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Optimizing patient care and outcome measures: which complaints are most burdensome to patients and their parents?
Mitochondrion 12 (2012) 550–591
Contents lists available at SciVerse ScienceDirect
Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
Mitochondrial Medicine 2012: Capitol Hill Abstract 1 Mutagenesis of mouse mitochondrial DNA Presenter: Mikhail Alexeyev Mikhail Alexeyev, Rafik Fayzulin University of South Alabama, Dr. North, Mobile, AL 5851, USA Mitochondrial diseases can be caused by mutations in both nuclear and mitochondrial DNA (nDNA and mtDNA, respectively) and have estimated prevalence of at least 1:5000. While many anecdotal reports describe positive prognoses for an occasional patient or patients treated with various vitamins, cofactors or reagents, there is no cure or reliable treatment for these often fatal disorders. Animal (mouse) models of human disease are instrumental in developing and testing new therapeutic modalities. Although the development of mouse models of human mitochondrial diseases caused by mutations in nuclear DNA is relatively straightforward, the routine development of animal models for the diseases caused by mtDNA mutations is not yet feasible. Recently, several groups demonstrated their ability to transfer mutant mtDNA from cultured cells into mice thus creating transmitochondrial mice. However, routine modeling of mitochondrial disorders caused by mtDNA mutations remains limited by the unavailability of a reliable supply of mouse cell lines bearing mutations in mtDNA. To date, some mutations in mouse mtDNA were isolated employing selection for resistance to various inhibitors, or by chemical mutagenesis. These strategies are inherently biased (e.g., by the spectrum of mtDNA mutations that can be induced by a given mutagen), and do not allow for the isolation of the full spectrum of mtDNA mutations. We developed a mtDNA mutagenesis strategy that utilizes proofreadingdeficient mutant of the mitochondrial DNA-polymerase gamma (mPolGexo−). Mouse cells are engineered for inducible (Tet-On) expression of the floxed mPolGexo−. These cells are induced with doxycycline, and mutagenesis is terminated at about one mutation per mtDNA by excising mPolGexo− with Cre recombinase. Following excision, cells are subjected to depletion to a level of about one mtDNA molecule per cell followed by repletion to normal copy number and cloning. Resulting clones are expanded and their mtDNA is sequenced to determine the nature of the mutation. The distribution of mutations obtained using this technique is fairly unbiased, which enables systematic mutagenesis and generation of banks of mtDNA mutations, which may be useful for both modeling of the human mitochondrial disease in mice and for the functional analysis of the Electron Transfer Chain. Using this approach, we isolated more than 40 homoplasmic mouse cell lines with single homoplasmic nonsynonymous substitutions in mtDNA. This exceeds 1567-7249/$ – see front matter.
the combined number of similar cell lines generated to date using all other approaches.
doi:10.1016/j.mito.2012.07.002
3 Optimizing patient care and outcome measures: which complaints are most burdensome to patients and their parents? Presenter: Saskia Koene Saskia Koene, Saskia Wortmann, Eva Morava, Maaike de Vries, Jan Smeitink Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6500 HB, PO BOX 9101, Nijmegen, The Netherlands To optimize patient care and to target supportive care efficiently, it is important to know what disabilities patients experience in daily life. The obtained information will also be useful in identifying outcome measure domains for future clinical trials. We investigated which complaints and symptoms in pediatric mitochondrial disease patients and their families are present and which of these complaints are most disabling. A questionnaire, designed to assess which symptoms are most burdensome to patients and their parents, was sent to all known Dutch-speaking patients with a mitochondrial disorder. Inclusion criteria: one or more enzyme complex deficiencies, a decreased ATP and PCr production in fresh muscle tissue, and/or a confirmed pathogenic mtDNA mutation. The questionnaire contained three main questions. The main questions were: i) Which complaints or symptoms are present?; ii) Which symptoms are most burdensome and wanted to improve most, indicated by the child or expected by the parent(s); iii) Which three symptoms would the parents like to change? The questionnaire response rate was 56% (83 out of 149). Eighty-nine percent (74 out of 83) of the parents and their children filled main questions ii) and iii). Thirty-two percent would (think that the child would) like to change the lack of energy, followed by tiredness (31%), and reduced muscle power (22%). Symptoms the parents would like to change include tiredness (25%), lack of energy (23%), and behavioural problems (23%). Of the 54 children with tiredness, 43% of the children and 39% of their parents rate this symptom as one of the three problems they
