelic and also that {varepsilon}IVS9-1G->C causes retention of intron 9 (Fig. 1B). RT–PCR spanning exons 9–11 also showed that {varepsilon}IVS9-1G->C does not cause skipping of exon 10 (data not shown). Heteroallelic distribution of {varepsilon}EF157V and {varepsilon}IVS4-2A->C in Pt 9 was established by cloning a PCR fragment spanning the two mutations.



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  		Figure 1. (A) Family analysis. For each family, affected members carry two mutant alleles (closed symbol), whereas unaffected members carry no (open symbol) or one (half-shaded symbol) mutant allele, indicating that each mutation is recessive. F1–F8 denote families 1–8. No family samples are available from Pts 5 and 9. Arrows point to propositi. Small symbols in family 1 indicate that no DNA is available. (B) Allele-specific RT–PCR to show heteroallelic distribution of {varepsilon}553del7 and {varepsilon}IVS9-1G->C in Pt 1. The {varepsilon}553del7-specific primer would exclusively amplify a transcript with {varepsilon}553del7, whereas the wild-type-specific primer would exclusively amplify a transcript without {varepsilon}553del7. Direct sequencing of a fragment amplified by wild-type-specific primer reveals that the fragment retains intron 9 and carries {varepsilon}IVS9-1G->C, indicating heteroallelic distribution of two mutations. Absence of a normal-sized fragment in the right lane also indicates that {varepsilon}IVS9-1G->C invariably causes retention of intron 9.

 
Aberrant transcripts in patients 1 and 2
During optimization of the allele-specific RT–PCR in Pt 1, we noticed amplification of unexpected fragments. To examine the origins of the unexpected fragments, we amplified and cloned the entire coding region of CHRNE cDNA by RT–PCR using muscle mRNA in Pt 1. We characterized 56 clones and identified seven different transcripts (Fig. 2). Transcripts I–V originated from the {varepsilon}553del7 allele, whereas transcripts VI and VII originated from the {varepsilon}IVS9-1G->C allele. Transcript V was the dominant species and was the only transcript that harbored no PTC. Retention of intron 11 observed in transcript IV was also present in control cDNA samples (transcript VIII in Fig. 2).



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  		Figure 2. Identified CHRNE transcripts in Pt 1 (I–VII) and Pt 2 (I, IV and V), and an aberrant transcript in a normal control (VIII). Exons (boxes) and introns (gaps) are drawn to the scale. Thick horizontal lines indicate retained introns. Thick splicing marks indicate skipped exons. Arrowheads point to mutations. PTCs, if any, are indicated with their sequences. Transcripts I–V arise from an allele with {varepsilon}553del7, whereas transcripts VI and VII are from an allele with {varepsilon}IVS9-1G->C. As exon 6 comprises 101 nucleotides, transcript V restores the ORF after {varepsilon}553del7, and is the only species that harbors no PTC. Right columns show the number of identified clones in Pts 1 and 2. n.a., not applicable.

 
We also cloned CHRNE cDNA in Pt 2, characterized 33 clones, and identified three transcripts that were identical with transcripts I, IV and V in Pt 1 (Fig. 2). Transcript V was again the dominant species in Pt 2 as in Pt 1.

Expression studies of aberrant transcripts in HEK cells
To examine the effects of the identified aberrant transcripts on AChR expression, we expressed each mutant CHRNE transcript with wild-type CHRNA1, CHRNB1 and CHRND cDNAs in HEK cells. As a control, we coexpressed wild-type CHRNA1, CHRNB1 and CHRND cDNAs with or without wild-type CHRNE cDNA.

The mutant CHRNE transcripts reduced the surface expression of [125I]{alpha}-bgt binding sites on HEK cell to 9–29% of normal (data not shown). As {alpha}-bgt binding sites of the {varepsilon}-omitted {alpha}2ß{delta}2 pentamers were 26±4% of normal, the mutant CHRNE transcripts may not be incorporated into cell surface pentamers.

To confirm this, we measured ACh binding by competition against the initial rate of [125I]{alpha}-bgt binding (50). Wild-type {alpha}2ß{delta}{varepsilon} pentamers bind ACh in a monophasic manner, whereas {varepsilon}-omitted {alpha}2ß{delta}2 pentamers bind ACh in a biphasic manner (51). Transcript I demonstrated a biphasic ACh binding as we observed with the {varepsilon}-omitted {alpha}2ß{delta}2 pentamers, indicating that transcript I is not incorporated into cell surface pentamers (data not shown). A very low expression level of transcript V precluded using this transcript in the ACh competition assay, but lack of 40 essential residues (codons 148–187) that constitute the F-loop in the extracellular agonist binding domain (52,53) predicts that gene product of transcript V will not assemble into cell surface pentamers.

