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Neuromuscular Disorders 23 (2013) 43–51

Cell models for McArdle disease and aminoglycoside-inducedread-through of a premature termination codon

Kathryn E. Birch a,b,1, Ros M. Quinlivan a,2, Glenn E. Morris a,b,⇑

a Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UKb Institute for Science and Technology in Medicine, Keele University, UK

Received 12 March 2012; received in revised form 29 May 2012; accepted 19 June 2012

Abstract

McArdle disease results from mutations in the gene encoding muscle glycogen phosphorylase (PYGM) protein and the two mostcommon mutations are a premature termination codon (R50X) and a missense mutation (G205S). Myoblasts from patients cannotbe used to create a cell model of McArdle disease because even normal myoblasts produce little or no PYGM protein in cell culture.We therefore created cell models by expressing wild-type or mutant (R50X or G205S) PYGM from cDNA integrated into the genomeof Chinese hamster ovary cells. These cell lines enable the study of McArdle mutations in the absence of nonsense-mediated decay ofmRNA transcripts. Although all cell lines produced stable mRNA, only wild-type produced detectable PYGM protein. Our data suggestthat the G205S mutation affects PYGM by causing misfolding and accelerated protein turnover. Using the N-terminal region of PYGMcontaining the R50X mutation fused to green fluorescent protein, we were able to demonstrate both small amounts of truncated proteinproduction and read-through of the R50X premature termination codon induced by the aminoglycoside, G418.� 2012 Elsevier B.V. All rights reserved.

Keywords: Glycogen phosphorylase; PYGM; McArdle disease; Glycogen storage disease V; Aminoglycoside read-through; Premature termination codon;Cell model

1. Introduction

McArdle disease (glycogen storage disease V) is causedby a defect in glycogenolysis within skeletal muscle cells[1]. It results in an inability to utilise glycogen as an energysource for anaerobic muscle contraction and aerobic gly-colysis. Affected people complain of exercise intolerancewith fatigue and myalgia and are at risk of acute episodesof rhabdomyolysis [2]. McArdle disease is caused by muta-tions in the PYGM gene that encodes the muscle glycogen

0960-8966/$ - see front matter � 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.nmd.2012.06.348

⇑ Corresponding author at: Wolfson Centre for Inherited Neuromus-cular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK.Tel.:+44 1691 404155; fax: +44 1691 404170.

E-mail address: glenn.morris@rjah.nhs.uk (G.E. Morris).1 Present address: Medical Research Council, Harwell Science and

Innovation Campus, Oxfordshire, OX11 0RD, UK.2 Present address: National Hospital for Neurology and Neurosurgery,

MRC Centre for Neuromuscular Diseases, London, WC1N 3BG, UK.

phosphorylase enzyme. To date, over 150 causative muta-tions in the PYGM gene have been identified. The R50Xpremature termination codon is the most common muta-tion and the G205S missense mutation is the second mostcommon mutation in Caucasians in Europe and NorthAmerica. Allele frequencies vary between countries, withrecent reports of allele frequencies of 54–77% for R50Xand of 3–10% for G205S [3,4]. The R50X mutation resultsin an absence of mRNA due to nonsense-mediated decay[5–7]. The G205S mutation is located within a domaininvolved in glycogen binding and tetramerisation [8]. Itresults in stable mRNA, but the mutation prevents PYGMenzymatic function [9–11], although the exact mechanismof this is not known.

At present there is no effective treatment for the disor-der, although regular aerobic exercise is not harmful andmay be beneficial through aerobic conditioning [3].Enzyme replacement therapy is a successful treatment

44 K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51

for some lysosomal storage diseases, including Type 1Gaucher disease and Pompe disease (glycogen storage dis-ease II) [12,13]. However, enzyme replacement therapy isnot a suitable treatment for McArdle disease as wild-typePYGM protein is cytoplasmic and the replacementenzyme would be directed to the lysosome rather thanthe cytoplasm. If nonsense-mediated decay could be sup-pressed, then read-through of the R50X premature termi-nation codon in order to produce full-length PYGMprotein would be a potential treatment strategy for McAr-dle disease patients with this mutant allele providing theresultant protein was enzymatically active. Aminoglyco-side antibiotics such as gentamicin, or novel non-amino-glycoside compounds (e.g. Ataluren/PTC124) have beenshown to induce read-through of premature terminationcodons in clinical trials in cystic fibrosis and Duchennemuscular dystrophy patients [14–16]. Some aminogly-coside read-through drugs may have the dual effect ofsuppressing nonsense-mediated decay and inducing read-through of the premature termination codon [17–19].The exact mechanism of how the G205S mutation disruptsPYGM enzyme activity is not known, but elucidating thiscould suggest potential treatments for patients with thismutation.