Abstracts
would like to change most. In the 55 patients with lack of energy, these percentages are 42 and 35%, respectively. In the 53 patients with developmental delay, 32% of the parents rate this as one of the most important problems. Of the 39 patients with speech difficulties, 36% of the children and 30% of the parents rate this symptom in the top three. For the 27 children with epilepsy, these percentages are 37 and 41% respectively. For the 26 children with behavioural problems, 66% of the parents rate this symptom as a major point they would like to change. In conclusion, behavioural, speech and language problems are more cumbersome to parents and children than we expected. We advise to take these aspects into account more frequently in supportive care management. doi:10.1016/j.mito.2012.07.003
4 MELAS and l-arginine therapy —therapeutic timing and long term effects Presenter: Yasutoshi Koga Yasutoshi Kogaa, Toshi Abeb, Takayuki Taniwakic, Povalko Nataliyaa a Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan b Department of Radiology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan c Department of Neurology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan Investigator-initiated clinical trial of l-arginine to cure the symptoms of acute phase stroke-like episodes (SLE) and to prevent SLE in the interictal phase of MELAS finished on June 30, 2010. We are now cleaning the date and are processing the date for approval. However we still do not know what is the best therapeutic timing of IV on SLE, and whether it can prevent the disease progression of MELAS during long term administration. We used l-arginine infusion at hyper-acute phase of SLE in 2 MELAS patients and evaluated the therapeutic effects by clinical and the serial neuroimaging analysis. Patient 1 is a 6-year-old girl who was diagnosed with MELAS/Leigh overlap syndrome based on a finding of clinical symptoms and 78% mutation in an A3243G in the mitochondrial tRNALeu(UUR) gene. Patient 2 is a 32-year-old girl who fulfilled the diagnostic criteria of MELAS and has a 55% mutation of A3243G in muscles. She has a bilateral sensorineural hearing loss and diabetes mellitus at the age of 31. She has two histories of stroke-like episodes and was then followed as MELAS. Within 2 h after the onset of SLE, we infused 0.5 g/kg/dose of l-arginine within 1 h after the onset and took the brain MRI seriously. All of the clinical symptoms disappeared within 30 min after l-arginine infusion without using anti-convulsants. The series of MRIs were performed first, seven days and one month from onset of stroke-like episodes. MRI obtained at 24 h after the onset showed high intensity signal in T2WI, and DWI and low in apparent diffusion coefficient (ADC), however those abnormal findings were completely normalized on MRIs taken at 7 days and one month later. We also administered l-arginine on MELAS for more than 5 years and evaluated its long term effects by duration of hospitalization and by Japanese Mitochondrial Disease Rating Scale (JMDRS) compared with the MELAS cohort study in Japan without using l-arginine. l-Arginine therapy significantly decreases the JMDRS and prevents the disease progression as compared with the cohort study. Our data indicated that l-arginine infusion at hyper-acute phase shows promise in the cure of strokelike episodes seen in MELAS, and the long term l-arginine therapy prevents the disease progression of MELAS. doi:10.1016/j.mito.2012.07.004
551
5 A novel sequence variant of tRNAThr in mtDNA, transferred by bone marrow transplantation in patient with ALL Presenter: Nataliya Povalko Nataliya Povalkoa, Yoshihisa Nagatoshib, Hiroko Inadaa, Shuichi Yatsugaa, Yasutoshi Kogaa a Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan b Section of Pediatrics, National Kyushu Cancer Center, 3-1-1 Notame, Minamiku, Fukuoka 811-1395, Japan Bone marrow transplantation (BMT) is one of the possible choices for the therapy of mitochondrial disorders such as MINGIE. However current donor matching system is only focused on the HLA typing based on the major histocompatibility antigens to avoid the graftversus-host disease (GVHD). We had a 5-year old patient with ALL (acute lymphoblastic leukemia) who showed recurrent stroke-like episodes before and after the BMT. We found the novel mtDNA sequence variant which has not been found in a patient mtDNA before BMT. Analysis of the entire sequence of mtDNA was performed twice (before and after BMT). First analysis was done after the first stroke-like episode. Patient under the chemotherapy suddenly showed severe headache, and a left tonic seizure with right hemiplegia. MRI of the brain showed high intensity areas on T2 and FLAIR in occipital, frontal, and parietal lobes. The