We also determined the ability of mutant {varepsilon} subunits to dimerize with wild-type {alpha} subunit, an early step in AChR assembly. When the mutant CHRNE transcripts were coexpressed with wild-type CHRNA1 cDNA, the number of {alpha}-bgt binding sites was reduced to 16–36% of normal (data not shown). As the number of {alpha}-bgt binding sites of wild-type {alpha} subunit alone was 35±3%, the mutant {varepsilon} subunit is unlikely to dimerize with the {alpha} subunit.

To summarize, the expression studies show that each mutant CHRNE transcript is a null or a functionally null mutation, in which {alpha}{varepsilon} dimerization and incorporation into cell surface pentamers are probably compromised.

Recapitulation of skipping of exon 6 in COS cells
Presence of the major transcript V in Pts 1 and 2 was unexpected, because {varepsilon}553del7 was in exon 7, whereas the preceding exon 6 was skipped. We first confirmed by RT–PCR that {varepsilon}553del7 is associated with skipping of exon 6 in muscle mRNA in Pts 1, 2, 3, and in a previously reported patient (case 3 in 9). As being predicted from cDNA cloning studies of Pts 1 and 2, both heterozygous (Pt 1 and the previously reported case 3) and homozygous (Pts 2 and 3) patients showed both normally spliced and exon 6-skipped transcripts (Fig. 3A). Real-time PCR revealed that homozygous patients (Pts 2 and 3) harbor more exon 6-skipped transcripts than heterozygous patients (Pt 1 and the previously reported case 3) and that even a normal control carries a small amount of an exon 6-skipped transcript (Fig. 3A).



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  		Figure 3. Skipping of exon 6 in muscle and transfected COS cells. (A) RT–PCR spanning CHRNE exons 5–7 of muscle mRNA shows skipping of exon 6 in Pts 1, 2 and 3, as well as in a previously reported patient (case 3 in 9) whose mRNA was not analyzed before. RT–PCR fails to amplify fragments retaining introns 5 and/or 6 (transcripts II, III and VI in Fig. 2) probably due to replication disadvantage of longer fragments. Ratios of the exon 6-skipped transcript are indicated by mean±SD below the gel. Ratios of total CHRNE cDNAs to ß-actin cDNA were 0.03±0.023, 0.006±0.003, 0.006±0.001, 0.021±0.005 and 0.0006±0.0002 for lanes 1–5, respectively, which probably represent variable numbers of EP regions included in muscle specimens. (B) Schematic diagrams of minigene and entire CHRNE. Exons (boxes) and introns (horizontal line) are drawn to the scale. Exon 6 is shaded. Arrowheads point to {varepsilon}553del7. (C) RT–PCR spanning CHRNE exons 5–7 of cytoplasmic RNA of transfected COS cells. Both wild-type and mutant minigenes predominantly result in an exon 6-skipped transcript. Wild-type entire CHRNE mostly yields a normally spliced transcript, whereas {varepsilon}553del7–CHRNE predominantly produces an exon 6-skipped transcript. Ratios of the exon 6-skipped transcript are indicated by mean±SD below the gel. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.03±0.002, 0.03±0.006, 0.95±0.31 and 0.94±0.15 for lanes 1–4, respectively.

 
To understand why {varepsilon}553del7 promotes accumulation of the exon 6-skipped transcript, we first constructed wild-type and mutant minigenes spanning CHRNE exons 4–8, and expressed each in COS cells. We chose COS cells, because expression of CHRNE was not detected in native COS cells. These constructs, however, failed to recapitulate the aberrant splicing: exon 6 was similarly skipped in both wild-type and {varepsilon}553del7-CHRNEs (lanes 1 and 2 in Fig. 3C). As different splicing patterns between a minigene and patient-derived cells have been previously reported (38,54), we next constructed wild-type and mutant entire CHRNEs spanning 12 exons and 11 introns, and expressed each in COS cells. These constructs successfully recapitulated the aberrant splicing (lanes 3 and 4 in Fig. 3C). In contrast to muscle mRNA of homozygous patients (Pts 2 and 3), cytoplasmic RNA of COS cells transfected with {varepsilon}553del7-CHRNE showed only a faint band arising from the normally spliced transcript.

Does {varepsilon}553del7 affect an exonic splicing enhancer?
We next asked whether {varepsilon}553del7 causes skipping of exon 6 by disrupting an ESE. Although ESEs are known to work on an ESE-bearing exon, disruption of an ESE possibly affects ordered removal of introns and subsequently results in skipping of exon 6. We thus scanned for the effect of a 7 bp deletion from four nucleotides upstream to four nucleotides downstream of {varepsilon}553del7 (Fig. 4A). This showed that none affected skipping of exon 6. We further deleted six- and four-nucleotides beginning at nucleotide 553 ({varepsilon}553del6 and {varepsilon}553del4), and found that an inframe 6 bp deletion resulted in normal splicing, whereas a frameshifting 4 bp deletion predominantly yielded an exon 6-skipped transcript (Fig. 4A). These results indicate that no specific nucleotides in exon 7 are required for inclusion of exon 6, but a shift in the ORF is causally associated with skipping of exon 6.