Cell models play a valuable role in improving under-standing of the molecular mechanisms of disease andfor testing treatments. A model of the R50X andG205S mutations is required to test and identify poten-tial therapies for McArdle disease. At present there areovine and bovine models of the disease [20–22], but nei-ther has the R50X or G205S mutations. For many mus-cle diseases, cultured muscle cells from patients can beused to produce an in vitro cell model. However, thisis not possible for McArdle disease because, althoughPYGM protein is expressed at high levels in skeletalmuscle in vivo, cultured wild-type muscle cells do notmature sufficiently and only express the protein at verylow levels [23].

Here we describe the creation of cell models express-ing wild-type PYGM, or the two most common mutants(R50X and G205S), from cDNA. To overcome problemsof low-level PYGM expression from muscle cells in cul-ture, our cell models were created by transient and stabletransfection with cDNA under the control of a humancytomegalovirus (CMV) promoter for high-level constitu-tive expression. We show that cloned cell lines with gen-ome-integrated cDNA produced stable mRNA from allthree cDNAs, since nonsense-mediated decay cannotoperate on intronless RNA transcripts. We demonstrateaminoglycoside-induced read-though of a construct con-taining the R50X premature termination mutation usingG418. We also provide experimental evidence that mis-folding and aggregation of mutant PYGM may partlyexplain why the G205S mutation causes McArdledisease.

2. Materials and methods

2.1. Plasmid constructs

pCMV-SPORT6 plasmid containing mouse Pygm

cDNA (NCBI Accession BC012961) was obtained fromthe I.M.A.G.E. consortium (I.M.A.G.E. ID 3989941)[24,25]. Mouse Pygm cDNA was used because full-lengthhuman PYGM cDNA was not available when these studieswere performed, and mouse and human PYGM aminoacid sequences have high amino acid identity (97%). ThecDNA was transferred to pCI-neo (Promega, Madison,WI) using compatible SalI and NotI restriction sites.The QuikChange XL site-directed mutagenesis kit(Stratagene-Agilent, Santa Clara, CA) was used to intro-duce the specific base changes. The R50X mutation wasintroduced into Pygm using forward primer 50-GTGGCT ACT CCG TGA GAT TAC TAT TTT GC-30 andreverse primer 50-GCA AAA TAG TAA TCT CTCGGA GTA GCC ACA TTG CG-30 to change AGA toTGA. The G205S mutation was introduced into Pygm

using forward primer 50-CTG CCT GTG CAT TTCTAT AGC CGA GTG GAG C-30 and reverse primer50-GCT CCA CTC GGC TAT AGA AAT GCA CAGGCA G-30 to change GGC to AGC.

To introduce N-terminal GFP-encoding sequences(Fig. 1), GFP cDNA was excised from pEGFP-C1 (Clon-tech, Mountain View, CA) and cloned into pCI-neo usingNheI and XhoI restriction sites on both plasmids. Wild-typeand mutant Pygm were then cloned from pCI-neo into theGFP-pCI-neo constructs with MluI and NotI.

For read-through studies, constructs encoding C-termi-nal fusions of GFP to the 50 amino acids of wild-type(R50R) or 49 amino acids of mutant (R50X) PYGM proteinwere produced (Fig. 1). PCR was performed using a com-mon forward primer (50-CGC TGC TAG CGC CATGTC CAG GCC TCT TTC-30) and either a reverse primerencoding a NheI site (50-CGG GGC TAG CTC TCTCGG AGT AGC CAC ATT-30) on the wild-type template,or a reverse primer encoding a NheI site and stop codon (50-CGG GGC TAG CTC TCA CGG AGT AGC CAC ATT-30) on the R50X Pygm template in pCI-neo. The PCR prod-ucts were digested with NheI and cloned into the NheI siteof pCI-neo. Plasmids from positive colonies were sequencedto confirm orientation, reading-frame and single insertion.To remove the neomycin cassette from these R50X-GFPand R50R-GFP constructs, they were digested with AvrIIand BstBI, blunt-ended and religated (Fig. 1).

