- Email: [email protected]
The molecular etiology of Progressive External Ophthalmoplegia (PEO) associated with mitochondrial myopathy
560
Abstracts
Mitochondrial genome manipulation is a potential therapy for many incurable mitochondrial diseases of children and adults resulting from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. We treated LHON and LS cells respectively harboring G11778A and T8993G mutant mtDNA by N90%, with a single dose of healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM). Our results showed improvement in mitochondrial respiration by ~ 1.2 fold in LHON cells and restoration of ~N50% ATP synthase function in LS cells. We also observed increases in mitochondrial replication, transcription and translation of key respiratory genes and proteins in the short term. Upregulation of NRF1, TFAMB1 and TFAMA expressions demonstrated that mitochondrial biogenesis likely accounted for the mechanism for improving mitochondrial respiration. While much work needs to be done in determining the factors that might influence stability, transmission and maintenance of the introduced mtDNA; these results represent opportunities for therapeutic interventions for LHON and LS patients in the near future. doi:10.1016/j.mito.2012.07.026
29 First tier molecular diagnosis of mitochondrial disorders — The experience of a mitochondrial diagnostic laboratory pre‐NextGen era Presenter: Sha Tang Sha Tang, Jing Wang, Fangyuan Li, Victor Wei Zhang, Megan Landsverk, Hong Cui, Cavatina K. Truong, Guoli Wang, Eric S. Schmitt, William J. Craigen, Lee-Jun C. Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Mitochondrial disorders are a group of genetically and clinically heterogeneous diseases. For patients with suspected mitochondrial dysfunction, DNA-based molecular diagnosis generally starts with screening for mitochondrial DNA (mtDNA) common point mutations and large deletions (screening), followed by analysis of the entire mitochondrial genome (mtWGS) and/or POLG gene (POLG) by Sanger sequencing. These three tests are considered the “first tier” analyses in the routine molecular evaluation of mitochondrial diseases. From May 2005 to December 2011, the screening, mtWGS, and POLG tests were performed on 9260, 2851, and 4240 independent patients at the Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, respectively. A total of 343 patients (3.7%) were positive for the screening test, including 225 patients harboring a common mtDNA point mutation, 86 patients containing a single mtDNA large deletion, and 32 patients with mtDNA multiple deletions. Among common mtDNA point mutations, m.3243ANG remains the most frequent one (56.9%), followed by m.11778GNA (9.8%) and m.8993TNG (7.6%). 109 mtDNA mutations (3.8%), including 93 reported and 16 novel pathogenic changes, have been identified in the patient cohort with mtWGS. We imposed stringent criteria, including results from clinical, biochemical, and molecular genetic studies of matrilineal family members, in the assessment of pathogenicity for the novel mutations. Definitive molecular diagnosis of POLG-related disorder was made for 137 patients (3.2%) and a heterozygous POLG mutation, either reported (69) or novel (29), was detected in another 98 (2.3%) subjects. Conclusive molecular findings were detected in 23 out of 343 patients tested simultaneously for all the three tests. This detection rate (6.7%) almost doubled that of any single test alone (3.2–3.8%). In the remaining 320 patients negative for all three tests, no further molecular/biochemical tests were pursued in 40 (12.5%).