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  		Figure 4. Skipping of CHRNE exon 6 is not due to disruption of an ESE but to a shift in the ORF. (A) RT–PCR spanning CHRNE exons 5–7 of cytoplasmic RNA of transfected COS cells. Scanning of a 7 bp deletion from positions 549–557 (lanes 1–6) using the entire CHRNE construct reveals that all constructs predominantly result in skipping of exon 6. Position of {varepsilon}553del7 is indicated by a bar under the wild-type sequence. {varepsilon}554del7 and {varepsilon}556del7 are not tested, because they result in the same sequence as {varepsilon}553del7 and {varepsilon}555del7, respectively. An inframe 6 bp deletion ({varepsilon}553del6) yields a normally spliced transcript (lane 7), whereas a frameshifting {varepsilon}553del4 results in skipped exon 6 (lane 8), indicating that a shift in the open reading frame rather than disruption of an ESE determines the major transcript. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.06±0.02, 0.04±0.02, 0.13±0.04, 0.12±0.06, 0.02±0.004, 0.02±0.005, 0.11±0.05 and 0.29±0.12 for lanes 1–8, respectively. (B) Partial cDNA sequence of CHRNE from middle of exon 5 to middle of exon 8. Mutated nucleotides are underlined. Fr1–Fr3 represent three reading frames, where Fr1 is the native frame. Skipping of exon 6 shifts a reading frame from Fr1 to Fr3. {varepsilon}553del7 shifts a reading frame from Fr1 to Fr2, and {varepsilon}578delTG restores the frame from Fr2 back to Fr1. Asterisks indicate a stop codon. Exon boundaries are indicated by arrows. Three digit numbers at left shows position of the leftmost nucleotide, where position +1 represents the initial nucleotide of the mature protein. (C) RT–PCR spanning CHRNE exons 5–7 of cytoplasmic RNA of COS cells transfected with the indicated constructs to confirm that a shift in the ORF determines the major transcript. Ratios of the exon 6-skipped transcript are indicated by mean±SD below the gel. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.95±0.31, 0.94±0.15 and 0.68±0.19 for lanes 1–3, respectively.

 
To confirm that a shift in the ORF accounts for skipping of exon 6, we deleted two nucleotides at 578 and 579 in exon 7 ({varepsilon}578delTG) in the {varepsilon}553del7–CHRNE construct so that a normally spliced transcript should have no PTC, whereas skipping of exon 6 should place a PTC in exon 7 (Fig. 4B). As expected, {varepsilon}578delTG efficiently prevented accumulation of exon 6-skipped transcript in the {varepsilon}553del7–CHRNE construct, which provides additional evidence that a shift in the ORF is associated with skipping of exon 6 (Fig. 4C).

Does nonsense-mediated mRNA decay contribute to skipping of exon 6?
We next examined if NMD contributes to exon 6 skipping. We expressed wild-type and {varepsilon}553del7–CHRNEs in COS cells and treated the transfected COS cells with 100 µg/ml of anisomycin for 0.5, 1.0, 1.5 and 2.0 h, which inhibits protein synthesis as well as NMD (55,56). Anisomycin treatment revealed that both normally spliced and exon 6-skipped transcripts were produced from both wild-type and {varepsilon}553del7–CHRNEs (Fig. 5). This indicates that exon 6 is spliced out even in wild-type CHRNE, but the exon 6-skipped transcript is probably degraded by NMD. {varepsilon}553del7 removes a PTC from an exon 6-skipped transcript and makes the transcript immune to NMD, whereas a normally spliced transcript harboring {varepsilon}553del7 is likely degraded by NMD.



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  		Figure 5. Inhibition of protein synthesis and NMD by anisomycin.(A) RT–PCR spanning CHRNE exons 5–7 of cytoplasmic RNA of COS cells transfected with the indicated constructs. Treatment of transfected COS cells with 100 µg/ml of anisomycin for 2 h results in production of both normally spliced and exon 6-skipped transcripts from both wild-type and {varepsilon}553del7-CHRNEs. (B) Ratios of the exon 6-skipped transcript after anisomycin treatment are shown by mean±SD. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.95±0.31 (0 h), 1.08±0.37 (0.5 h), 2.08±0.13 (1.0 h), 2.03±0.06 (1.5 h) and 1.28±0.19 (2.0 h) for wild-type CHRNE, and 0.94±0.15 (0 h), 2.27±0.20 (0.5 h), 1.61±0.38 (1.0 h), 3.34±0.56 (1.5 h) and 1.31±0.04 (2.0 h) for {varepsilon}553del7. Note that the ratios increase up to 1.5 h after anisomycin treatment.

