- Email: [email protected]
Analysis of mtDNA multiple deletions in 209 muscle specimens using massively parallel sequencing (MPS)
932
Abstracts
Other hiPSC lines are currently being driven to formation of neurons, skeletal myotubes and cardiomyocytes. Conclusions: hiPSCs have been successfully prepared from skin fibroblasts of patients with both mtDNA and nuclear DNA abnormalities. Further studies are in progress to determine if differentiation to myotubes and neurons faithfully reproduces markers of mitochondrial dysfunction present in the patients. doi:10.1016/j.mito.2013.07.086
95 Mitochondrial gene therapy approaches for understanding and treating Leber's hereditary optic neuropathy Presenter: Shilpa Iyer Shilpa Iyera, Erich Gnaigerb, Raj R. Raoc a Center for the Study of Biological Complexity, Virginia Commonwealth University, United States b Medical University of Innsbruck, Department of Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Laboratory, Austria c Chemical and Life Science Engineering, Virginia Commonwealth University, United States Body of abstract: One of the most extensively studied mitochondrial disorder, Leber's hereditary optic neuropathy (LHON) causes blindness due to death of the optic nerve cells. The most important factor for visual recovery in patients with LHON is influenced by reduction in the heteroplasmic burden of abnormal mtDNA or improved bioenergetics in affected cells over time. The abnormal mtDNA is primarily caused by mutations in the mitochondrial genome affecting the respiratory chain complexes. An optimal cure would be gene therapy, which involves introducing the missing gene(s) into the mitochondria to complement the defect. Our earlier research results indicate the potential of an innovative protein-transduction technology, consisting of a recombinant mitochondrial transcription factor A (TFAM) that avidly binds mtDNA and permits efficient targeting into mitochondria in situ and in vivo. Our studies in LHON cybrid models indicate that mitochondrial gene therapy increased (a) mtDNA copy number and gene expression; (b) respiration and respiratory protein levels in 3 weeks and (c) triggers mitochondria biogenesis by 3 weeks. Additional studies in human neural progenitor stem cells indicate potential for introducing and expressing pathogenic LHON G11778A mtDNA towards understanding mitochondrial DNA dynamics in LHON neuronal lineages. It is expected that this mitochondrial genome manipulation approach based on introduction of exogenous normal or pathogenic mtDNA provides hope for LHON patients afflicted with other mutations in the mitochondrial genome. doi:10.1016/j.mito.2013.07.087
96 Analysis of mtDNA multiple deletions in 209 muscle specimens using massively parallel sequencing (MPS) Presenter: Victor Wei Zhang Fang-Yuan Lia, Xia Tiana, Margherita Miloneb, Hong Cuia, Victor Wei Zhanga, Lee-Jun 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 55905, United States Background: Large mitochondrial DNA (mtDNA) deletions in muscle have been found to be associated with human diseases
including Kearns–Sayre syndrome (KSS), Pearson syndrome, diabetes and hearing loss, myopathy, and progressive external ophthalmoplegia (PEO). Patients with mtDNA multiple deletions often present with various myopathic phenotype. MtDNA deletions are traditionally detected by using Southern blot analysis. However, it does not detect low levels of mtDNA deletions, and it does not provide the breakpoints information. The newly developed massively parallel sequencing (MPS) with deep coverage was able to detect and distinguish between mtDNA single and multiple deletions. Objective: To develop an algorithm for the analysis of the MPS sequence data to detect mtDNA large deletions and to determine deletion breakpoints. Method: The intact circular mitochondrial genome was amplified as a single amplicon followed by MPS analysis, which provides a coverage depth of ~20,000X. The breakpoint detection (BPD) algorithm aligns imperfectly mapped sequence reads to the reference sequence with modified and less stringent parameters using NextGENe software (SoftGenetics, State College, PA). A total of 209 muscle specimens from patients of various age with mostly myopathic presentations of mitochondrial disorders were analyzed by MPS and BPD algorithm. Result: MtDNA deletions at various levels of heteroplasmies were detected