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Exome sequencing and functional biology reveal novel mitochondrial disease genes
Abstracts
can elucidate a patient's pathophysiology and guide targeted treatment. The results obtained may also assist with the enrollment of patients into research and drug treatment trials that benefit from the improved understanding of defined disease mechanisms. About 25% of our previous MitoNucleomeDx tests analyzing 312 genes are definitely/very likely to be positive. In addition, for the MitoDx test, about 20% of the samples analyzed are definitely positive or very likely to be positive in the mitochondrial genome. This is consistent with the mitochondrial genome having a mutation rate that is 10-20 fold higher than that of the nuclear genome. The original 312 genes tested (of which 166 are known to cause mitochondrial diseases) include (1) those coding for the proteins in Complexes I–V, (2) OXPHOS Complex assembly factors, (3) OXPHOS co-enzyme synthesis, (4) factors involved in mtDNA replication/ transcription/translation (including the 86 ribosomal protein genes), (5) genes involved in maintaining mitochondrial copy number (mutations in which cause mitochondrial DNA depletion), (6) genes involved in the urea cycle, Krebs cycle, and fatty acid oxidation pathways, which prepare molecules for input into the OXPHOS pathway, and (7) genes coding for additional likely relevant mitochondrial proteins that are not yet associated with specific diseases. An additional 197 genes have been added to the new MitoNuc2Dx test (of which 81 are known to be mitochondrial disease-associated). These genes include (1) mitochondrial tRNA synthesis genes, (2) solute carriers, (3) mitochondrial membrane transporters, (4) mitochondrial maintenance genes, and (5) additional genes that include those discovered in the last 2 years. In conclusion, analysis of 104 recent samples for the initial 312 genes indicates that an estimated 25% are positive; a higher percentage is expected for the 509 genes in MitoNuc2Dx. Examples of some of the cases positive for a mutation in nuclear mitochondrial genes will be discussed. doi:10.1016/j.mito.2012.07.092
105 Exome sequencing and functional biology reveal novel mitochondrial disease genes Presenter: Penelope E. Bonnen Penelope E. Bonnen, Arnaud Besse, Tarak Donti, Adithya Raghavan, Seema Lalani, Fernando Scaglia, William J. Craigen, Brett H. Graham Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Mitochondrial disease is a diverse group of disorders that are estimated to occur with a combined incidence of 1/5000. Approximately ninety percent of pediatric onset cases are caused by high penetrance recessive mutations in the nuclear genome, however after exhausting all available diagnostic tests most patients remain without a molecular diagnosis. Using a combination of sequencing, bioinformatics, and cellular and molecular biology we have discovered highconfidence pathogenic mutations. The Mitochondrial Respiratory Chain Disorders Collection at Baylor College of Medicine (MRCDBCM) contains over 15,000 patients with suspected MRCD with accompanying DNA, diagnostic and clinical information. From this database we identified pediatric patients who have been prescreened and shown to be negative for mitochondrial and known nuclear gene candidates but have overwhelming evidence supporting a mitochondrial disease diagnosis. A battery of mitochondrial functional assays have been performed on all samples with available tissues including electron transport chain analysis, FACS based analysis of relative mitochondrial membrane potential and mitochondrial DNA copy number. Exome sequencing has been completed on 20 patients. We have discovered and validated the pathogenic
587
mutation in five patients. A causal relationship has been established using cDNA complementation and mitochondrial functional assays. This work has resulted in the discovery of novel disease genes, improved molecular diagnosis, and new insights into the pathogenetic mechanisms underlying mitochondrial disease. doi:10.1016/j.mito.2012.07.093