 
Why is CHRNE exon 6 easily skipped?
We inspected splicing signals spanning CHRNE exon 6 to understand why exon 6 is easily skipped. The polypyrimidine tract of CHRNE intron 5 has 19 pyrimidines and is interrupted by multiple trains of Gs (Fig. 6A) to give a pyrimidine to total nucleotide ratio of 19/31 (61.3%), which is the lowest among 11 introns of CHRNE (mean±SD, 78.0±10.1%; range 61.3–100%). The consensus value (CV) is an indicator of the similarity of any 8 bp sequence to the consensus splice-site sequences deduced from a comprehensive collation of human genes (22,57,58). An optimal splice site should have a CV of 1.0, whereas the lowest CV value would be 0.0. The 5' splice site of intron 6 harbors four mismatches against the RNA binding site of U1 snRNP (Fig. 6A), and has a CV of 0.736, which is the second lowest among 11 introns of CHRNE (mean±SD, 0.802±0.052; range, 0.717–0.868).



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  		Figure 6. Optimization of splicing signals flanking exon 6 prevents its skipping. (A) Native polypyrimidine tract of intron 5 has 19 pyrimidines (Y) interrupted by multiple trains of Gs at the YnNCAG consensus sequence between the putative branch point (underlined) and the 3' splice site. We substituted T for each G (arrows) to make uninterrupted stretch of 30 pyrimidines (‘Int 5 Plus’ construct). The native 5' splice site of intron 6 harbors four mismatches against the RNA binding site (5'-ACUUACCUG-3') of U1 snRNP. We engineered optimized nucleotides (arrows) that are complementary to U1 snRNP (‘Int 6 Plus’ construct). (B) RT–PCR spanning CHRNE exons 5–7 of cytoplasmic RNA of COS cells transfected with indicated constructs with or without anisomycin. Alleles-specific RT–PCR faintly detected the exon 6-skipped transcript only in the ‘Int 6 Plus, {varepsilon}553del7’ construct in the presence of anisomycin (not shown), and the estimated ratio of the exon 6-skipped transcript was 0.10±0.07% by real-time PCR. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.75±0.06, 0.29±0.04, 1.63±0.20, 2.36±0.16, 5.20±1.42, 5.46±0.75, 8.11±1.07 and 1.98±0.48 for lanes 1–8, respectively.

 
We optimized the splicing signals flanking CHRNE exon 6 either by introducing a stretch of 30 pyrimidines into intron 5 (‘Int 5 Plus’ in Fig. 6A), or by introducing a complementary sequence of U1 snRNP into intron 6 (‘Int 6 Plus’ in Fig. 6A), and expressed each construct in COS cells in the presence or absence of anisomycin. We found that optimization of either of the flanking splice sites prevented skipping of exon 6 even in the presence of anisomycin and/or {varepsilon}553del7 (Fig. 6B), indicating that unstable splicing of exon 6 is due to weak splicing signals at the flanking introns. This is analogous to CFTR exon 9, in which an extended stretch of U residues in the polypyrimidine tract in intron 8 (24) or optimization of the 5' splice site of intron 9 (59) prevents skipping of exon 9.

Do mutations in CHRNE exon 6 affect its splicing?
Weak splicing signals and the consequent unstable splicing of CHRNE exon 6 imply that ESEs could be involved in recognition of exon 6 (60). We thus tested if any of naturally occurring mutations in exon 6 affects its splicing. We analyzed one polymorphism and four mutations in exon 6 (Fig. 7A). The polymorphism was a C-to-T substitution at nucleotide 459 predicting no amino acid substitution ({varepsilon}459C/T); this polymorphism was observed in seven out of 208 alleles in our series. The four mutations were {varepsilon}E154X in Pt 8, {varepsilon}EF157V in Pt 9, and the previously reported {varepsilon}T159P (61) and {varepsilon}D175N (62) (Fig. 7A). {varepsilon}T159P engineered into a normally spliced CHRNE transcript does not express on cell surface (61). {varepsilon}D175N engineered into a normally spliced CHRNE impairs AChR channel opening efficiency (62).



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  		Figure 7. Mutations in CHRNE exon 6 that affect splicing of exon 6. (A) One hundred one nucleotides of CHRNE exon 6 (capital letters) with deduced amino acids and flanking intronic regions (lower case letters). Positions of one polymorphism and four CMS mutations are indicated by arrows. Hyphens indicate deletions. Arrowhead points to a cryptic 3' splice site activated by {varepsilon}E154X and {varepsilon}EF157V. Boxes enclose putative branch point sequences for the native and cryptic 3' splice sites. Pyrimidines in the polypyrimidine tract for the native and cryptic 3' splice sites are underlined. (B) RT–PCR spanning exons 5–7 of cytoplasmic RNA of COS cells transfected with indicated CHRNE constructs. {varepsilon}459C/T, {varepsilon}T159P and {varepsilon}D175N have no effect on splicing of exon 6. {varepsilon}E154X predominantly activates a cryptic 3' splice site in exon 6 (arrowhead in A) leading to a frameshifting deletion of 47 nucleotides. The identity of a faint top band in {varepsilon}E154X is not determined. {varepsilon}EF157V predominantly causes exon 6 skipping (94±6%), but also shows normal splicing and activation of a cryptic 3' splice site. Ratios of total CHRNE cDNA to ß-actin cDNA were 0.26±0.01, 0.27±0.03, 0.89±0.12, 3.29±0.08 and 1.29±0.26 for lanes 1–6 respectively. (C) RT–PCR spanning exons 5–7 of muscle mRNA in a control and Pt 8, who is homozygous for {varepsilon}E154X.