in 29% of muscle specimens. Among them, 90% had multiple deletions. The frequency of mtDNA multiple deletions in muscle specimens appears to increase with age. None of the muscle specimens from 102 patients younger than 10 years of age had mtDNA multiple deletions, while all 16 samples from patients age 60 and above harbored mtDNA multiple deletions. Although most of low level multiple deletions cannot be detected by Southern analysis, all deletion breakpoints identified by BPD algorithm could be confirmed by PCR/sequencing. Conclusion: MPS is a sensitive and effective method to detect mtDNA multiple deletions. Not only the low levels of mtDNA multiple deletions could be detected but also the multiple breakpoint sequences could be determined. The mtDNA multiple deletions appear to occur often in elder patients with mitochondrial myopathy. doi:10.1016/j.mito.2013.07.088
97 Improved diagnosis of mitochondrial disorders by next generation sequencing approach Presenter: Victor Wei Zhang Lee-Jun Wong, Xia Tian, Yanming Feng, Jing Wang, Victor Wei Zhang Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States Background: Diagnosis of mitochondrial disorders is challenging due to the extreme genetic and clinical heterogeneity. Step-wise analyses of the two genomes are time consuming and cost ineffective. Next generation high throughput (NGS) technology allowing simultaneous sequence analysis of any number of target genes is a solution for the diagnosis of complex dual genome mitochondrial disorders. Purpose: To apply clinically validated NGS approach to the diagnosis of mitochondrial disorders. Methods: The intact circular mitochondrial genome is enriched by long range PCR as a single amplicon. Target nuclear genes are enriched by probe capture in solution. Sequence was performed on HiSeq2000. Results: At an average coverage of 20,000X and 1,000X for mtDNA and nuclear genes respectively, mtDNA point mutations anywhere in the genome can be detected at a low limit of 1.5% heteroplasmy. MtDNA large single and multiple deletions can be detected with breakpoint sequences determined. Poorly covered coding regions of nuclear target genes, about 0–3%, are identified and filled in by specific PCR/Sanger sequencing to ensure 100% coverage.
Abstracts
Other hiPSC lines are currently being driven to formation of neurons, skeletal myotubes and cardiomyocytes. Conclusions: hiPSCs have been successfully prepared from skin fibroblasts of patients with both mtDNA and nuclear DNA abnormalities. Further studies are in progress to determine if differentiation to myotubes and neurons faithfully reproduces markers of mitochondrial dysfunction present in the patients. doi:10.1016/j.mito.2013.07.086
95 Mitochondrial gene therapy approaches for understanding and treating Leber's hereditary optic neuropathy Presenter: Shilpa Iyer Shilpa Iyera, Erich Gnaigerb, Raj R. Raoc a Center for the Study of Biological Complexity, Virginia Commonwealth University, United States b Medical University of Innsbruck, Department of Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Laboratory, Austria c Chemical and Life Science Engineering, Virginia Commonwealth University, United States Body of abstract: One of the most extensively studied mitochondrial disorder, Leber's hereditary optic neuropathy (LHON) causes blindness due to death of the optic nerve cells. The most important factor for visual recovery in patients with LHON is influenced by reduction in the heteroplasmic burden of abnormal mtDNA or improved bioenergetics in affected cells over time. The abnormal mtDNA is primarily caused by mutations in the mitochondrial genome affecting the respiratory chain complexes. An optimal cure would be gene therapy, which involves introducing the missing gene(s) into the mitochondria to complement the defect. Our earlier research results indicate the potential of an innovative protein-transduction technology, consisting of a recombinant mitochondrial transcription factor A (TFAM) that avidly binds mtDNA and permits efficient targeting into mitochondria in situ and in vivo. Our studies in LHON cybrid models indicate that mitochondrial gene therapy increased (a) mtDNA copy number and gene expression; (b) respiration and respiratory protein levels in 3 weeks and (c) triggers mitochondria biogenesis by 3 weeks. Additional studies in human neural progenitor stem cells indicate potential for introducing and expressing pathogenic LHON G11778A mtDNA towards understanding mitochondrial DNA dynamics in LHON neuronal lineages. It is expected that this mitochondrial genome manipulation approach based on introduction of exogenous normal or pathogenic mtDNA provides hope for LHON patients afflicted with other mutations in the mitochondrial genome. doi:10.1016/j.mito.2013.07.087
