G.P.3.14 Comparative human mitochondrial genome analysis using the affymetrix Mitochip v2 and conventional cycle sequencing

Abstracts / Neuromuscular Disorders 18 (2008) 724–833 Neurology, New York, United States; 3 Kaohsiung Medical University, Graduate Institute of Medicine, Kaohsiung, Taiwan Multiple acyl-CoA dehydrogenase deficiency (MADD) is a clinically heterogeneous disorder mainly caused by mutations in the genes for electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETF-QO). Mutations in ETFDH, which encodes for ETF-QO has been associated with myopathic form of CoQ10 deficiency, riboflavin-responsive MADD, and riboflavin-nonresponsive MADD, although the underlying mechanism of these different phenotypes is still unclear. We identified three novel mutations in ETFDH in four Taiwanese riboflavin-responsive MADD patients from three unrelated families. All patients harbored p.A84T. Patient 1 and 2, who are siblings, had compound heterozygosity for p.A84T and p.R175L. Patient 3 had homozygosity for p.A84T and patient 4 had compound heterozygosity for p.A84T and p.L127H. Our mutation analysis suggests that p.A84T may be a common cause for MADD in Taiwan. CoQ10 level in skeletal muscle was normal in two, indicating that not all MADD patients have CoQ10 deficiency, even though CoQ10 supplementation was reported to be probably effective. Accordingly, except for riboflavin, CoQ10 should still be considered as a choice of treatment depending on its level. Since MADD is often a treatable disease, genetic confirmation of the disease will be beneficial for patients. However, more studies are necessary to establish the genotype-phenotype correlation in terms of therapeutic effectiveness. doi:10.1016/j.nmd.2008.06.107

G.P.3.12 Riboflavin-Responsive multiple Acyl-CoA dehydrogenation deficiency (MADD-RR): Clinical, biochemical, molecular genetic and 31 P-MRS studies A. Toscano 1; I. Nishino 2; B. Garavaglia 3; O. Musumeci 1; R. Lodi 4; B. Barbiroli 4; W.C. Liang 5; G. Vita 1 1 University of Messina, Department of Neurosciences Psychiatry and Anesthesiology, Messina, Italy; 2 National Institute of Neuroscience, Department of Neuromuscular Research, Kodaira, Japan; 3 Istituto Neurologico C. Besta, Milan, Italy; 4 University of Bologna, Bologna, Italy; 5 National Institute of Neuroscience, Kodaira, Japan Multiple Acyl-CoA dehydrogenation deficiency (MADD) is a rare autosomal recessive disorder of the electron transfer flavoprotein or ubiquinone oxidoreductase, resulting in abnormal fatty acid, amino acid and choline metabolism.Three clinical forms have been described: A neonatal-onset form with congenital anomalies (type I), a neonatal-onset form without congenital anomalies (type II) and a late-onset form (type III). Many patients with MADD have been reported with mutations in ETFA, ETFB or ETFDH genes. Some of these patients are responsive to a riboflavin treatment: in these cases underlying disorders of mitochondrial FAD transport or metabolism have been proposed. Recently, it has been demonstrated that MADD Riboflavin-Responsive (MADDRR) is associated with defects in the ETFDH gene. Herein, we report two unrelated patients (44 yrs and 14 yrs at onset respectively), with MADD-RR: both patients presented with myalgias, exercise intolerance and proximal muscle weakness. Serum CK was elevated (8000 IU/L/ 5000 IU/L) as well lactacidemia before and after cycloergometer exercise. Muscle biopsy showed a florid vacuolar myopathy with lipid storage, especially in type I fibers. Total and free carnitine were low in muscle. The activities of SCAD, MCAD and LCAD in isolated muscle mitochondria were greatly reduced. In vivo 31 P-MRS evidenced a reduced brain phosphorylation potential with a more marked deficiency of skeletal muscle mitochondrial respiration. After 2 months of riboflavin treatment (200 mg/die) clinical, morphological, biochemical and spectroscopic investigations revealed a marked improvement of their clinical condition. At 10 years follow-up, they are still on riboflavin

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and their improvement is stable. Recently, a molecular genetic analysis of ETFDH gene identified three novel mutations: one homozygous mutation c1531 G > A (D511N) in exon 12 in the first patient whereas a compound heterozygosity c. 1366 (p.P456S) in exon 11 and c. 1828 G > A (p.G610R) in exon 13 was detected in the younger patient. In these cases, clinical and molecular results confirm the presence of ETFDH mutations as major causes of MADD-RR. doi:10.1016/j.nmd.2008.06.108