parameters of endothelial function, including plasma level of l-arginine, and FMD (flow mediated dilatation), were abnormal. No pathogenic mtDNA mutations were found at his original mitochondrial DNA. The second entire sequencing of mtDNA was performed when he had the second episode of stroke 5 months after BMT. He showed cortical blindness, vomiting and headache. Since the parameters of endothelial function were abnormal, infusion of l-arginine was immediately started. No abnormalities were found on MRI taken after the stroke-like episode. We found a novel A15929G in the mitochondrial tRNAThr gene. The heteroplasmy of this mutation was 90% in white blood cell, and 8% in nail, however this mutation was not detected in urine, and recipient's bone marrow before BMT, WBCs from mother, brother and sister of the proband, suggesting that this mutation was transferred by BMT. According to the haplogroup analysis of mtDNA, original mtDNA belonged to haplogroup D4a (superhaplogroup M), on the other hand, donor's mtDNA belonged to haplogroup F1 (superhaplogroup N). Since mtDNA has a very high mutation rate and has at least 30 polymorphic sites, minor antigen mismatching caused by the amino acid sequence polymorphism may occur leading to the GVHD. On the other hand, many oligo-symptomatic or asymptomatic carriers having a pathogenic mtDNA mutation may become a potential donor for BMT and thus transmit the mutation to the recipient. Current donor matching system may have a risk of transmission of pathogenic mtDNA mutation and may contribute the GVHD by the proteins which are generated by the transmitted mtDNA.
doi:10.1016/j.mito.2012.07.005
6 The comparative study of actin and myosin genes in Molgula manhattensis, Styela clava, and Limulus polyphemus: Implication on mitochondrial DNA maintenance Presenter: Meghan Buckley Caitlin A. Baker, Meghan F. Buckley, Taylor L. DeRosa, Jenna A. Hernandez, Erin L. Hillis, Caroline R. Luciani, Rita Matta, Alexandra R. Novak, Christine C. Smith, Qingyu Xu, Mary Jane Paolella Sacred Heart Academy, 265 Benham St. Hamden, CT 06514, United States
Contents lists available at SciVerse ScienceDirect
Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
Mitochondrial Medicine 2012: Capitol Hill Abstract 1 Mutagenesis of mouse mitochondrial DNA Presenter: Mikhail Alexeyev Mikhail Alexeyev, Rafik Fayzulin University of South Alabama, Dr. North, Mobile, AL 5851, USA Mitochondrial diseases can be caused by mutations in both nuclear and mitochondrial DNA (nDNA and mtDNA, respectively) and have estimated prevalence of at least 1:5000. While many anecdotal reports describe positive prognoses for an occasional patient or patients treated with various vitamins, cofactors or reagents, there is no cure or reliable treatment for these often fatal disorders. Animal (mouse) models of human disease are instrumental in developing and testing new therapeutic modalities. Although the development of mouse models of human mitochondrial diseases caused by mutations in nuclear DNA is relatively straightforward, the routine development of animal models for the diseases caused by mtDNA mutations is not yet feasible. Recently, several groups demonstrated their ability to transfer mutant mtDNA from cultured cells into mice thus creating transmitochondrial mice. However, routine modeling of mitochondrial disorders caused by mtDNA mutations remains limited by the unavailability of a reliable supply of mouse cell lines bearing mutations in mtDNA. To date, some mutations in mouse mtDNA were isolated employing selection for resistance to various inhibitors, or by chemical mutagenesis. These strategies are inherently biased (e.g., by the spectrum of mtDNA mutations that can be induced by a given mutagen), and do not allow for the isolation of the full spectrum of mtDNA mutations. We developed a mtDNA mutagenesis strategy that utilizes proofreadingdeficient mutant of the mitochondrial DNA-polymerase gamma (mPolGexo−). Mouse cells are engineered for inducible (Tet-On) expression of the floxed mPolGexo−. These cells are induced with doxycycline, and mutagenesis is terminated at about one mutation per mtDNA by excising mPolGexo− with Cre recombinase. Following excision, cells are subjected to depletion to a level of about one mtDNA molecule per cell followed by repletion to normal copy number and cloning. Resulting clones are expanded and their mtDNA is sequenced to determine the nature of the mutation. The distribution of mutations obtained using this technique is fairly unbiased, which enables systematic mutagenesis and generation of banks of mtDNA mutations, which may be useful for both modeling of the human mitochondrial disease in mice and for the functional analysis of the Electron Transfer Chain. Using this approach, we isolated more than 40 homoplasmic mouse cell lines with single homoplasmic nonsynonymous substitutions in mtDNA. This exceeds 1567-7249/$ – see front matter.