Predicted sizes of recombinant proteins are: (a) GFP-PYGM and GFP-G205S: GFP + 20aa linker + PYGM =127 kDa, (b) GFP only: GFP + 36aa linker to first pCI-neo stop codon = 31 kDa, (c) GFP-R50X: GFP + 20aalinker + 6 kDa (49aa) = 35 kDa, (d) R50R-GFP: 50aa ofPYGM + 8aa linker + GFP + 36aa linker to first pCI-neo stop codon = 37.5 kDa.

Fig. 1. Constructs encoding wild-type and mutant PYGM and GFP fusion proteins were used to study expression of PYGM protein. Thecytomegalovirus immediate early promoter (CMV) and neomycin resistance cassette (neo) are provided by the pCI-neo vector. Full details of the cloningand construction of the constructs are given in methods (see Section 2).

K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51 45

2.2. Production of stably-transfected cell lines

To introduce plasmid constructs by electroporation,CHO-K1 cells (ATCC, CCL-61) (106) in Ham’s F-10 weremixed with 10 lg plasmid DNA in a 0.2 cm cuvette andsubjected to a 1.5 kV pulse in a Bio-Rad Gene Pulser(200 X and 25 lF). The cells were grown for 48 h in Ham’sF-12 plus 10% fetal bovine serum before adding G418(Invitrogen, Paisley, UK) for a further 5–10 days to selectfor neomycin resistance. Genomic integration of Pygm

cDNA was confirmed by PCR and cells were then sub-jected to three rounds of cloning by limiting dilution. Tenclones were selected for each of the three constructs(wild-type, R50X and G205S).

2.3. Transient transfection

Cells were grown on glass coverslips or in 24-wellplates overnight in Ham’s F-12 plus 10% fetal bovineserum then changed to F-12 with 5% serum. For transfec-tion, GeneJuice (Novagen) was incubated with serum-freeF-12 for 7 min. before adding endotoxin-free plasmid fora further 7 min. at a ratio of 3:1 (GeneJuice:plasmid).This mixture was added dropwise to the coverslip or welland cells were grown for 24 h before fixing with 1:1acetone:methanol, or for 48 h before treatment withaminoglycosides.

2.4. Treatment of cells with aminoglycosides

Following transient transfection, cells were incubatedfor 72 h with Ham’s F-12 with 5% fetal bovine serumand an aminoglycoside. Aminoglycosides used were genta-micin C sulfate salt (50 lg/ml), sisomicin (10 lg/ml) andtobramycin (25 lg/ml) (all from Sigma), G418 (G418sulphate; 50 lg/ml) and hygromycin B (10 lg/ml) (bothfrom Invitrogen).

2.5. Immunofluorescence microscopy of cells

Fixed cells on coverslips were incubated with mousemonoclonal anti- PYGB/PYGM (HyTest, Turku, Finland,clone 3G1) primary antibody. This was detected with AlexaFluor 546-conjugated goat anti-mouse IgG (Invitrogen).Nuclei were counter-stained with 40,6-Diamidino-2-phenyl-indole (DAPI). The coverslip was mounted in Hydromountand imaged with a Leica SP5 confocal microscope.

2.6. Western blot analysis

Cytoplasmic proteins were prepared by lysing cell pelletsin 20 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.5%NP-40, pH 7.4, plus Protease Inhibitor Cocktail (Sigma,P8340). Protein samples were separated by SDS–polyacryl-amide gel electrophoresis using a 7% stacking gel and a 7%