G.P.3.13 Supplementation studies in primary human muscle cells with mtDNA depletion caused by mutations in the DGUOK and POLG1 genes S. Bulst 1; A. Abicht 1; C. Thirion 1; H. Lochmu¨ller 2; R. Horvath 3 1 Ludwig-Maximilians-University, Friedrich-Baur-Institute, Munich, Germany; 2 University of Newcastle upon Tyne, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom; 3 University of Newcastle upon Tyne, Mitochondrial Research Group, Newcastle upon Tyne, United Kingdom Mitochondrial DNA depletion syndrome (MDS), a frequent cause of severe childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA in different tissues which leads to insufficient synthesis of respiratory chain complexes. Mutations of eight nuclear genes (DGUOK, POLG1, MVP17. ECGF1, TK2, SUCLA2, SUCLG1 and RRM2B), all involved in the synthesis or maintenance of mitochondrial nucleotide pools, were identified. It was previously shown that mtDNA depletion can be prevented by dGMP and dAMP supplementation in culture of deoxyguanosine kinase-deficient fibroblasts. Since then, similar experiments were not published. We performed experiments on human primary myoblasts of patients carrying pathogenic mutations in DGUOK and POLG1. After supplementation with dGMP and dAMP alone and in combination, mtDNA copy number and biochemical analysis of the cytochrome c oxidase (COX) were tested. Serum deprivation and myotube formation triggered a decrease in the mtDNA copy number in primary myotubes of patients carrying pathogenic mutations in DGUOK or POLG1. MtDNA copy number decreased significantly in myotubes of patients with both gene defects but not in controls. Supplementation of the cell culture medium with dGMP and dAMP alone and in combinations rescued the mtDNA depletion in DGUOK deficient cells, but not in myotubes carrying compound heterozygous mutations in POLG1. The effect of the administration of dGMP and dAMP were significant and reproducable. Our results show that supplementation with dGMP/dAMP can increase mtDNA copy number in DGUOK deficient myotubes. The lack of an improvement of mtDNA depletion in POLG1 deficiency reflects a heterogeneous pathomechanism of mtDNA depletion. No adverse effect was observed on high dose supplementation. Further studies are needed to decide about the possible therapeutic implications of dAMP/dGMP supplementation in DGUOK deficiency. doi:10.1016/j.nmd.2008.06.109

G.P.3.14 Comparative human mitochondrial genome analysis using the affymetrix Mitochip v2 and conventional cycle sequencing A.J. Duncan 1; M.G. Sweeney 2; E. Stern 1; R. Taylor 3; C. Woodward 2; M.B. Davis 2; M.G. Hanna 2; S. Rahman 2 1 UCL Institute of Child Health,Mitochondrial Research Group, London, United Kingdom; 2 National Hospital for Neurology and Neurosurgery, Centre for Neuromuscular Disease, London, United Kingdom; 3 Newcastle University, Mitochondrial Research Group, Newcastle, United Kingdom

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Abstracts / Neuromuscular Disorders 18 (2008) 724–833

Introduction: Human mitochondrial DNA (mtDNA) is a circular molecule of 16,569 base pairs encoding 37 genes. Previously we have sequenced the entire mitochondrial genome using 45 overlapping PCR fragments, a time-consuming and costly procedure. Recently affymetrix have updated their mitochondrial resequencing chip. The Mitochip v2 is an array based sequencing platform that offers the potential of rapid and high throughput analysis of mtDNA. Here, we sought to compare the effectiveness and accuracy of MitoChip v2 compared to cycle sequencing, by paired analysis of the mtDNA from 40 patients with suspected mtDNA disease. Methods: A cohort of 40 patients with clinical, histological or biochemical evidence of mitochondrial disease, but without a genetic diagnosis, was identified through referral to the National Commissioning Group Mitochondrial Genetics laboratories in London and Newcastle. Sequence analysis was undertaken by both methods described above, and the resulting data sets compared to determine the effectiveness of the Mitochip v2 in the identification of pathogenic mutations, and the frequency of false positives and false negatives. In addition, we aimed to identify any regions that were consistently mis-called by the Affymetrix software. Results and discussion: To date, 20 patients have been analysed using the Mitochip v2. Six known pathogenic mutations and four novel and potentially pathogenic mutations (2 probably pathogenic, 2 of uncertain status) have been identified. All changes were confirmed by cycle sequencing. 13 of these 20 Mitochips generated between one and three false positives; 19 of the 20 Mitochips contained up to four false negatives. This preliminary data suggests that the Mitochip v2 is a potentially useful tool in the identification of mtDNA mutations, but further analysis is needed to determine whether all types of mutation are detected by this method. doi:10.1016/j.nmd.2008.06.110