the combined number of similar cell lines generated to date using all other approaches.
doi:10.1016/j.mito.2012.07.002
3 Optimizing patient care and outcome measures: which complaints are most burdensome to patients and their parents? Presenter: Saskia Koene Saskia Koene, Saskia Wortmann, Eva Morava, Maaike de Vries, Jan Smeitink Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6500 HB, PO BOX 9101, Nijmegen, The Netherlands To optimize patient care and to target supportive care efficiently, it is important to know what disabilities patients experience in daily life. The obtained information will also be useful in identifying outcome measure domains for future clinical trials. We investigated which complaints and symptoms in pediatric mitochondrial disease patients and their families are present and which of these complaints are most disabling. A questionnaire, designed to assess which symptoms are most burdensome to patients and their parents, was sent to all known Dutch-speaking patients with a mitochondrial disorder. Inclusion criteria: one or more enzyme complex deficiencies, a decreased ATP and PCr production in fresh muscle tissue, and/or a confirmed pathogenic mtDNA mutation. The questionnaire contained three main questions. The main questions were: i) Which complaints or symptoms are present?; ii) Which symptoms are most burdensome and wanted to improve most, indicated by the child or expected by the parent(s); iii) Which three symptoms would the parents like to change? The questionnaire response rate was 56% (83 out of 149). Eighty-nine percent (74 out of 83) of the parents and their children filled main questions ii) and iii). Thirty-two percent would (think that the child would) like to change the lack of energy, followed by tiredness (31%), and reduced muscle power (22%). Symptoms the parents would like to change include tiredness (25%), lack of energy (23%), and behavioural problems (23%). Of the 54 children with tiredness, 43% of the children and 39% of their parents rate this symptom as one of the three problems they
Abstracts
would like to change most. In the 55 patients with lack of energy, these percentages are 42 and 35%, respectively. In the 53 patients with developmental delay, 32% of the parents rate this as one of the most important problems. Of the 39 patients with speech difficulties, 36% of the children and 30% of the parents rate this symptom in the top three. For the 27 children with epilepsy, these percentages are 37 and 41% respectively. For the 26 children with behavioural problems, 66% of the parents rate this symptom as a major point they would like to change. In conclusion, behavioural, speech and language problems are more cumbersome to parents and children than we expected. We advise to take these aspects into account more frequently in supportive care management. doi:10.1016/j.mito.2012.07.003
4 MELAS and l-arginine therapy —therapeutic timing and long term effects Presenter: Yasutoshi Koga Yasutoshi Kogaa, Toshi Abeb, Takayuki Taniwakic, Povalko Nataliyaa a Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan b Department of Radiology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan c Department of Neurology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan Investigator-initiated clinical trial of l-arginine to cure the symptoms of acute phase stroke-like episodes (SLE) and to prevent SLE in the interictal phase of MELAS finished on June 30, 2010. We are now cleaning the date and are processing the date for approval. However we still do not know what is the best therapeutic timing of IV on SLE, and whether it can prevent the disease progression of MELAS during long term administration. We used l-arginine infusion at hyper-acute phase of SLE in 2 MELAS patients and evaluated the therapeutic effects by clinical and the serial neuroimaging analysis. Patient 1 is a 6-year-old girl who was diagnosed with MELAS/Leigh overlap syndrome based on a finding of clinical symptoms and 78% mutation in an A3243G in the mitochondrial tRNALeu(UUR) gene. Patient 2 is a 32-year-old girl who fulfilled the diagnostic criteria of MELAS and has a 55% mutation of A3243G in muscles. She has a bilateral sensorineural hearing loss and diabetes mellitus at the age of 31. She has two histories of stroke-like episodes and was then followed as MELAS. Within 2 h after the onset of SLE, we infused 0.5 g/kg/dose of l-arginine within 1 h after the onset and took the brain MRI