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Targeted massively parallel sequencing detects exonic copy number changes in patients with mitochondrial disorders
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Abstracts
We additionally present the hypothesis that acute metformin challenge can provoke measurable, graded autonomic responses in healthy individuals. In rats acute metformin challenge reduces blood pressure and increases heart rate. Preliminary human data suggests subtle effects on heart rate variability/resting sympathetic tone even in the absence of subjective effects. An inexpensive, safe, and sensitive test of aggregate systemic mitochondrial complex 1 functional reserve would provide a useful tool for community screening, constraining genomic searches, stratifying patients for novel mitochondrial treatments, and possibly identifying genotype-phenotype correlations associated with augmented complex 1 activity/metformin insensitivity. doi:10.1016/j.mito.2013.07.067
73 Enhanced detection sensitivity and diagnostic yield for mtDNA deletion syndrome by massively parallel sequencing Presenter: Guo-Li Wang Guo-Li Wang, Fang-Yuan Li, Victor Wei Zhang, Jing Wang, Lee-Jun Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Body of Abstract: Background: Mitochondrial DNA (mtDNA) deletions have been associated with heterogeneous types of mtDNA deletion syndromes. Unlike nuclear gene mutations, deleterious mtDNA mutations, including large mtDNA deletions, often occur at variable degrees of heteroplasmy in differentially affected tissues leading to greatly variable clinical phenotype. The ability to detect low levels of mtDNA mutation heteroplasmy is particularly important in counseling the inheritance risk to family members. Detection of mtDNA deletions by Southern blot is limited to not only the amount of DNA required for such analysis but also its detection sensitivity. Objective: To evaluate the advantages of massively parallel sequencing (MPS) in the detection of mtDNA deletions by comparison with traditional Southern blot analysis. Methods: The intact circular mitochondrial genome was amplified as a single amplicon using a long-range PCR-based enrichment approach followed by massively parallel sequencing (mitoNGS). Southern blot analysis was carried out by RFLP using digoxigenin (DIG) labeled mtDNA probes. Results: A total of 1193 and 11,329 specimens from patients with clinical indication of mitochondrial related disorders were analyzed by mitoNGS and Southern blot analysis, respectively. MtDNA deletions were detected in 46 (3.86%) and 132 (1.17%) samples, respectively. Forty three MitoNGS positive samples have also been analyzed by Southern method, which could detect mtDNA deletions unambiguously in only 15 (35%) samples. These results are consistent with the observation that diagnostic yield of mitoNGS is 3 times higher than that of Southern analysis. The majority of low levels of mtDNA multiple deletions were not detected by Southern. In addition, using our analytical algorithm, the deletion junction sequences can be easily determined. Conclusions: mitoNGS analysis is a far more sensitive method than Southern in the detection of mtDNA deletions. Not only the low levels of mtDNA deletions can be detected, but also the multiple deletion junction sequences can be unequivocally determined. Thus, mitoNGS can be considered as the first line testing for mtDNA related disorders with a much improved diagnostic yield. doi:10.1016/j.mito.2013.07.068
74 Targeted massively parallel sequencing detects exonic copy number changes in patients with mitochondrial disorders Presenter: Victor Wei Zhang Yanming Feng, Guoli Wang, Victor Wei Zhang, Lee-Jun Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Body of Abstract: Massively parallel sequencing (MPS) technology has now been applied to clinical molecular diagnosis. Since 85% of all known mutations are in the coding regions and the splice sites, capture-based exon targeted sequencing, including a panel of related genes or whole exome, has become a cost-effective way to identify genetic alteration. Clinical laboratories have established validated next generation sequencing (NGS) approaches for many of genetic disease tastings for clinical utility. The NGS-based approaches can provide target sequence information of multiple genes simultaneously. However, most analytical algorithms used for NGS do not take large exonic deletions into consideration. An extra step, such as targeted array comparative genomic hybridization (aCGH), has often been employed to detect copy number variants (CNVs). We proposed a CNV detection algorithm, in which a reference sequence coverage file was generated using the normalized average coverage of each exon from a group of normal samples, followed by the comparison of the normalized coverage file of the testing sample with that of normal samples. The differences then allow the CNV calls. We validated our algorithm with 8 patients' samples with mitochondrial disorders in which CNVs were identified previously by aCGH. All CNVs were accurately detected, including both homozygous and heterozygous single-exons or multiple exon deletions. In addition, NGS may detect apparently homozygous point mutations, in which the second mutant allele is actually an exonic deletion encompassing the exon containing the point mutation. Thus, the validated MPS approach allows comprehensive analysis of point mutations and copy number changes. doi:10.1016/j.mito.2013.07.069
75 Pyrrole-imidazole polyamide, a synthetic DNA-binding compound, is effective at increasing levels of wild-type mtDNA in both cybrid cells and MELAS patient-derived fibroblast cells with the MELAS A3243G mutation by a selective promotion of wild-type replication Presenter: Takamitsu Yano Takamitsu Yanoa,b,c, Nono Takeuchi-Tomitac, Takuya Uedac, Richard H. Haasa,b a Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA b The Mitochondrial and Metabolic Disease Center, University of California San Diego, La Jolla, CA 92093, USA c Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-856, Japan Mutations in mtDNA cause a wide spectrum of human diseases. However, there is currently no effective cure for patients with mtDNA mutations. In the majority of mtDNA disease patients, wild-type and mutant molecules coexist in the same cells, a condition termed heteroplasmy. Oxidative phosphorylation function is impaired below certain threshold levels of wild-type mtDNA. Therefore, the selective increase of wild-type mtDNA levels above the threshold can be an