G.P.3.15 Waking the sleeping giant; habitual physical inactivity in people with mitochondrial disease M.I. Trenell; S. Apabhai; D.M. Turnbull Newcastle University, Mitochondrial Research Group, Newcastle upon Tyne, United Kingdom Aims/hypothesis: Physical inactivity reduces mitochondrial density and compromises mitochondrial function. It is not known whether the exercise intolerance characteristic of mitochondrial disease impacts daily habitual physical activity and thus exacerbates any genetic mitochondrial deficiency. The aim of this ongoing study is to characterise habitual physical activity in people with known mitochondrial disease and relate these to normative values and mitochondrial disease rating scales. Methods: 40 people with mitochondrial disease were assessed. Habitual physical activity was assessed objectively using a validated multi-sensor array over 3 days and using the international physical activity questionnaire. Clinical presentations were assessed using the neuromuscular disease rating scale. Mitochondrial disease was identified from biopsy. Results: 85% of the sample failed to achieve the recommended daily physical activity target of 10,000 steps. Physical activity (steps per day) was negatively associated with disease severity, assessed by NMDAS (P < 0.05). Daily energy expenditure (calories) was negatively correlated with disease severity, assessed by NMDAS (P < 0.05). Subjective and objective reports of physical activity were not correlated (R2 = 0.2, P > 0.05). Conclusions/interpretation: Physical inactivity is prominent in people with mitochondrial disease and relates to disease severity. Subjective reports of physical activity are ineffective. Greater physical inactivity may hold a significant, and potentially manageable, negative influence upon mitochondrial function in people with mitochondrial disease. doi:10.1016/j.nmd.2008.06.112

ANTISENSE AND CELLULAR APPROACHES TO THERAPY DEVELOPMENT; POSTER PRESENTATIONS T.P.2.01 Antisense oligomer design: Targeting and assay systems S.D. Wilton; C. Mitrpant; P.L. Meloni; A.M. Adams; S. Fletcher University of Western Australia, Centre for Neuromuscular & Neurological Disorders, Perth, Australia Antisense oligonucleotides (AOs) can disrupt exon recognition and splicing during intron removal from the mature mRNA. We, and others, have found that approximately two out of three AOs can induce some exon skipping. However, the efficiency of exon removal varies greatly within and between exons. Some AOs may induce weak exon removal only after transfection at high concentrations, while other more optimised compounds induce substantial exon skipping at much lower concentrations. An empirical approach to AO design is to evaluate a ”first pass” panel of AOs to identify the most promising target in cultured human cells, and then design a series of overlapping AOs for further evaluation. Oligomer length and target annealing site are critical parameters in AO design. Despite strong homology between the mouse and human dystrophin genes, and the observation that a normal dystrophin gene is processed correctly in transgenic mice, we have identified several examples where targeting the same AO annealing sites leads to different exon skipping outcomes. Targeting the exon 23 donor splice site leads to efficient exon skipping in the mouse, while the human equivalent site was totally unresponsive. An oligomer designed to excise exon 16 from the normal dystrophin pre-mRNA was found to be more than an order of magnitude more efficient when applied to a dystrophic cell line with an exon 16 splice motif mutation. When such variation in AO efficacy is observed in cell lines expressing different dystrophin transcripts, the validity of using artificial systems, such as a plasmid containing a single dystrophin exon in the context of limited flanking sequence in a reporter gene, for oligomer design must be questioned. We propose that the most valid system in which to evaluate clinically relevant AOs is one in which dystrophin mRNA is processed in the presence of all normal cis and trans splicing elements. doi:10.1016/j.nmd.2008.06.113

T.P.2.02 Development of antisense oligonucleotides for dystrophin exon skipping based on target site accessibility predicted by dynamic co-transcriptional pre-mRNA secondary structure analysis Z.A.D. Pramono 1; D.K.B. Wee 2; X. Qianbin 1; P.S. Lai 3; W.C. Yee 1 1 National Neuroscience Institute (New Address: Div. of Research, Singapore General Hospital), Singapore, Singapore; 2 Bioinformatics Institute, A*STAR, Singapore, Singapore; 3 Yong Loo Lin School of Medicine, National University of Singapore, Dept. of Paediatrics, Singapore, Singapore Antisense oligonucleotide (AON) mediated exon skipping has considerable potential for therapy of Duchenne muscular dystrophy (DMD) and is currently undergoing clinical trials in DMD. In an in-silico study of published AONs, we recently confirmed that the efficacy and efficiency of AONs correlate with AON target site accessibility as determined by analysis of dynamic co-transcriptional pre-mRNA secondary structures (Wee et al, 2008). Using target site accessibility as predicted by our analysis together with exon splicing enhancing (ESE) sites determined by ESE predicting software, we designed a panel of AONs to induce skipping of nine individual dystrophin gene exons. The AONs were designed to skip exons to correct the reading frame for a majority of DMD mutations, including exons previously reported as ”unskippable” or difficult to skip. Testing of these AONS in human fibroblast cells confirmed that these AONs were efficacious in inducing specific exon skipping, even at