seriously. All of the clinical symptoms disappeared within 30 min after l-arginine infusion without using anti-convulsants. The series of MRIs were performed first, seven days and one month from onset of stroke-like episodes. MRI obtained at 24 h after the onset showed high intensity signal in T2WI, and DWI and low in apparent diffusion coefficient (ADC), however those abnormal findings were completely normalized on MRIs taken at 7 days and one month later. We also administered l-arginine on MELAS for more than 5 years and evaluated its long term effects by duration of hospitalization and by Japanese Mitochondrial Disease Rating Scale (JMDRS) compared with the MELAS cohort study in Japan without using l-arginine. l-Arginine therapy significantly decreases the JMDRS and prevents the disease progression as compared with the cohort study. Our data indicated that l-arginine infusion at hyper-acute phase shows promise in the cure of strokelike episodes seen in MELAS, and the long term l-arginine therapy prevents the disease progression of MELAS. doi:10.1016/j.mito.2012.07.004
551
5 A novel sequence variant of tRNAThr in mtDNA, transferred by bone marrow transplantation in patient with ALL Presenter: Nataliya Povalko Nataliya Povalkoa, Yoshihisa Nagatoshib, Hiroko Inadaa, Shuichi Yatsugaa, Yasutoshi Kogaa a Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan b Section of Pediatrics, National Kyushu Cancer Center, 3-1-1 Notame, Minamiku, Fukuoka 811-1395, Japan Bone marrow transplantation (BMT) is one of the possible choices for the therapy of mitochondrial disorders such as MINGIE. However current donor matching system is only focused on the HLA typing based on the major histocompatibility antigens to avoid the graftversus-host disease (GVHD). We had a 5-year old patient with ALL (acute lymphoblastic leukemia) who showed recurrent stroke-like episodes before and after the BMT. We found the novel mtDNA sequence variant which has not been found in a patient mtDNA before BMT. Analysis of the entire sequence of mtDNA was performed twice (before and after BMT). First analysis was done after the first stroke-like episode. Patient under the chemotherapy suddenly showed severe headache, and a left tonic seizure with right hemiplegia. MRI of the brain showed high intensity areas on T2 and FLAIR in occipital, frontal, and parietal lobes. The parameters of endothelial function, including plasma level of l-arginine, and FMD (flow mediated dilatation), were abnormal. No pathogenic mtDNA mutations were found at his original mitochondrial DNA. The second entire sequencing of mtDNA was performed when he had the second episode of stroke 5 months after BMT. He showed cortical blindness, vomiting and headache. Since the parameters of endothelial function were abnormal, infusion of l-arginine was immediately started. No abnormalities were found on MRI taken after the stroke-like episode. We found a novel A15929G in the mitochondrial tRNAThr gene. The heteroplasmy of this mutation was 90% in white blood cell, and 8% in nail, however this mutation was not detected in urine, and recipient's bone marrow before BMT, WBCs from mother, brother and sister of the proband, suggesting that this mutation was transferred by BMT. According to the haplogroup analysis of mtDNA, original mtDNA belonged to haplogroup D4a (superhaplogroup M), on the other hand, donor's mtDNA belonged to haplogroup F1 (superhaplogroup N). Since mtDNA has a very high mutation rate and has at least 30 polymorphic sites, minor antigen mismatching caused by the amino acid sequence polymorphism may occur leading to the GVHD. On the other hand, many oligo-symptomatic or asymptomatic carriers having a pathogenic mtDNA mutation may become a potential donor for BMT and thus transmit the mutation to the recipient. Current donor matching system may have a risk of transmission of pathogenic mtDNA mutation and may contribute the GVHD by the proteins which are generated by the transmitted mtDNA.
doi:10.1016/j.mito.2012.07.005
6 The comparative study of actin and myosin genes in Molgula manhattensis, Styela clava, and Limulus polyphemus: Implication on mitochondrial DNA maintenance Presenter: Meghan Buckley Caitlin A. Baker, Meghan F. Buckley, Taylor L. DeRosa, Jenna A. Hernandez, Erin L. Hillis, Caroline R. Luciani, Rita Matta, Alexandra R. Novak, Christine C. Smith, Qingyu Xu, Mary Jane Paolella Sacred Heart Academy, 265 Benham St. Hamden, CT 06514, United States