Abstracts
We additionally present the hypothesis that acute metformin challenge can provoke measurable, graded autonomic responses in healthy individuals. In rats acute metformin challenge reduces blood pressure and increases heart rate. Preliminary human data suggests subtle effects on heart rate variability/resting sympathetic tone even in the absence of subjective effects. An inexpensive, safe, and sensitive test of aggregate systemic mitochondrial complex 1 functional reserve would provide a useful tool for community screening, constraining genomic searches, stratifying patients for novel mitochondrial treatments, and possibly identifying genotype-phenotype correlations associated with augmented complex 1 activity/metformin insensitivity. doi:10.1016/j.mito.2013.07.067
73 Enhanced detection sensitivity and diagnostic yield for mtDNA deletion syndrome by massively parallel sequencing Presenter: Guo-Li Wang Guo-Li Wang, Fang-Yuan Li, Victor Wei Zhang, Jing Wang, Lee-Jun Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Body of Abstract: Background: Mitochondrial DNA (mtDNA) deletions have been associated with heterogeneous types of mtDNA deletion syndromes. Unlike nuclear gene mutations, deleterious mtDNA mutations, including large mtDNA deletions, often occur at variable degrees of heteroplasmy in differentially affected tissues leading to greatly variable clinical phenotype. The ability to detect low levels of mtDNA mutation heteroplasmy is particularly important in counseling the inheritance risk to family members. Detection of mtDNA deletions by Southern blot is limited to not only the amount of DNA required for such analysis but also its detection sensitivity. Objective: To evaluate the advantages of massively parallel sequencing (MPS) in the detection of mtDNA deletions by comparison with traditional Southern blot analysis. Methods: The intact circular mitochondrial genome was amplified as a single amplicon using a long-range PCR-based enrichment approach followed by massively parallel sequencing (mitoNGS). Southern blot analysis was carried out by RFLP using digoxigenin (DIG) labeled mtDNA probes. Results: A total of 1193 and 11,329 specimens from patients with clinical indication of mitochondrial related disorders were analyzed by mitoNGS and Southern blot analysis, respectively. MtDNA deletions were detected in 46 (3.86%) and 132 (1.17%) samples, respectively. Forty three MitoNGS positive samples have also been analyzed by Southern method, which could detect mtDNA deletions unambiguously in only 15 (35%) samples. These results are consistent with the observation that diagnostic yield of mitoNGS is 3 times higher than that of Southern analysis. The majority of low levels of mtDNA multiple deletions were not detected by Southern. In addition, using our analytical algorithm, the deletion junction sequences can be easily determined. Conclusions: mitoNGS analysis is a far more sensitive method than Southern in the detection of mtDNA deletions. Not only the low levels of mtDNA deletions can be detected, but also the multiple deletion junction sequences can be unequivocally determined. Thus, mitoNGS can be considered as the first line testing for mtDNA related disorders with a much improved diagnostic yield. doi:10.1016/j.mito.2013.07.068
74 Targeted massively parallel sequencing detects exonic copy number changes in patients with mitochondrial disorders Presenter: Victor Wei Zhang Yanming Feng, Guoli Wang, Victor Wei Zhang, Lee-Jun Wong Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, United States Body of Abstract: Massively parallel sequencing (MPS) technology has now been applied to clinical molecular diagnosis. Since 85% of all known mutations are in the coding regions and the splice sites, capture-based exon targeted sequencing, including a panel of related genes or whole exome, has become a cost-effective way to identify genetic alteration. Clinical laboratories have established validated next generation sequencing (NGS) approaches for many of genetic disease tastings for clinical utility. The NGS-based approaches can provide target sequence information of multiple genes simultaneously. However, most analytical algorithms used for NGS do not take large exonic deletions into consideration. An extra step, such as targeted array comparative genomic hybridization (aCGH), has often been employed to detect copy number variants (CNVs). We proposed a CNV detection algorithm, in which a reference sequence coverage file was generated using the normalized average coverage of each exon from a group of normal samples, followed by the comparison of the normalized coverage file of the testing sample with that of normal samples. The differences then allow the CNV calls. We validated our algorithm with 8 patients' samples with mitochondrial disorders in which CNVs were identified previously by aCGH. All CNVs were accurately detected, including both homozygous and heterozygous single-exons or multiple exon deletions. In addition, NGS may detect apparently homozygous point mutations, in which the second mutant allele is actually an exonic deletion encompassing the exon containing the point mutation. Thus, the validated MPS approach allows comprehensive analysis of point mutations and copy number changes. doi:10.1016/j.mito.2013.07.069
75 Pyrrole-imidazole polyamide, a synthetic DNA-binding compound, is effective at increasing levels of wild-type mtDNA in both cybrid cells and MELAS patient-derived fibroblast cells with the MELAS A3243G mutation by a selective promotion of wild-type replication Presenter: Takamitsu Yano Takamitsu Yanoa,b,c, Nono Takeuchi-Tomitac, Takuya Uedac, Richard H. Haasa,b a Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA b The Mitochondrial and Metabolic Disease Center, University of California San Diego, La Jolla, CA 92093, USA c Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-856, Japan Mutations in mtDNA cause a wide spectrum of human diseases. However, there is currently no effective cure for patients with mtDNA mutations. In the majority of mtDNA disease patients, wild-type and mutant molecules coexist in the same cells, a condition termed heteroplasmy. Oxidative phosphorylation function is impaired below certain threshold levels of wild-type mtDNA. Therefore, the selective increase of wild-type mtDNA levels above the threshold can be an