Additional test(s) were performed for the other 280 negatives. The most common sequential tests are analyses of other mtDNA depletion genes (54.6%), ETC enzyme activity (49.3%), and mtDNA copy number (41.1%). These additional tests confirmed the molecular defects in only two (MPV17 and PDHA1) patients (0.7%), demonstrating the low detection rate of pre-NextGen sequential approach. In summary, each of the screening, mtWGS, and POLG tests has a positive detection rate of ~3.5% and the combination of the three (6.7%) significantly enhances the capability of making a definitive molecular diagnosis. We expect the flourishing application of the NextGen technology investigating multiple loci in parallel to really usher in a more efficient epoch of molecular diagnosis of mitochondrial disorders. doi:10.1016/j.mito.2012.07.027
30 The molecular etiology of Progressive External Ophthalmoplegia (PEO) associated with mitochondrial myopathy Presenter: Sha Tang Sha Tanga, Yu-Wen Huanga, Margherita Miloneb, Xia Tiana, Hong Cuia, Victor Wei Zhanga, Jing Wanga, Lee-Jun C. Wonga a Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States b Department of Neurology, Mayo Clinic, Rochester, MN, United States Objective: To elucidate the molecular etiology of Progressive External Ophathalmoplegia (PEO) with associated mitochondrial myopathy syndrome and to determine the prevalence of different gene defects causing PEO. Background: Progressive External Ophthalmoplegia (PEO) with associated mitochondrial myopathy can be caused by autosomal dominant mutations in POLG1 (adPEOA1), ANT1 (adPEOA2), and TWINKLE (adPEOA3). Two autosomal recessive POLG1 mutations can also result in autosomal recessive PEO (arPEO). Recently, mutations in POLG2 (adPEOA4) and RRM2B (adPEOA5) have been reported to be associated with PEO. Autosomal dominant optic atrophy caused by mutations in the OPA1 gene can also present as PEO. The encoded products of these genes are involved in mitochondrial DNA (mtDNA) replication (POLG1, POLG2, and TWINKLE), distribution (OPA1), and the maintenance of deoxynucleotide pools (ANT1 and RRM2B). Methods: The coding regions and exon–intron boundaries of POLG1, ANT1, and TWINKLE were sequenced in 187 patients (N10 years) suspected of PEO. RRM2B gene was sequence analyzed in 25 patients negative for mutations in the above three genes. Southern blot and/or PCR were used to detect mtDNA deletions in muscle specimens of 7 patients, 6 of which with two POLG1 mutant alleles and the 7th one harboring a dominant TWINKLE mutation. Results: In this cohort, 21 patients (11.2%) had a definite molecular diagnosis of PEO and mitochondrial myopathy. These include 12 patients with arPEO caused by 2 mutant alleles in POLG1 and 9 patient with adPEO caused by a heterozygous mutation in POLG1 (1 patient), ANT1 (1 patient), or TWINKLE (7 patients). In addition, 5 patients were heterozygous for a POLG1 mutation usually found in compound heterozygosity with another mutation. No mutation was detected in the RRM2B gene in 25 patients negative for POLG1, ANT1, and TWINKLE mutations. In 6 out of the 7 patients with muscle specimen available for analysis, multiple mtDNA deletions were detected (85.7%). Conclusions: Our data suggest that POLG1 (ar) and TWINKLE (ad) represent the most frequent molecular defects in PEO patients associated with mitochondrial myopathy. Multiple mtDNA deletion in the muscle is a cardinal feature for these patients, indicating that compromised mtDNA integrity plays an important role in the etiology of PEO.
Abstracts
Perspective: We are currently using the NextGen platform to simultaneously investigate all the 6 loci in patients with clinical symptoms consistent with PEO and multiple mtDNA deletions. We expect such approach to be more effective for molecular diagnosis. doi:10.1016/j.mito.2012.07.028
561
32 Functional analysis of astrocytes in a complex I deficient mouse model Presenter: Matthew J. Bird Matthew Birda,b, Ann Fraziera, Adrienne Laskowskia, David Thorburna,b a Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Melbourne, Australia b University of Melbourne Department of Pediatrics, Melbourne, Australia