46 K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51

separating gel. Proteins were transferred onto nitrocellu-lose by electrophoresis at 10 mA overnight using Bio-RadTransBlot equipment, with a cold water circulating system.The membrane was then blocked with 4% skimmed milkpowder in PBS with 0.05% Tween-20. The blot was incu-bated with mouse monoclonal anti-GFP (Sigma, G1546or GSN149), anti-b-actin (Abcam, Cambridge, UK,Ab8226), mouse monoclonal anti-PYGB/PYGM (HyTest,Turku, Finland, clone 3G1), or rabbit polyclonal anti-PYGM antibody [26]. Mouse monoclonal primary anti-bodies were detected with goat anti-mouse Ig HRP(DAKO, Glostrup, Denmark, P0260). Rabbit polyclonalantibody was detected with VECTASTAIN ABC kit(Vector Labs, Peterborough, UK). Peroxidase activityupon SuperSignal West Pico or West Femto (Pierce Bio-technology, Rockford, IL) substrate produced chemilumi-nescence that was detected using a Bio-Rad imagingcamera and software. Purified glycogen phosphorylasefrom rabbit muscle (Sigma, P-1261) was used to producePYGM protein standards. Protein expression was quanti-fied by densitometry using ImageJ (NIH freeware).

2.7. Relative quantification (RQ) of mRNA with TaqMan

For each of the 30 stably-transfected clones, mRNAexpression was assayed using TaqMan reagents in theABI 7500 Real-Time PCR System. Total RNA was puri-fied from CHO-K1 cell clones using the RNeasy Plus Minikit (QIAGEN, Crawley, UK) and was used to synthesisecDNA using the High-Capacity cDNA Reverse Transcrip-tion kit (ABI, Carlsbad, CA). The mouse Pygm primer-probe kit Mm00478582_m1 (ABI, Cat 4331182) generateda PCR product of 71 bp spanning exons 17 and 18. Pygm

mRNA expression was normalised to the endogenous geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH;ABI, Cat 4308313).

In each reaction, the threshold cycle (CT; the cyclenumber where the fluorescence generated during the Taq-Man reaction reaches a fixed threshold), was determinedfor Pygm and GAPDH. RQ of the TaqMan data wasperformed using the 2�DDC

T method where �DDCT =�(DCT, sample�DCT, reference) [27]. mRNA expression wasquantified relative to untransfected CHO-K1 cells andexpressed as arbitrary units.

3. Results

Pygm mRNA could be detected from R50X and G205Sclones, as well as from wild-type clones. Production ofPygm mRNA was very variable between different clonesand not affected by the mutations. Although expressionwas highest in wild-type clone G12, some R50X andG205S clones had higher levels of Pygm mRNA than manyother wild-type clones. The range of mRNA levels (arbi-trary units) was 50–14,000 for wild-type, 200–9000 forG205S and 100–5000 for R50X. When Pygm mRNAexpression from 10 wild-type clones was compared to 10

G205S or R50X clones using the Mann–Whitney U test,there was no significant difference.

Variable Pygm mRNA expression levels from genome-integrated cDNA could be due either to gene copy numberor to differences in transcriptional activity at the site ofintegration. Copy number for the integrated Pygm cDNAwas measured using a standard qPCR method; no correla-tion was found between copy number and Pygm mRNAlevels in different clones (data not shown). We concludethat the integration site is the most likely explanation forvariable expression.

The three wild-type clones with the highest level ofPygm mRNA were C10 with 1800 units, F10 with 2500units and G12 with 14,000 units. The western blot inFig. 2 shows that these three clones also have the highestPYGM protein expression, as expected. The G205S andR50X stable transfectants did not produce any detectableprotein (data not shown), even when they had high levelsof Pygm mRNA. The anti-PYGM antibody availablewas raised to the C-terminal region of the PYGM protein[26], so would not detect R50X mutant protein consistingof only the first 49 amino acids of PYGM (which wouldbe unlikely to be functional even if it were produced). Cellswere therefore transiently transfected with cDNAs encod-ing wild-type and mutant PYGM with an N-terminalGFP tag, which enabled use of an anti-GFP antibody todetect protein production. Transient transfection was usedto achieve higher cDNA levels. A prominent band of wild-type GFP-PYGM and a fainter band of GFP-G205Smutant PYGM were observed at the expected size of127 kDa by western blot with anti-PYGB/PYGM antibody(Fig. 3A). This antibody also detected the endogenousPYGB isoform at 97 kDa in all cells and this provided aconvenient control for equal loading of the extracts. Den-sitometry of replicates showed that the amount of G205Smutant protein was about 12% of the amount of wild-typeGFP-PYGM (Fig. 3C). There was a statistically significantdifference between the amount of GFP-PYGM (wild-type)and amount of GFP-G205S mutant protein detected bywestern blot (Mann Whitney U test, p < 0.01). The bindingsite for the anti-PYGM/PYGB antibody is not presentwithin the first 50 amino-acids of PYGM, so we used ananti-GFP antibody to detect GFP-R50X protein at theexpected size (for GFP plus 49 amino acids of PYGM) of35 kDa (Fig. 3B). The smallest band (and a minor bandat 34 kDa) was also observed after transfection with GFPcDNA only (Fig. 3B).