106 Two-dimensional intact mitochondrial DNA agarose electrophoresis demonstrates the complexity of mtDNA structural forms and in vivo supercoiling by TFAM Presenter: Brett A. Kaufman Jill E. Kolesar, Catherine Y. Wang, Shih-Hsuan Chou, Brett A. Kaufman Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce St. VET220E, Philadelphia, PA 19104, United States The circular mitochondrial genome's structural and topological conformations make the study of mitochondrial DNA (mtDNA) compaction notoriously difficult. The principal double-stranded DNA binding protein in the mitochondria is TFAM, which has been shown to supercoil and compact circular DNA in vitro, but not yet in vivo due to a lack of suitable methods to resolve myriad mtDNA structures that vary in both shape and compaction state. Here we describe two-dimensional intact mtDNA agarose gel electrophoresis (2D-IMAGE) profiling, which detected approximately two-dozen whole genome structures, which we have characterized through multiple molecular approaches. The major topoisomer forms were well conserved across many cell and tissue types, but differences unique to certain cells and tissues were also present. Linear mtDNA, which is not adequately resolved from circular DNA by conventional 1D electrophoresis, is increased by oxidative stress. Increased steady-state expression levels of TFAM altered the relative distribution and absolute abundance of topoisomers, by increasing covalently closed circles and extent of their supercoiling. Additional studies are underway to examine mtDNA by 2D-IMAGE in mouse models of human disease. doi:10.1016/j.mito.2012.07.094
107 Evidence for mitochondrial adaptation to chronic glucose exposure in beta cells Presenter: Brett A. Kaufman Jill E. Kolesara, Yumiko V. Taguchia, Rebecca L. Cramera, Catherine Wanga, Shih-Hsuan Choua, Scott A. Soleimanpourb, Doris A. Stoffersb, Brett A. Kaufmana a Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States b Department of Medicine and the Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania, Philadelphia, PA 19104, United States Pancreatic beta cells increase in number, size, and insulin synthesis/secretion to compensate for higher endocrine demands driven by increased circulating glucose. We have found that mitochondrial DNA (mtDNA) increases in response to chronic high glucose exposure of pancreatic insulinoma cells, and developed TAQMAN expression arrays to examine changes in mitochondrial factors in these cells. We identified PIF1 and FEN1 as the candidate factors regulating the observed increase in mtDNA content; the full
can elucidate a patient's pathophysiology and guide targeted treatment. The results obtained may also assist with the enrollment of patients into research and drug treatment trials that benefit from the improved understanding of defined disease mechanisms. About 25% of our previous MitoNucleomeDx tests analyzing 312 genes are definitely/very likely to be positive. In addition, for the MitoDx test, about 20% of the samples analyzed are definitely positive or very likely to be positive in the mitochondrial genome. This is consistent with the mitochondrial genome having a mutation rate that is 10-20 fold higher than that of the nuclear genome. The original 312 genes tested (of which 166 are known to cause mitochondrial diseases) include (1) those coding for the proteins in Complexes I–V, (2) OXPHOS Complex assembly factors, (3) OXPHOS co-enzyme synthesis, (4) factors involved in mtDNA replication/ transcription/translation (including the 86 ribosomal protein genes), (5) genes involved in maintaining mitochondrial copy number (mutations in which cause mitochondrial DNA depletion), (6) genes involved in the urea cycle, Krebs cycle, and fatty acid oxidation pathways, which prepare molecules for input into the OXPHOS pathway, and (7) genes coding for additional likely relevant mitochondrial proteins that are not yet associated with specific diseases. An additional 197 genes have been added to the new MitoNuc2Dx test (of which 81 are known to be mitochondrial disease-associated). These genes include (1) mitochondrial tRNA synthesis genes, (2) solute carriers, (3) mitochondrial membrane transporters, (4) mitochondrial maintenance genes, and (5) additional genes that include those discovered in the last 2 years. In conclusion, analysis of 104 recent samples for the initial 312 genes indicates that an estimated 25% are positive; a higher percentage is expected for the 509 genes in MitoNuc2Dx. Examples of some of the cases positive for a mutation in nuclear mitochondrial genes will be discussed. doi:10.1016/j.mito.2012.07.092