31 PDHA1 mutations and continued clinical and genetic heterogeneity: Are there gender differences? Presenter: Lisa Emrick Sha Tanga, Lisa Emricka, Inn-Chi Leea,b, Guoli Wanga, Fangyuan Lia, Shao-Wen Wengc, William J. Craigena, Lee-Jun C. Wonga a Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States b Department of Pediatrics, Division of Pediatric Neurology, College of Medicine, Chung-Shan Medical University Hospital, Institute of Medicine of Chung-Shan Medical University, Taichung, Taiwan c Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan Pyruvate dehydrogenase complex (PDHC) deficiency is a genetically and clinically heterogeneous disorder. Mutations in the PDHA1 gene are responsible for the majority of cases of PDHC deficiency. The PDHA1 gene is on the X chromosome, however, PDHC deficiency appears to affect males and females equally due to skewed X chromosome inactivation in the affected females. Sequence analyses of the PDHA1 gene were performed for 624 patients, including 323 females and 301 males, with suspected PDHC deficiency. Targeted sequence analyses for selected variants in relevant family members were also performed to aid in the assessment of pathogenicity. From this cohort, causative mutations were detected in 13.6% of females (44/323) and 8.3% of males (25/301) and 24 novel mutations have been identified. In addition, we have used aCGH to investigate the possibility of large deletions in 7 female patients with biochemically proven PDHC deficiency without identifiable mutations by sequencing. However, copy number changes were not found. The genetics and clinical features of the 69 positive cases were reviewed. The average age of onset was 3.5 years for females and 5.9 years for males. We have clinical information on 44/69 (32 females and 12 males) patients with mutations. Most cases presented with neurological and other system involvement. Seven of 44 patients, all females, had only neurological symptoms. Nine of 44 patients had abnormal PDHC activity (7/32 females and 2/12 males). The most common reason for testing was lactic acidosis (31/44). Developmental delay was the most common neurological finding (19/32 females and 6/12 males). Microcephaly was recorded in 14/44 positive cases (12/32 females and 2/12 males). Among 16 patients with brain MRI results, 4 had cortical atrophy (3 females and 1 male), 3 had brain malformations (all females), 2 had abnormalities of the basal ganglia (1 female and 1 male), and 3 had undisclosed MRI abnormalities (all females). Female patients tend to have more severe mutations (34% with null mutations) while males have milder mutations (8% with null mutations). In summary, a higher mutation detection rate was observed in females compared to males tested. Females tend to have more severe mutations than males, probably because null mutations are better tolerated due to X inactivation. In those patients where information is available, female patients tended to have more problems related to brain developments, in particular there was a higher rate of microcephaly and brain malformations in females in comparison to males. doi:10.1016/j.mito.2012.07.029
Mitochondrial dysfunction is strongly associated with a range of neurological conditions. However the mechanisms underlying mitochondrial neuropathology are poorly understood. Accessing biologically relevant neurological tissue has traditionally been difficult, until the recent development of animal models. Our lab has characterized a mouse model of complex I deficiency, the most common mitochondrial respiratory chain defect. Ndufs4fky/fky mice have a retroviral insertion resulting in a complete knockout of the Ndufs4 subunit of complex I. The mice exhibit a progressive neurological disease and we have isolated and characterized a number of relevant primary cell lines including astrocytes, a glial cell type which supports neuronal cells. Likewise, making mouse embryonic fibroblasts is a useful reference population as it is typical to receive human fibroblasts in a diagnostic lab for pathological analysis and they are accordingly well characterized. To this end, astrocytes and MEFs have been isolated from Ndufs4fky/fky pups and embryos respectively. Primary cell cultures were established, then grown in media containing either glucose or galactose as primary carbon source for 24 h prior to analysis. Galactose catabolism is rate limiting as compared to glucose, and it is expected that its utilization could force the cells to rely more heavily on oxidative phosphorylation. Enzymatically, complex I activity is equally impaired in all cell types tested. Examination of the rates of ATP synthesis and reactive oxygen species generation in the primary cell lines suggests that the Ndufs4 knockout astrocytes have an underlying defect regardless of the carbon source. This is in contrast to the MEFs, which appear phenotypically normal when cultured on glucose, only presenting a compromised phenotype when cultured on galactose as the primary carbon source. Membrane potential is apparently conserved in both cell types tested, as is gross mitochondrial morphology.