96 Analysis of mtDNA multiple deletions in 209 muscle specimens using massively parallel sequencing (MPS) Presenter: Victor Wei Zhang Fang-Yuan Lia, Xia Tiana, Margherita Miloneb, Hong Cuia, Victor Wei Zhanga, Lee-Jun 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 55905, United States Background: Large mitochondrial DNA (mtDNA) deletions in muscle have been found to be associated with human diseases
including Kearns–Sayre syndrome (KSS), Pearson syndrome, diabetes and hearing loss, myopathy, and progressive external ophthalmoplegia (PEO). Patients with mtDNA multiple deletions often present with various myopathic phenotype. MtDNA deletions are traditionally detected by using Southern blot analysis. However, it does not detect low levels of mtDNA deletions, and it does not provide the breakpoints information. The newly developed massively parallel sequencing (MPS) with deep coverage was able to detect and distinguish between mtDNA single and multiple deletions. Objective: To develop an algorithm for the analysis of the MPS sequence data to detect mtDNA large deletions and to determine deletion breakpoints. Method: The intact circular mitochondrial genome was amplified as a single amplicon followed by MPS analysis, which provides a coverage depth of ~20,000X. The breakpoint detection (BPD) algorithm aligns imperfectly mapped sequence reads to the reference sequence with modified and less stringent parameters using NextGENe software (SoftGenetics, State College, PA). A total of 209 muscle specimens from patients of various age with mostly myopathic presentations of mitochondrial disorders were analyzed by MPS and BPD algorithm. Result: MtDNA deletions at various levels of heteroplasmies were detected in 29% of muscle specimens. Among them, 90% had multiple deletions. The frequency of mtDNA multiple deletions in muscle specimens appears to increase with age. None of the muscle specimens from 102 patients younger than 10 years of age had mtDNA multiple deletions, while all 16 samples from patients age 60 and above harbored mtDNA multiple deletions. Although most of low level multiple deletions cannot be detected by Southern analysis, all deletion breakpoints identified by BPD algorithm could be confirmed by PCR/sequencing. Conclusion: MPS is a sensitive and effective method to detect mtDNA multiple deletions. Not only the low levels of mtDNA multiple deletions could be detected but also the multiple breakpoint sequences could be determined. The mtDNA multiple deletions appear to occur often in elder patients with mitochondrial myopathy. doi:10.1016/j.mito.2013.07.088
97 Improved diagnosis of mitochondrial disorders by next generation sequencing approach Presenter: Victor Wei Zhang Lee-Jun Wong, Xia Tian, Yanming Feng, Jing Wang, Victor Wei Zhang Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States Background: Diagnosis of mitochondrial disorders is challenging due to the extreme genetic and clinical heterogeneity. Step-wise analyses of the two genomes are time consuming and cost ineffective. Next generation high throughput (NGS) technology allowing simultaneous sequence analysis of any number of target genes is a solution for the diagnosis of complex dual genome mitochondrial disorders. Purpose: To apply clinically validated NGS approach to the diagnosis of mitochondrial disorders. Methods: The intact circular mitochondrial genome is enriched by long range PCR as a single amplicon. Target nuclear genes are enriched by probe capture in solution. Sequence was performed on HiSeq2000. Results: At an average coverage of 20,000X and 1,000X for mtDNA and nuclear genes respectively, mtDNA point mutations anywhere in the genome can be detected at a low limit of 1.5% heteroplasmy. MtDNA large single and multiple deletions can be detected with breakpoint sequences determined. Poorly covered coding regions of nuclear target genes, about 0–3%, are identified and filled in by specific PCR/Sanger sequencing to ensure 100% coverage.