After transient transfection, the localization within the cellof GFP-fusions to wild-type or G205S PYGM was com-pared (Fig. 4). In 65% of cells transfected with G205S, exten-sive aggregation was observed, especially in the perinuclearregion. This was significantly greater than the 25% of cellswith aggregates after wild-type PYGM transfection and 6%with GFP alone (Fig. 4) (Mann Whitney U test, p < 0.01).

To study read-through, a new construct was made withGFP located immediately after the R50X mutation, so thatany production of GFP-containing protein of the expected

Fig. 2. PYGM protein production by wild-type cell lines. (A) Expression of PYGM protein from 10 wild-type Pygm CHO-K1 clones was detected bywestern blot using rabbit anti-PYGM or anti-b-actin (loading control). Clone names are shown under the two western blots which together show 10different clones plus controls. CHO-K1 indicates untransfected CHO-K1 cells. (B) Nanogram (ng) amounts of muscle glycogen phosphorylase proteinproduced per 5 � 105 cells. Protein expression was quantified by densitometry comparison with purified PYGM protein standards; 100 ng (S1), 50 ng (S2),25 ng (S3) using ImageJ software (mean ± SEM, n = 3). The wild-type clones with the highest level of Pygm mRNA also had the highest levels of PYGMprotein expression.

K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51 47

size would indicate read-through. Following incubationwith G418, CHO-K1 cells transfected with the R50X-GFP construct produced a 4 kDa higher molecular weightband, as predicted and with the same size as the positivecontrol protein R50R-GFP (Fig. 5). No convincing read-through was seen with the other four aminoglycoside drugstested: tobramycin, hygromycin B, gentamicin and sisomi-cin. A fainter band of similar size in the untreated R50X-GFP may have a different origin, but we cannot rule outthe possibility of some spontaneous read-through. AGFP-only band was detected in all transfected cells. Thishas been observed by other researchers and is due to trans-lation from the start codon of the GFP sequence [28,29].This was the major product even when the 50 amino acidwild-type PYGM-GFP (labelled R50R-GFP, Fig. 5) wasused, so it was not a consequence of the R50X prematuretermination codon. It is unclear why both “read-through”and “GFP-only” bands migrate faster, relative to Mr.markers, than the expected sizes of 37.5 kDa and 31 kDa,though the last 36 amino-acids (4 kDa) of both are encodedby the plasmid and unstructured, so some or all may havebeen removed by proteolysis.

4. Discussion

In our cell models, mRNA containing the R50X muta-tion was produced at similar levels to the wild-type Pygm

mRNA, in contrast to in vivo, where PYGM mRNA con-taining the R50X mutation is undetectable because it hasbeen degraded by nonsense-mediated decay [5–7]. Severalpotential therapeutic treatments (such as read-through orexon skipping) require the presence of stable mRNA. Toapply such therapies to McArdle patients with the R50Xmutation in the PYGM gene, it would be necessary to sup-press nonsense-mediated decay. Existing pharmaceuticalapproaches which may suppress nonsense-mediated decayand stabilise mRNA include small molecules [30] or siRNA[31]. Studies have shown that some aminoglycoside read-through drugs, such gentamicin [17], negamicin [18] orG418 [19] may have a dual effect in stabilising the mRNAthat would normally be subject to nonsense-mediateddecay and inducing read-through of the premature termi-nation codon. A different system that includes intronswould be needed to study such stabilisation in model celllines.

For nonsense-mediated decay to occur, several criteriamust be met, including presence of an intron downstreamof the premature termination codon (reviewed in [32]).Our use of cDNA (which does not contain introns) enabledproduction of cell lines expressing intronless mRNA whichcan escape the nonsense-mediated decay pathway andenabled us to investigate translation of the stable mRNAand possible read-through of a premature terminationcodon.