doi:10.1016/j.mito.2012.07.030
33 Thymidine kinase 2 deficiency-induced mtDNA depletion in liver leads to defect β-oxidation and insufficient supply of ketone bodies and glucose for brain function Presenter: Sophie Curbo Sophie Curboa,⁎, Xiaoshan ZhouIa, Kristina Kannistob, Ulrika von Döbelnc, Kjell Hultenbyd, Sindra Isetunc, Mats Gåfvelsb, Anna Karlssona a Division of Clinical Microbiology F-68, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden b Division of Clinical Chemistry, C1-72, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden c Division of Metabolic Diseases, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden d Division of Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden ⁎Contact information: Sophie Curbo, Karolinska Institutet, Division of Clinical Microbiology F-68, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. Tel.: +46 8 52483616; fax: +46 8 58587933. E-mail: [email protected]
Abstracts
Mitochondrial genome manipulation is a potential therapy for many incurable mitochondrial diseases of children and adults resulting from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. We treated LHON and LS cells respectively harboring G11778A and T8993G mutant mtDNA by N90%, with a single dose of healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM). Our results showed improvement in mitochondrial respiration by ~ 1.2 fold in LHON cells and restoration of ~N50% ATP synthase function in LS cells. We also observed increases in mitochondrial replication, transcription and translation of key respiratory genes and proteins in the short term. Upregulation of NRF1, TFAMB1 and TFAMA expressions demonstrated that mitochondrial biogenesis likely accounted for the mechanism for improving mitochondrial respiration. While much work needs to be done in determining the factors that might influence stability, transmission and maintenance of the introduced mtDNA; these results represent opportunities for therapeutic interventions for LHON and LS patients in the near future. doi:10.1016/j.mito.2012.07.026
29 First tier molecular diagnosis of mitochondrial disorders — The experience of a mitochondrial diagnostic laboratory pre‐NextGen era Presenter: Sha Tang Sha Tang, Jing Wang, Fangyuan Li, Victor Wei Zhang, Megan Landsverk, Hong Cui, Cavatina K. Truong, Guoli Wang, Eric S. Schmitt, William J. Craigen, Lee-Jun C. Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Mitochondrial disorders are a group of genetically and clinically heterogeneous diseases. For patients with suspected mitochondrial dysfunction, DNA-based molecular diagnosis generally starts with screening for mitochondrial DNA (mtDNA) common point mutations and large deletions (screening), followed by analysis of the entire mitochondrial genome (mtWGS) and/or POLG gene (POLG) by Sanger sequencing. These three tests are considered the “first tier” analyses in the routine molecular evaluation of mitochondrial diseases. From May 2005 to December 2011, the screening, mtWGS, and POLG tests were performed on 9260, 2851, and 4240 independent patients at the Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, respectively. A total of 343 patients (3.7%) were positive for the screening test, including 225 patients harboring a common mtDNA point mutation, 86 patients containing a single mtDNA large deletion, and 32 patients with mtDNA multiple deletions. Among common mtDNA point mutations, m.3243ANG remains the most frequent one (56.9%), followed by m.11778GNA (9.8%) and m.8993TNG (7.6%). 109 mtDNA mutations (3.8%), including 93 reported and 16 novel pathogenic changes, have been identified in the patient cohort with mtWGS. We imposed stringent criteria, including results from clinical, biochemical, and molecular genetic studies of matrilineal family members, in the assessment of pathogenicity for the novel mutations. Definitive molecular diagnosis of POLG-related disorder was made for 137 patients (3.2%) and a heterozygous POLG mutation, either reported (69) or novel (29), was detected in another 98 (2.3%) subjects. Conclusive molecular findings were detected in 23 out of 343 patients tested simultaneously for all the three tests. This detection rate (6.7%) almost doubled that of any single test alone (3.2–3.8%). In the remaining 320 patients negative for all three tests, no further molecular/biochemical tests were pursued in 40 (12.5%).