 
We engineered each nucleotide change into the cloned entire CHRNE and expressed each mutant in COS cells. RT–PCR of cytoplasmic RNA of transfected COS cells revealed that {varepsilon}459C/T, {varepsilon}T159P and {varepsilon}D175N did not affect splicing of exon 6; {varepsilon}E154X activated a cryptic 3' splice site located 47 nucleotides downstream of the native 3' splice site; and {varepsilon}EF157V caused skipping of exon 6 (Fig. 7B). RT–PCR of muscle mRNA of Pt 8, who was homozygous for {varepsilon}E154X, was almost identical to that of transfected COS cells (Fig. 7C). Muscle mRNA of Pt 9, who carried {varepsilon}EF157V, was not available.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
PATIENTS AND METHODS
 REFERENCES
 
Skipping of a preceding exon in CHRNE
Cloning of the entire coding region of CHRNE cDNA in Pts 1 and 2 (Fig. 2) and RT–PCR analysis of muscle mRNA in Pts 1–3, and a previously reported patient (Fig. 3A) demonstrate that {varepsilon}553del7 in exon 7 is unexpectedly associated with skipping of the preceding exon 6. The exon 6-skipped transcript restores the ORF after {varepsilon}553del7 and is the most abundant species (transcript V in Fig. 2).

To understand the mechanism of skipping of exon 6 in CHRNE, we constructed a series of site-directed mutants using the cloned entire CHRNE and analyzed cytoplasmic RNA of transfected COS cells. We found the following: (i) scanning mutagenesis in and around {varepsilon}553del7 established that the remote splicing effect is not owing to disruption of an ESE in exon 7, but to a shift in the ORF (Fig. 4); (ii) treatment of transfected COS cells with anisomycin resulted in production of both normally spliced and exon 6-skipped transcripts from both wild-type and {varepsilon}553del7-CHRNEs (Fig. 5); however, the exon 6-skipped transcript arising from wild-type CHRNE and the normally spliced transcript arising from {varepsilon}553del7-CHRNE are probably eliminated by NMD; and (iii) boundaries of exon 6 carry weak splicing signals, and optimization of the splicing signals prevented skipping of exon 6 (Fig. 6). On the basis of these observations, we attribute skipping of exon 6 associated with {varepsilon}553del7 to inherently weak splicing signals at the boundaries of exon 6 and transcript selection driven by NMD. Therefore, {varepsilon}553del7 does not directly affect splicing of exon 6, but unmasks skipping of exon 6 owing to its weak splicing signals.

Swapping of the NMD target is also observed in ‘nonsense-associated altered splicing’ driven by disruption of an ESE, in which a nonsense mutation alters splice site selection of the mutant exon by disrupting an ESE. Here the alternatively spliced transcript is immune to NMD because of lack of a PTC, whereas a normally spliced transcript carries a PTC and is degraded by NMD (56,63,64). In the present study, however, skipping of CHRNE exon 6 is not owing to disruption of an ESE, but to inherently weak splicing signals at the boundaries of the skipped exon 6.

A recent report shows that ~20% of human genes undergo alternative splicing, and one-third of alternative transcripts harbor a PTC and therefore are targets of NMD (65). In glutaminase (66) and fibroblast growth factor receptor 2 (67), production of PTC-bearing alternative transcripts regulates gene expression levels. Skipping of exon 6 in wild-type CHRNE may or may not have the similar functional significance at the motor endplate.

Skipping of remote exons in other genes
Skipping of remote exons has been reported in other genes. Four exonic and two intronic disease-causing mutations in four genes are known to affect splicing of remote exons. In SLC25A20 encoding the solute carrier family 25 (carnitine–acylcarnitine translocase) member 20, a single nucleotide deletion in exon 1 results in skipping of exon 3. Here, an exon 3-skipped transcript is the major transcript and restores the ORF (47).

In DBT encoding the dihydrolipoamide branched chain transacylase (E2 component of branched chain keto acid dehydrogenase complex), a 2 bp deletion in exon 2 results in skipping of exon 4 or 6. In the same patient, a heteroallelic nonsense mutation in exon 6 gives rise to skipping of exon 4 or 6. The patient also carries a transcript with skipped exons 2–8, which is the only transcript that restores the ORF (49).

In BTK encoding the Bruton agammaglobulinemia tyrosine kinase, a 3' splice site mutation in intron 17 deletes the first nucleotide of exon 18 by shifting the 3' splice site, and also shows skipping of remote exon 16, which fails to restore the ORF (48). In the same patient, a heteroallelic nonsense mutation in exon 18 gives rise to three aberrant transcripts: (i) skipping of exon 16; (ii) skipping of exons 15 and 16, and (iii) creation of a new 3' splice acceptor site in exon 18 that also skips exons 16 and 17. Only the last transcript restores the ORF.