Fig. 3. Expression of R50X and G205S mutant PYGM fusion protein in cell models of McArdle disease. CHO-K1 cells were transiently transfected withplasmid constructs encoding N-terminal GFP fusion proteins with full-length wild-type or G205S or R50X mutant PYGM or with plasmid constructsencoding GFP only (see Fig. 1). CHO-K1 indicates untransfected CHO-K1 cells. Two parallel western blots were developed with (A) anti-PYGB/PYGMantibody and (B) anti-GFP. In (C), full-length GFP-PYGM protein (127 kDa) from CHO-K1 cells transiently transfected with plasmids encoding GFPfusion proteins was quantified by densitometry using ImageJ software (mean ± SEM, n = 3). Endogenous PYGB from CHO cells in (A) served as aloading marker. The amount of GFP-G205S protein was significantly reduced compared to the wild-type GFP-PYGM (p < 0.01). GFP-R50X truncatedprotein was detected.

48 K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51

If R50X mRNA were stabilised, then it would be pre-dicted that translation of the mRNA up to the prematuretermination codon would produce a truncated protein of49 amino acids instead of the native 841 amino acids. Thisis supported by our experimental data showing that ourGFP-PYGM fusion protein was truncated when the codingsequence contained the R50X mutation. The truncatedprotein appeared to be stable, although the GFP moietymay have increased the stability of the N-terminal PYGMfragment. Both the N- and C-terminal domains are neces-sary for PYGM enzyme activity [33], so it is unlikely thata protein made up of only a small part of the N-terminaldomain would be functional.

A potential therapy for McArdle disease would be theuse of aminoglycosides (or similar treatments) to read-through the premature termination codon and produce afull-length protein. We demonstrated read-through of theR50X premature termination codon in transient transfec-tants treated with G418. Read-through approaches mightbe explored further as a possible therapy for McArdledisease patients with the R50X mutant allele. Although

aminoglycosides can be effective at inducing read-throughof premature termination codons, their use in vivo is limitedby ototoxic and nephrotoxic side-effects. Recently, therehas been interest in identifying and developing novel non-aminoglycoside compounds which can read-through pre-mature termination codons [34–36] whilst having fewertoxic effects than aminoglycosides. Ataluren/PTC124 isone non-aminoglycoside compound which is currently inclinical trials in patients with Duchenne muscular dystro-phy and cystic fibrosis who have disease-causing prematuretermination codons [14,15,37].

We did not observe read-through when cells were trea-ted with gentamicin. This is consistent with the results ofSchroers et al. [38], who used gentamicin to treat fourMcArdle patients with the R50X mutant allele. They didnot observe any noticeable change in energy metabolism(which would indicate expression of functional PYGMenzyme) following the treatment. Treatment with tobramy-cin, hygromycin B, and sisomicin also failed to induceread-through in our cell model. Differences in read-throughmay be due to the aminoglycosides exhibiting sequence

Fig. 4. The G205S mutation leads to aggregation of the GFP-G205S fusion protein. CHO-K1 cells were transiently transfected with the GFP constructs.Fixed cells were counterstained with anti-PYGB/PYGM antibody (red) and DAPI (blue) as described in methods (see Section 2). Images shown aremerged from GFP (green), anti-PYGB/PYGM (red) and DAPI (blue). Co-localisation appears yellow (although the antibody also detects endogenousPYGB). GFP-G205S mutant protein accumulated in aggregates within the cytoplasm (top right) or aggregates which appeared to mislocalise to thenucleus (top left). GFP-PYGM wild-type protein and GFP protein alone had diffused cytoplasmic localisation with few aggregates. (B) Percentage oftransfected cells displaying intracellular aggregates of GFP fusion protein (mean ± SEM, n = 11). Significantly greater levels of aggregation were observedin cells expressing GFP-G205S protein compared to GFP-PYGM wild-type or GFP protein alone (p < 0.01).

Fig. 5. Aminoglycoside-induced read-through of the PYGM R50X mutation. CHO-K1 cells were transiently transfected with R50X-GFP plasmidconstructs, and treated with tobramycin (Tob), hygromycin B (Hyg), G418, gentamicin C (Gen) or sisomicin (Sis). CHO-K1 indicates untransfected CHO-K1 cells; R50X-GFP and R50R-GFP indicate cells that were not treated with aminoglycosides. Western blot was performed with anti-GFP antibody.Production of a band about 4 kDa larger than GFP indicated read-through of the R50X stop codon following incubation with G418. This band was notseen after treatment with any of the other aminoglycosides.