Additional test(s) were performed for the other 280 negatives. The most common sequential tests are analyses of other mtDNA depletion genes (54.6%), ETC enzyme activity (49.3%), and mtDNA copy number (41.1%). These additional tests confirmed the molecular defects in only two (MPV17 and PDHA1) patients (0.7%), demonstrating the low detection rate of pre-NextGen sequential approach. In summary, each of the screening, mtWGS, and POLG tests has a positive detection rate of ~3.5% and the combination of the three (6.7%) significantly enhances the capability of making a definitive molecular diagnosis. We expect the flourishing application of the NextGen technology investigating multiple loci in parallel to really usher in a more efficient epoch of molecular diagnosis of mitochondrial disorders. doi:10.1016/j.mito.2012.07.027
30 The molecular etiology of Progressive External Ophthalmoplegia (PEO) associated with mitochondrial myopathy Presenter: Sha Tang Sha Tanga, Yu-Wen Huanga, Margherita Miloneb, Xia Tiana, Hong Cuia, Victor Wei Zhanga, Jing Wanga, Lee-Jun C. Wonga a Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States b Department of Neurology, Mayo Clinic, Rochester, MN, United States Objective: To elucidate the molecular etiology of Progressive External Ophathalmoplegia (PEO) with associated mitochondrial myopathy syndrome and to determine the prevalence of different gene defects causing PEO. Background: Progressive External Ophthalmoplegia (PEO) with associated mitochondrial myopathy can be caused by autosomal dominant mutations in POLG1 (adPEOA1), ANT1 (adPEOA2), and TWINKLE (adPEOA3). Two autosomal recessive POLG1 mutations can also result in autosomal recessive PEO (arPEO). Recently, mutations in POLG2 (adPEOA4) and RRM2B (adPEOA5) have been reported to be associated with PEO. Autosomal dominant optic atrophy caused by mutations in the OPA1 gene can also present as PEO. The encoded products of these genes are involved in mitochondrial DNA (mtDNA) replication (POLG1, POLG2, and TWINKLE), distribution (OPA1), and the maintenance of deoxynucleotide pools (ANT1 and RRM2B). Methods: The coding regions and exon–intron boundaries of POLG1, ANT1, and TWINKLE were sequenced in 187 patients (N10 years) suspected of PEO. RRM2B gene was sequence analyzed in 25 patients negative for mutations in the above three genes. Southern blot and/or PCR were used to detect mtDNA deletions in muscle specimens of 7 patients, 6 of which with two POLG1 mutant alleles and the 7th one harboring a dominant TWINKLE mutation. Results: In this cohort, 21 patients (11.2%) had a definite molecular diagnosis of PEO and mitochondrial myopathy. These include 12 patients with arPEO caused by 2 mutant alleles in POLG1 and 9 patient with adPEO caused by a heterozygous mutation in POLG1 (1 patient), ANT1 (1 patient), or TWINKLE (7 patients). In addition, 5 patients were heterozygous for a POLG1 mutation usually found in compound heterozygosity with another mutation. No mutation was detected in the RRM2B gene in 25 patients negative for POLG1, ANT1, and TWINKLE mutations. In 6 out of the 7 patients with muscle specimen available for analysis, multiple mtDNA deletions were detected (85.7%). Conclusions: Our data suggest that POLG1 (ar) and TWINKLE (ad) represent the most frequent molecular defects in PEO patients associated with mitochondrial myopathy. Multiple mtDNA deletion in the muscle is a cardinal feature for these patients, indicating that compromised mtDNA integrity plays an important role in the etiology of PEO.
Abstracts
Perspective: We are currently using the NextGen platform to simultaneously investigate all the 6 loci in patients with clinical symptoms consistent with PEO and multiple mtDNA deletions. We expect such approach to be more effective for molecular diagnosis. doi:10.1016/j.mito.2012.07.028
561
32 Functional analysis of astrocytes in a complex I deficient mouse model Presenter: Matthew J. Bird Matthew Birda,b, Ann Fraziera, Adrienne Laskowskia, David Thorburna,b a Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Melbourne, Australia b University of Melbourne Department of Pediatrics, Melbourne, Australia
31 PDHA1 mutations and continued clinical and genetic heterogeneity: Are there gender differences? Presenter: Lisa Emrick Sha Tanga, Lisa Emricka, Inn-Chi Leea,b, Guoli Wanga, Fangyuan Lia, Shao-Wen Wengc, William J. Craigena, Lee-Jun C. Wonga a Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States b Department of Pediatrics, Division of Pediatric Neurology, College of Medicine, Chung-Shan Medical University Hospital, Institute of Medicine of Chung-Shan Medical University, Taichung, Taiwan c Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan Pyruvate dehydrogenase complex (PDHC) deficiency is a genetically and clinically heterogeneous disorder. Mutations in the PDHA1 gene are responsible for the majority of cases of PDHC deficiency. The PDHA1 gene is on the X chromosome, however, PDHC deficiency appears to affect males and females equally due to skewed X chromosome inactivation in the affected females. Sequence analyses of the PDHA1 