105 Exome sequencing and functional biology reveal novel mitochondrial disease genes Presenter: Penelope E. Bonnen Penelope E. Bonnen, Arnaud Besse, Tarak Donti, Adithya Raghavan, Seema Lalani, Fernando Scaglia, William J. Craigen, Brett H. Graham Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Mitochondrial disease is a diverse group of disorders that are estimated to occur with a combined incidence of 1/5000. Approximately ninety percent of pediatric onset cases are caused by high penetrance recessive mutations in the nuclear genome, however after exhausting all available diagnostic tests most patients remain without a molecular diagnosis. Using a combination of sequencing, bioinformatics, and cellular and molecular biology we have discovered highconfidence pathogenic mutations. The Mitochondrial Respiratory Chain Disorders Collection at Baylor College of Medicine (MRCDBCM) contains over 15,000 patients with suspected MRCD with accompanying DNA, diagnostic and clinical information. From this database we identified pediatric patients who have been prescreened and shown to be negative for mitochondrial and known nuclear gene candidates but have overwhelming evidence supporting a mitochondrial disease diagnosis. A battery of mitochondrial functional assays have been performed on all samples with available tissues including electron transport chain analysis, FACS based analysis of relative mitochondrial membrane potential and mitochondrial DNA copy number. Exome sequencing has been completed on 20 patients. We have discovered and validated the pathogenic
587
mutation in five patients. A causal relationship has been established using cDNA complementation and mitochondrial functional assays. This work has resulted in the discovery of novel disease genes, improved molecular diagnosis, and new insights into the pathogenetic mechanisms underlying mitochondrial disease. doi:10.1016/j.mito.2012.07.093
106 Two-dimensional intact mitochondrial DNA agarose electrophoresis demonstrates the complexity of mtDNA structural forms and in vivo supercoiling by TFAM Presenter: Brett A. Kaufman Jill E. Kolesar, Catherine Y. Wang, Shih-Hsuan Chou, Brett A. Kaufman Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce St. VET220E, Philadelphia, PA 19104, United States The circular mitochondrial genome's structural and topological conformations make the study of mitochondrial DNA (mtDNA) compaction notoriously difficult. The principal double-stranded DNA binding protein in the mitochondria is TFAM, which has been shown to supercoil and compact circular DNA in vitro, but not yet in vivo due to a lack of suitable methods to resolve myriad mtDNA structures that vary in both shape and compaction state. Here we describe two-dimensional intact mtDNA agarose gel electrophoresis (2D-IMAGE) profiling, which detected approximately two-dozen whole genome structures, which we have characterized through multiple molecular approaches. The major topoisomer forms were well conserved across many cell and tissue types, but differences unique to certain cells and tissues were also present. Linear mtDNA, which is not adequately resolved from circular DNA by conventional 1D electrophoresis, is increased by oxidative stress. Increased steady-state expression levels of TFAM altered the relative distribution and absolute abundance of topoisomers, by increasing covalently closed circles and extent of their supercoiling. Additional studies are underway to examine mtDNA by 2D-IMAGE in mouse models of human disease. doi:10.1016/j.mito.2012.07.094
107 Evidence for mitochondrial adaptation to chronic glucose exposure in beta cells Presenter: Brett A. Kaufman Jill E. Kolesara, Yumiko V. Taguchia, Rebecca L. Cramera, Catherine Wanga, Shih-Hsuan Choua, Scott A. Soleimanpourb, Doris A. Stoffersb, Brett A. Kaufmana a Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States b Department of Medicine and the Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania, Philadelphia, PA 19104, United States Pancreatic beta cells increase in number, size, and insulin synthesis/secretion to compensate for higher endocrine demands driven by increased circulating glucose. We have found that mitochondrial DNA (mtDNA) increases in response to chronic high glucose exposure of pancreatic insulinoma cells, and developed TAQMAN expression arrays to examine changes in mitochondrial factors in these cells. We identified PIF1 and FEN1 as the candidate factors regulating the observed increase in mtDNA content; the full