K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51 49

specificity in their ability to induce read-through of thesequence of the premature termination codon [39,40].

We have shown that in our cell models, mRNA contain-ing the G205S mutation was detectable and was producedat similar levels to the wild-type Pygm mRNA. This is con-sistent with previous reports of detectable full-lengthPYGM mRNA from McArdle patients with the G205Smutant allele [9,41]. McArdle patients homozygous forthe G205S mutation do not have detectable PYGM enzy-matic activity [9–11], demonstrating that G205S missensemutation prevents functioning of the PYGM enzyme, butthe mechanism has not yet been elucidated. In our cellmodels, G205S PYGM protein levels were greatly reduced

compared to wild-type controls, even though their mRNAlevels were similar, suggesting that the G205S mutant pro-tein is less stable. Transient transfection studies with GFPconstructs showed that protein levels in the mutant weregreatly decreased and that aggregation of the protein wasincreased compared with wild-type. In PYGM protein,amino acid 205 is located within the b8 strand. In the enzy-matically active tetrameric PYGM structure, the b8 and b9strands interact together in the region of the glycogen stor-age sites (reviewed in [33]). It has previously been suggestedthat the G205S mutation may disrupt these functions[8,42]. We performed three-dimensional modelling ofPYGM and found that the G205S mutation introduces a

50 K.E. Birch et al. / Neuromuscular Disorders 23 (2013) 43–51

larger side chain and a hydroxyl group into the regionbetween the b8 and b9 strands (data not shown), which islikely to destabilise the interaction between these strandsand lead to misfolding of the protein. Our data suggest thatdisruption of PYGM structure by the G205S mutationleads to aggregation and accelerated turnover, as well asloss of enzymatic activity. If prevention of misfolding andaggregation does improve PYGM enzyme activity inMcArdle patients, then compounds (such as pharmacolog-ical chaperones) that stabilise the protein could be poten-tially therapeutic for McArdle patients with the G205Smissense mutation. Research into identifying pharmaco-logical chaperones to treat other diseases is currently inprogress [43,44] and could provide insights into the devel-opment of a similar therapy for McArdle disease.

A limitation to research into McArdle disease is thatmuscle cells from McArdle patients cannot be used as a cellmodel due to the absence of PYGM protein expressionfrom wild-type cells in culture. We have demonstrated thathigh-level expression of wild-type and mutant Pygm

mRNA can be achieved by transfection of a plasmid con-taining cDNA into CHO-K1 cells. Our cell models demon-strated high levels of wild-type PYGM protein expression,and an absence or reduction of mutant protein. These cellshave enabled study of the effect of mutations upon thePYGM protein. These R50X and G205S mutant cells arefreely available for use as a model of McArdle disease,alongside wild-type clones as positive controls. The stabil-ity of the mRNA in the mutant cell lines may make themsuitable to test correction of the mutations at the mRNAlevel.

In summary, we have shown that a transfection-basedcell model can be used as a model of McArdle disease inorder to avoid problems with use of muscle cells. We dem-onstrate the use of a cell based model to investigate theexpression of mutant and wild-type PYGM protein, andto test potential therapies for McArdle disease. We provideevidence that aminoglycoside read-though of the R50Xpremature termination mutation might be used therapeuti-cally if a method could be found to stabilise PYGM

mRNA. We provide experimental results to suggest thatthe presence of the G205S missense mutation leads to mis-folding and aggregation of the PYGM protein, which maycontribute to loss of enzymatic function. This insight maylead to the development of new strategies, such as the useof pharmacological chaperones, for treating McArdlepatients with a missense mutation in the PYGM gene.

Acknowledgements

We thank the Association for Glycogen Storage Disease(UK), the Muscular Dystrophy Association (USA) and theOswestry Institute of Orthopaedics for financial supportand Dr. Brigitte Pfeiffer-Guglielmi (Tubingen) for agenerous gift of isoform-specific rabbit anti-PYGMantibody.

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