In MLH1, which is defective in human nonpolyposis colorectal cancer, IVS1-11T->A is associated with skipping of exon 2 along with six different combinations of skipping of exons 6, 9 and 10, and none restored the ORF (46).

All remotely affected exons in the above genes carry weak splicing signals in the polypyrimidine tract, the 3' splice site, and/or the 5' splice site. Low polypyrimidine ratios are observed in SLC25A20 introns 2 and 3, DBT intron 5, BTK introns 14 and 15, and MLH1 introns 5 and 8. Low CVs for the 3' splice site are observed at SLC25A20 intron 3 and MLH1 introns 8 and 9. Low CVs for the 5' splice site are recognized at DBT intron 4, BTK intron 15, and MLH1 intron 6. Indeed, in SCL25A20, DBT and MLH1, some affected exons with weak splicing signals are skipped even in normal subjects.

In all the above mutations, each exonic mutation introduces a PTC into a normally spliced transcript, and each intronic mutation primarily affects splicing of the neighboring exon and results in a PTC. Accelerated degradation of normally spliced transcripts or of transcripts with a primary splicing error is likely prerequisite for detecting aberrant transcripts affecting remote exons. Three out of four genes yield a transcript with restored ORF. The predominant species is determined only in SLC25A20, in which a transcript with restored ORF is predominant, as in our patients. Thus, in other genes as well as in CHRNE, skipping of remote exons likely owes to weak splicing signals that are unmasked by transcript selection driven by NMD.

Mutations in CHRNE exon 6 that affect splicing of exon 6
Weak intronic splicing signals predict presence of ESEs in the flanked exon (60). Analysis of one polymorphism and four CMS mutations in CHRNE exon 6 revealed that two mutations affect splicing of exon 6 and yield frameshifting transcripts: {varepsilon}EF157V in Pt 8 causes exon 6 skipping and {varepsilon}E154X in Pt 9 activates a cryptic 3' splice site in exon 6 (see Fig. 7).

{varepsilon}EF157V ({varepsilon}470delAGT) mutates a putative ESE motif (named 5A/3G) of TGGA (60) from TGGAGT to TGGTCA, which is possibly recognized by an ESE-binding SR protein, SRp40 (68). The ESE finder release 2.0 (http://exon.cshl.org/ESE/) (69) predicts that {varepsilon}E154X ({varepsilon}460G->T) mutates a putative SF2/ASF-binding site of CCGAAGA to CCTAAGA. SF2/ASF is another ESE-transactivating SR protein (68). The RESCUE-ESE server (http://genes.mit.edu/burgelab/rescue-ese/) (60) similarly predicts that {varepsilon}E154X affects two candidate ESE hexamers of CGAAGA and GAAGAG, which also probably bind to SF2/ASF (60). Altered splicing caused by {varepsilon}E154X is unlikely due to nuclear scanning of pre-mRNA as exemplified in other genes (70–74), because the activated cryptic 3' splice site also results in a frameshifting transcript.

The cryptic 3' splice site activated by {varepsilon}E154X has a CV of 0.742, whereas that of the native 3' splice site at intron 5 is 0.864, which is against the notion that a CV of a cryptic splice site is better than, or similar to, that of a native site (32,58). This indicates that other factors including disruption of an ESE should drive activation of a cryptic 3' splice site. The putative branch point for the cryptic 3' splice site is CGAAG at nucleotides 459–463 in exon 6, which is mutated to CTAAG by {varepsilon}E154X (see Fig. 7A). The mutant CTAAG conforms to the branch point consensus sequence of CTRAY better than the wild-type CGAAG. On the other hand, {varepsilon}EF157V shortens the polypyrimidine tract for the cryptic site and mutates a polypyrimidine stretch of TTC at nucleotides 472–474 in exon 6 to TC (see Fig. 7A). Differential alteration of these cis-acting elements likely accounts for differential splicing consequences between {varepsilon}E154X and {varepsilon}EF157V.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 PATIENTS AND METHODS
REFERENCES
 
Patients
Pts 1–9 (years of age and gender: 6F, 18F, 10M, 39F, 16M, 30F, 8F, 10M and 33F) have had moderate to severe myasthenic symptoms since birth or infancy. All patients have decremental EMG responses and no AChR antibodies, and respond partially to pyridostigmine. Parents of Pts 5 and 8 are first cousins. Pts 2 and 4 have an affected sibling. Pt 5 also has phenylketonuria. The gene for phenylalanine hydroxylase (PAH), however, is on 12q24.1, whereas CHRNE is on 17p13.

Endplate studies
Specimens of intercostal muscle were obtained intact from origin to insertion from Pts 1–3 and 8 and from control subjects without muscle disease undergoing thoracic surgery. All human studies were in accord with the guidelines of the Institutional Review Board of the Mayo Clinic. EPs were localized for electron microscopy and analyzed as previously described (75,76). The number of AChRs per EP was measured with [125I]{alpha}-bungarotoxin ({alpha}-bgt) (77).