gene were performed for 624 patients, including 323 females and 301 males, with suspected PDHC deficiency. Targeted sequence analyses for selected variants in relevant family members were also performed to aid in the assessment of pathogenicity. From this cohort, causative mutations were detected in 13.6% of females (44/323) and 8.3% of males (25/301) and 24 novel mutations have been identified. In addition, we have used aCGH to investigate the possibility of large deletions in 7 female patients with biochemically proven PDHC deficiency without identifiable mutations by sequencing. However, copy number changes were not found. The genetics and clinical features of the 69 positive cases were reviewed. The average age of onset was 3.5 years for females and 5.9 years for males. We have clinical information on 44/69 (32 females and 12 males) patients with mutations. Most cases presented with neurological and other system involvement. Seven of 44 patients, all females, had only neurological symptoms. Nine of 44 patients had abnormal PDHC activity (7/32 females and 2/12 males). The most common reason for testing was lactic acidosis (31/44). Developmental delay was the most common neurological finding (19/32 females and 6/12 males). Microcephaly was recorded in 14/44 positive cases (12/32 females and 2/12 males). Among 16 patients with brain MRI results, 4 had cortical atrophy (3 females and 1 male), 3 had brain malformations (all females), 2 had abnormalities of the basal ganglia (1 female and 1 male), and 3 had undisclosed MRI abnormalities (all females). Female patients tend to have more severe mutations (34% with null mutations) while males have milder mutations (8% with null mutations). In summary, a higher mutation detection rate was observed in females compared to males tested. Females tend to have more severe mutations than males, probably because null mutations are better tolerated due to X inactivation. In those patients where information is available, female patients tended to have more problems related to brain developments, in particular there was a higher rate of microcephaly and brain malformations in females in comparison to males. doi:10.1016/j.mito.2012.07.029
Mitochondrial dysfunction is strongly associated with a range of neurological conditions. However the mechanisms underlying mitochondrial neuropathology are poorly understood. Accessing biologically relevant neurological tissue has traditionally been difficult, until the recent development of animal models. Our lab has characterized a mouse model of complex I deficiency, the most common mitochondrial respiratory chain defect. Ndufs4fky/fky mice have a retroviral insertion resulting in a complete knockout of the Ndufs4 subunit of complex I. The mice exhibit a progressive neurological disease and we have isolated and characterized a number of relevant primary cell lines including astrocytes, a glial cell type which supports neuronal cells. Likewise, making mouse embryonic fibroblasts is a useful reference population as it is typical to receive human fibroblasts in a diagnostic lab for pathological analysis and they are accordingly well characterized. To this end, astrocytes and MEFs have been isolated from Ndufs4fky/fky pups and embryos respectively. Primary cell cultures were established, then grown in media containing either glucose or galactose as primary carbon source for 24 h prior to analysis. Galactose catabolism is rate limiting as compared to glucose, and it is expected that its utilization could force the cells to rely more heavily on oxidative phosphorylation. Enzymatically, complex I activity is equally impaired in all cell types tested. Examination of the rates of ATP synthesis and reactive oxygen species generation in the primary cell lines suggests that the Ndufs4 knockout astrocytes have an underlying defect regardless of the carbon source. This is in contrast to the MEFs, which appear phenotypically normal when cultured on glucose, only presenting a compromised phenotype when cultured on galactose as the primary carbon source. Membrane potential is apparently conserved in both cell types tested, as is gross mitochondrial morphology.
doi:10.1016/j.mito.2012.07.030
33 Thymidine kinase 2 deficiency-induced mtDNA depletion in liver leads to defect β-oxidation and insufficient supply of ketone bodies and glucose for brain function Presenter: Sophie Curbo Sophie Curboa,⁎, Xiaoshan ZhouIa, Kristina Kannistob, Ulrika von Döbelnc, Kjell Hultenbyd, Sindra Isetunc, Mats Gåfvelsb, Anna Karlssona a Division of Clinical Microbiology F-68, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden b Division of Clinical Chemistry, C1-72, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden c Division of Metabolic Diseases, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden d Division of Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden ⁎Contact information: Sophie Curbo, Karolinska Institutet, Division of Clinical Microbiology F-68, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. Tel.: +46 8 52483616; fax: +46 8 58587933. E-mail: [email protected]