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P49 Manipulation of human abnormally fertilized pronuclear stage zygotes following vitrification
S20
Posters, UK Neuromuscular Translational Research Conference 2011 / Neuromuscular Disorders 21S1 (2011) S7–S30
the co-existence of wild-type and mutant mtDNA in the same cell. Matters are further complicated by a lack of understanding regarding how these mutant mtDNA species segregate among tissues in the developing embryo. Therefore at risk couples planning a family often pursue genetic counseling, to determine their likely hood of conceiving a child severely affected by an mtDNA disorder. Here we have examined the mutant load for two mtDNA mutations; 14709T>C and 14487T>C in both oocytes and cleavage stage embryos for two patients undergoing in vitro fertilization (IVF) procedures. Mutant load analysis performed on individual blastomeres for the 14709T>C mtDNA mutation revealed overall heteroplasmy levels which ranged from as low as 35% to as high as 45%. For the 14487T>C mutation, low levels of heteroplasmy (<10%) were detected in all individual blastomeres analysed, for a total of five separate embryos. We also examined the mtDNA copy number in individual blastomeres and found the number of mtDNA molecules to be similar between cells of the same embryo. In summary these results successfully demonstrate the even segregation of mutant mtDNA species between cells of the same embryo, for these two specific mtDNA mutations. Furthermore they reveal the existence of similar levels of heteroplasmy between embryos sampled from the same patient. This therefore provides invaluable data for use in genetic counseling for these two mtDNA mutations (14709T>C and 14487T>C). P49 Manipulation of human abnormally fertilized pronuclear stage zygotes following vitrification L. Irving, L. Craven, M. Herbert, D.M. Turnbull, Mitochondrial Research Group. Centre for Brain Ageing and Vitality, Institute for ageing and Health, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK So far there has been limited success in developing effective treatments for mtDNA disease therefore as mtDNA is transmitted maternally, nuclear transfer techniques have been suggested as a means by which to prevent transmission of mtDNA disease to future offspring. Studies conducted so far in the lab using human abnormally fertilized multipronuclear stage zygotes have demonstrated the feasibility of such techniques for preventing transmission (Craven et al., 2010). However it has previously been shown that abnormally fertilised embryos have a limited potential for development. Therefore we propose to create normally fertilized (2PN) embryos by activating donated metaphase II stage oocytes, received by donors to determine the potential of the pronuclear transfer in normally fertilized embryos. However it is possible that we will be unable to synchronize each of the donors cycles therefore we require a means by which to cryopreserve these oocytes following activation. Here we sought to determine the feasibility of the novel cryopreservation technique known as vitrification as a means by which to store human pronuclear stage zygotes. So far we have currently vitrified a number (n = 72) of abnormally fertilised multipronuclear PN stage embryos, using the McGill cryoleaf. Here post-thaw survival rates were determined and embryo morphology assessed using a pre-determined criteria. Embryos were then either cultured to assess developmental potential following vitrification or considered for manipulation procedures. Those embryos which we did attempt to manipulate went onto to develop to cleavage stage embryos, demonstrating the feasibility of vitrification as a means by which to successfully store embryos.