Recordings of the miniature EP potentials and currents, and estimates of the number of transmitter quanta released by nerve impulse were performed as described elsewhere (77,78). Single channel currents were performed in the cell-attached mode as described (79).

Sequencing procedures
PCR-amplified fragments were purified by the QIAquick PCR Purification Kit (Qiagen). Plasmids were purified by the QIAprep Spin Miniprep Kit (Qiagen). PCR products and plasmids were sequenced with an ABI 377 DNA sequencer (Applied Biosystems) using fluorescently labeled dideoxy terminators.

Mutation analysis
Genomic DNA and mRNA were isolated from muscle specimens as described (80). Genomic DNA was also isolated from blood using the QIAamp DNA Blood kit (Qiagen). Using genomic DNA, we directly sequenced CHRNA1, CHRNB1, CHRND and CHRNE in Pts 1 and 2, and CHRNE in Pts 3–9 as described (80).

To trace mutations in family members, we used HinfI, EcoNI, and MboII restriction enzymes (New England Biolabs) for {varepsilon}553del7, {varepsilon}IVS9-1G->C, and {varepsilon}E154X, respectively. {varepsilon}553del7 gains a HinfI site; {varepsilon}IVS9-1G->C loses an EcoNI site, and {varepsilon}E154X loses an MboII site. For {varepsilon}1021ins20, {varepsilon}1098insG, {varepsilon}EF157V and {varepsilon}IVS4-2A->C, we directly sequenced genomic DNA of family members.

To determine allelic distribution of {varepsilon}553del7 and {varepsilon}IVS9-1G->C in Pt 1, we employed allele-specific RT–PCR. The allele-specific forward primers were 5'-GAACGGCGAG·TCGACTTC-3' for the mutant allele with {varepsilon}553del7 (a dot indicates a 7 bp deletion), and 5'-GAACGGCGAGTGGGCCAT-3' for the normal allele without {varepsilon}553del7. The reverse primer was 5'-GTGCCTCTGCCCCTCAAA-3' in exon 10. Allele-specific RT–PCR would yield 607 and 614 bp fragments from {varepsilon}553del7 and wild-type transcripts, respectively.

Cloning of aberrant transcripts in muscle of patients 1 and 2
To determine aberrant transcripts associated with the identified mutations, we cloned RT–PCR products from muscle specimens of Pts 1 and 2. A 1579 bp fragment of CHRNE cDNA spanning positions -61 to 1518 was amplified by nested RT–PCR, where position +1 represents the initial nucleotide of the mature protein and the coding region is from -60 to 1419. We introduced an EcoRI site at the 5' end of nested primers. The RT–PCR product was ligated into pBlueScript II SK(-) (Stratagene) using an EcoRI site. We screened for aberrant transcripts by double digestion of isolated clones with DdeI and HinfI, and determined aberrant transcripts by sequencing the entire inserts. We characterized 56 and 33 clones for Pts 1 and 2, respectively.

Construction of human wild-type and mutant AChR subunit cDNAs and expression in HEK cells
Sources of human CHRNA1, CHRNB1, CHRND, and CHRNE cDNAs were as previously described (51,81,82). All four cDNAs were cloned into the CMV-based expression vector pRBG4 (83) for expression in 293 HEK cells.

Among seven different transcripts obtained from Pts 1 and 2 (transcripts I–VII in Fig. 2), transcripts II, III and VI predicted the same truncated protein. Thus, five transcripts (I, II, IV, V and VII) were cloned into the expression vector pRBG4 for expression in HEK cells. Absence of PCR artifacts was confirmed by sequencing the entire inserts.

HEK cells were transfected with a mutant or wild-type CHRNE cDNA along with the complementary wild-type CHRNA1, CHRNB1 and CHRND cDNAs using the calcium phosphate precipitation method as described previously (80). Total number of [125I]{alpha}-bgt sites of intact or saponin-permeabilized HEK cells and the ACh competition measurements were determined as described (51).

Cloning of minigene and the entire gene of CHRNE
To recapitulate the aberrant splicing in transfected COS cells, we first PCR-amplified wild-type and mutant DNA segments spanning nucleotide 176 in exon 4 to nucleotide 853 in exon 8, where position +1 represents the initial nucleotide of the mature protein, and cloned them into the CMV-based expression vector pRBG4. These minigene constructs, however, failed to recapitulate the aberrant splicing (Fig. 3C).

We next PCR-amplified the entire CHRNE spanning 12 exons and 11 introns and cloned it into pRBG4. The insert spanned 4392 bp from 29 nucleotides upstream of the translational start site in exon 1 down to 35 nucleotides downstream of a TAG stop codon in exon 12. Mutations were engineered into each construct using the QuikChange Site-Directed Mutagenesis kit. Absence of PCR artifacts was confirmed by sequencing the entire insert.