P50 Exercise training in patients with mitochondrial myopathy: the analysis of COX-intermediate fibres J.L. Murphy1 , E. Shang1 , T. Taivassalo2 , R.G. Haller3 , R.W. Taylor1 , D.M. Turnbull1 . 1 Mitochondrial Research Group, Newcastle University, UK; 2 Department of Kinesiology, McGill University, Montreal, Canada; 3 The Institute for Exercise and Environmental Medicine, Dallas, USA Mitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA). There has been significant progress in the management and diagnosis of patients, however there still remains a need for effective treatments. One proposed treatment is resistance exercise training which has been shown to induce physiological improvements in muscle function and lead to changes in mtDNA mutation load (Murphy et al. Brain 2008; 131: 2832–2840). During exercise training studies of patients with mitochondrial myopathy it was found that the analysis of patient biopsies during this study was complicated by the presence of what we have called “intermediate fibres”, which proved difficult to categorise in terms of their mitochondrial enzyme activity since they seem to have partial histochemical cytochrome c oxidase (COX) activity. Given these fibres appear neither fully COX-deficient or COX-positive, it is important to class these intermediate fibres separately rather than including them within the generic description of fibres exhibiting COX deficiency, because variations in the levels of these COXintermediate muscle fibres could indicate subtle and early stages in a response of the muscle biochemical phenotype to exercise. We have therefore further characterised these fibres by performing densitometric assessment of COX activity within each fibre, and correlating this to mtDNA mutation load. Our data highlight the limitations of the sequentialCOX/SDH reaction when applying downstream molecular testing including real time PCR protocols. P51 Diabetes is a risk factor for hypertension in adults with the m.3243A>G mitochondrial DNA mutation V. Nesbitt‡ 1 , M.G.D. Bates‡ 1 , D.M. Turnbull1,2 , R. McFarland1,2 . 1 Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE2 4HH, 2 Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, NE7 7DN, UK Background: Diabetes mellitus is a frequent clinical manifestation of mitochondrial disease due to the m.3243A>G mutation.1 The prevalence of hypertension in patients with diabetes mellitus is approximately 1.5–2.0 times greater than in matched nondiabetic individuals in the general population.2 Elevated body mass index (BMI) is associated with an increased risk of developing hypertension, yet the prevalence of these diagnoses in patients with mitochondrial disease is unknown. Aims: To determine the frequency of hypertension and abnormal body mass index in adult patients with mitochondrial disease due to the m.3243A>G mutation. Methods: Patients were identified from the Medical Research Council Centre for Neuromuscular Diseases Mitochondrial Diseases Patient Cohort Study UK. BMI and non-invasive blood pressure (NIBP) were recorded in all patients attending out-patients clinic between January 2009 and December 2010. Hypertension was defined a persistent systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg. BMI was calculated as weight (kg) / [height (m)]2 . Results: Complete data on 29 patients was available (13 male, 16 female). Hypertension was present in 5 unrelated patients: BMI < 20 = 1 (female), BMI 20–25 = 3 (2 male, 1 female), BMI > 25 = 1 (female). 8 patients had diabetes or impaired glucose tolerance; 4 of these had co-existent hypertension (BMI < 20 = 1, BMI 20–25 = 2, BMI > 25 = 1). Conclusion: Hypertension is no more common in patients with the m.3243A>G mutation than the general population, and does not
Posters, UK Neuromuscular Translational Research Conference 2011 / Neuromuscular Disorders 21S1 (2011) S7–S30
the co-existence of wild-type and mutant mtDNA in the same cell. Matters are further complicated by a lack of understanding regarding how these mutant mtDNA species segregate among tissues in the developing embryo. Therefore at risk couples planning a family often pursue genetic counseling, to determine their likely hood of conceiving a child severely affected by an mtDNA disorder. Here we have examined the mutant load for two mtDNA mutations; 14709T>C and 14487T>C in both oocytes and cleavage stage embryos for two patients undergoing in vitro fertilization (IVF) procedures. Mutant load analysis performed on individual blastomeres for the 14709T>C mtDNA mutation revealed overall heteroplasmy levels which ranged from as low as 35% to as high as 45%. For the 14487T>C mutation, low levels of heteroplasmy (<10%) were detected in all individual blastomeres analysed, for a total of five separate embryos. We also examined the mtDNA copy number in individual blastomeres and found the number of mtDNA molecules to be similar between cells of the same embryo. In summary these results successfully demonstrate the even segregation of mutant mtDNA species between cells of the same embryo, for these two specific mtDNA mutations. Furthermore they reveal the existence of similar levels of heteroplasmy between embryos sampled from the same patient. This therefore provides invaluable data for use in genetic counseling for these two mtDNA mutations (14709T>C and 14487T>C). P49 Manipulation of human abnormally fertilized pronuclear stage zygotes following vitrification L. Irving, L. Craven, M. Herbert, D.M. Turnbull, Mitochondrial Research Group. Centre for Brain Ageing and Vitality, Institute for ageing and Health, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK So far there has been limited success in developing effective treatments for mtDNA disease therefore as mtDNA is transmitted maternally, nuclear transfer techniques have been suggested as a means by which to prevent transmission of mtDNA disease to future offspring. Studies conducted so far in the lab using human abnormally fertilized multipronuclear stage zygotes have demonstrated the feasibility of such techniques for preventing transmission (Craven et al., 2010). However it has previously been shown that abnormally fertilised embryos have a limited potential for development. Therefore we propose to create normally fertilized (2PN) embryos by activating donated metaphase II stage oocytes, received by donors to determine the potential of the pronuclear transfer in normally fertilized embryos. However it is possible that we will be unable to synchronize each of the donors cycles therefore we require a means by which to cryopreserve these oocytes following activation. Here we sought to determine the feasibility of the novel cryopreservation technique known as vitrification as a means by which to store human pronuclear stage zygotes. So far we have currently vitrified a number (n = 72) of abnormally fertilised multipronuclear PN stage embryos, using the McGill cryoleaf. Here post-thaw survival rates were determined and embryo morphology assessed using a pre-determined criteria. Embryos were then either cultured to assess developmental potential following vitrification or considered for manipulation procedures. Those embryos which we did attempt to manipulate went onto to develop to cleavage stage embryos, demonstrating the feasibility of vitrification as a means by which to successfully store embryos.