Isolation of cytoplasmic RNA of transfected COS cells
For transfection into COS cells, ~1.5x105 cells were seeded into a six-well plate 24 h before transfection. Cells were transfected with 1 µg of each pRBG4 construct using 3 µl of FuGENE 6 transfection reagent (Roche) according to the manufacturer's recommendations. Fresh medium was added 16 h after transfection. Cytoplasmic RNA was extracted 48 h after transfection using the RNeasy Mini kit (Qiagen) including the DNase I treatment according to the manufacturer's instructions. To suppress translation and subsequently NMD, we added 100 µg/ml anisomycin (Sigma) 0.5, 1.0, 1.5 and 2.0 h before harvesting cells (55,56).

RT–PCR analysis of cytoplasmic RNA of transfected COS cells
For RT–PCR, one-third of the isolated cytoplasmic RNA was used for cDNA synthesis using an oligo-dT primer and the Superscript II reverse transcriptase (Invitrogen). One-twentieth of synthesized cDNA was used for each RT–PCR in 25 µl with primers, 5'-CTTCGATTGGCAGAACTGTT-3' in exon 5 and 5'-AGGAAGTAGGCGAGCAGCAC-3' in exon 7. With these primers, a normally spliced transcript would yield a 324 bp fragment, whereas a transcript skipping exon 6 (101 bp) would give rise to a 223 bp fragment. The PCR condition comprised 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min using the FastStart Taq DNA polymerase (Roche) and the DNA engine (MJ Research). We always included a negative control that is transfected only with FuGene 6 but without any plasmids to confirm that there were no cross-contaminations in experimental procedures and that RT–PCR products were not from the native COS cells.

Real-time PCR analysis
To estimate the ratio of normally spliced to exon 6-skipped transcripts in muscle specimens and in transfected COS cells, we compared amplification of each transcript with those of standards using the iCycler iQ Real-Time PCR Detection System (Bio-Rad). Each transcript was amplified using allele-specific RT–PCR. The allele-specific forward primers were 5'-TGTTCGCTTATTTTCCG·CTC-3' for the normally spliced transcript (a dot indicates the boundary of exons 5 and 6), and 5'-CTGTTCGCTTATTTTCCG·AGA-3' for the exon 6-skipped transcript (a dot indicates the boundary of exons 5 and 7). The reverse primer was 5'-GGGCACGATGATGTTAATGA-3' in exon 7. The normally spliced and exon 6-skipped transcripts would yield 267 and 167 bp fragments, respectively. One-twentieth or less of the above synthesized cDNA was used for each allele-specific RT–PCR in 25 µl, so that an estimated initial copy number fell within the range of standards. The PCR condition comprised 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min using the FastStart Taq DNA polymerase in the presence of x1/7.5 SYBR Green I (Molecular Probes). Each allele-specific RT–PCR amplified an expected single fragment and no primer dimers. Each sample was analyzed in triplicate.

We used the cloned CHRNE cDNA (pRBG4-CHRNE) and the cloned transcript V (pBluescript-transcript V) as standards. Copy numbers of two standards were matched by amplifying the same fragment with 5'-ATTGGCAGGATTACCGACTC-3' in exon 4 and 5'-AGCGGAAAATAAGCGAACAG-3' in exon 5 using the real-time PCR.

To monitor transfection efficiency, we estimated the copy number of ß-actin cDNA in COS cells. To this end, we amplified the entire 1128 bp coding region of ß-actin cDNA (accession number, AB004047) of African green monkey, from which COS cells were derived. The RT–PCR product was cloned into pGEM-T (Promega) and was used as a standard. We amplified a 213 bp ß-actin cDNA fragment using 5'-AACCTTCCTTCCTGG·GCAT-3' and 5'-CAGGAGGAGCAATGAT·CTTGAT-3', where dots indicate presumptive exon boundaries. The primers were complementary to both African green monkey and human ß-actin cDNAs, and amplified an expected single fragment from both species.

We amplified 1x103 to 1x107 copies of each standard in triplicate. Each standard was fitted to an equation: cycle threshold (Ct)=A·ln (initial copies) +B, where A=-1.57, B=37.4, correlation coefficient r=0.996 for the normally spliced transcript; A=-1.49, B=37.5, r=0.997 for the exon 6-skipped transcript; and A=-1.08, B=27.4, r=0.997 for ß-actin. Initial copy numbers of each CHRNE transcript and of ß-actin transcript were estimated by the respective equations.


   ACKNOWLEDGEMENTS
 
This work was supported by the National Institutes of Health grant NS6277 and by a Muscular Dystrophy Association research grant to A.G.E. We are grateful to Dr Susan T. Iannaccone for referral of Pts 1 and 7, Dr John N. Whittaker for Pt 4, Dr Shin J. Oh for Pts 5 and 9, and Dr John Chapin for Pt 6.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 5072845102; Fax: +1 5072845831; Email: ohnok{at}mayo.edu Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 PATIENTS AND METHODS
 REFERENCES
 

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