P50 Exercise training in patients with mitochondrial myopathy: the analysis of COX-intermediate fibres J.L. Murphy1 , E. Shang1 , T. Taivassalo2 , R.G. Haller3 , R.W. Taylor1 , D.M. Turnbull1 . 1 Mitochondrial Research Group, Newcastle University, UK; 2 Department of Kinesiology, McGill University, Montreal, Canada; 3 The Institute for Exercise and Environmental Medicine, Dallas, USA Mitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA). There has been significant progress in the management and diagnosis of patients, however there still remains a need for effective treatments. One proposed treatment is resistance exercise training which has been shown to induce physiological improvements in muscle function and lead to changes in mtDNA mutation load (Murphy et al. Brain 2008; 131: 2832–2840). During exercise training studies of patients with mitochondrial myopathy it was found that the analysis of patient biopsies during this study was complicated by the presence of what we have called “intermediate fibres”, which proved difficult to categorise in terms of their mitochondrial enzyme activity since they seem to have partial histochemical cytochrome c oxidase (COX) activity. Given these fibres appear neither fully COX-deficient or COX-positive, it is important to class these intermediate fibres separately rather than including them within the generic description of fibres exhibiting COX deficiency, because variations in the levels of these COXintermediate muscle fibres could indicate subtle and early stages in a response of the muscle biochemical phenotype to exercise. We have therefore further characterised these fibres by performing densitometric assessment of COX activity within each fibre, and correlating this to mtDNA mutation load. Our data highlight the limitations of the sequentialCOX/SDH reaction when applying downstream molecular testing including real time PCR protocols. P51 Diabetes is a risk factor for hypertension in adults with the m.3243A>G mitochondrial DNA mutation V. Nesbitt‡ 1 , M.G.D. Bates‡ 1 , D.M. Turnbull1,2 , R. McFarland1,2 . 1 Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE2 4HH, 2 Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, NE7 7DN, UK Background: Diabetes mellitus is a frequent clinical manifestation of mitochondrial disease due to the m.3243A>G mutation.1 The prevalence of hypertension in patients with diabetes mellitus is approximately 1.5–2.0 times greater than in matched nondiabetic individuals in the general population.2 Elevated body mass index (BMI) is associated with an increased risk of developing hypertension, yet the prevalence of these diagnoses in patients with mitochondrial disease is unknown. Aims: To determine the frequency of hypertension and abnormal body mass index in adult patients with mitochondrial disease due to the m.3243A>G mutation. Methods: Patients were identified from the Medical Research Council Centre for Neuromuscular Diseases Mitochondrial Diseases Patient Cohort Study UK. BMI and non-invasive blood pressure (NIBP) were recorded in all patients attending out-patients clinic between January 2009 and December 2010. Hypertension was defined a persistent systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg. BMI was calculated as weight (kg) / [height (m)]2 . Results: Complete data on 29 patients was available (13 male, 16 female). Hypertension was present in 5 unrelated patients: BMI < 20 = 1 (female), BMI 20–25 = 3 (2 male, 1 female), BMI > 25 = 1 (female). 8 patients had diabetes or impaired glucose tolerance; 4 of these had co-existent hypertension (BMI < 20 = 1, BMI 20–25 = 2, BMI > 25 = 1). Conclusion: Hypertension is no more common in patients with the m.3243A>G mutation than the general population, and does not