Mitochondrial Disorders: Biochemical and Molecular Analysis (Methods in Molecular Biology, Vol. 837)

Description

Due to the complexity of the mitochondrial system and the myriad genes involved in their function, the clinical manifestations of mitochondrial diseases are remarkably variable and heterogeneous. The laboratory and clinical methodologies used to evaluate dysfunction may vary widely, and thusly a systematic presentation of the numerous protocols that are applied to the assessment of these clinically and genetically heterogeneous disorders has proven to be essential.  Mitochondrial Disorders: Biochemical and Molecular Analysis strives to fill this need with a collection of key protocols provided by leading experts in the field.  Beginning with overviews of complexity of mitochondrial and nuclear genome disorders, the book continues with a section devoted to current biochemical protocols and a part focused on the DNA-based approaches used to identify molecular defects.  Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.   Detailed and authoritative, Mitochondrial Disorders: Biochemical and Molecular Analysis serves as a vital guide to researchers seeking to understand this incredibly complicated type of breakdown in cellular biology. Cover_978-1-61779-503-9 FrontMatter Mitochondrial Disorders Preface Contents Contributors Part I: Mitochondrial Disorder Chapter 1: Mitochondrial DNA Mutations: An Overview of Clinical and Molecular Aspects Chapter 1: Mitochondrial DNA Mutations: An Overview of Clinical and Molecular Aspects 1. Introduction 2. History 3. mtDNA Structure 4. mtDNA Mutations 5. mtDNA Rearrangements 6. mtDNA Point Mutations References Chapter 2: Nuclear Gene Defects in Mitochondrial Disorders Chapter 2: Nuclear Gene Defects in Mitochondrial Disorders 1. Introduction 2. Defects in Structural Respiratory Chain Genes 3. Defects in Genes Involved in the Assembly of Mitochondrial Respiratory Chain Complexes 4. Defects in Genes Involved in mtDNA Stability 5. Defects in Genes Involved in Mitochondrial Protein Synthesis 5.1. Molecular Defects in Genes Encoding Mitochondrial tRNA Modifying Enzymes 5.2. Molecular Defects in Genes Encoding Mitochondrial Elongation Factors 5.3. Molecular Defects in MRPS16 and MRPS22 5.4. Molecular Defects in Genes Encoding Mitochondrial Aminoacyl-Transfer RNA Synthetases ( RARS2 , DARS2 , and YARS2) 6. Molecular Defects in Genes Encoding Proteins Involved in Mitochondrial Motility, Fusion, and Fission 7. Molecular Defects in Genes Associated with Secondary Respiratory Chain Defects 8. Other Nuclear Genes Involved in Mitochondrial Function 9. Conclusions References Chapter 3: Diagnostic Challenges of Mitochondrial Disorders: Complexities of Two Genomes Chapter 3: Diagnostic Challenges of Mitochondrial Disorders: Complexities of Two Genomes 1. Introduction 2. Clinical Presentation Informs Approach to Molecular Testing 3. Recommen­dations for Molecular Evaluation of a Patient with Suspected Mitochondrial Disease (Figs.  1 – 3) 4. Conclusions References Part II: Biochemical Analysis of Mitochondrial Disorders Chapter 4: Biochemical Analyses of the Electron Transport Chain Complexes by Spectrophotometry Chapter 4: Biochemical Analyses of the Electron Transport Chain Complexes by Spectrophotometry 1. Introduction 2. Materials 2.1. Materials and Equipment Required for Sample Preparation 2.2. Equipment and Reagents for Enzymatic Assays 3. Methods 3.1. Enriched Mitochondrial Preparation from Cultured Cells 3.2. Tissue Homogenate Preparation 3.3. Citrate Synthase Assay 3.4. Complex I Assay 3.5. Complex II Assay 3.6. Complex II+III Assay 3.7. Complex III Assay 3.8. Complex IV Assay 3.9. Complex V Assay 3.10. Calculations 4. Notes References Chapter 5: Measurement of Mitochondrial Oxygen Consumption Using a Clark Electrode Chapter 5: Measurement of Mitochondrial Oxygen Consumption Using a Clark Electrode 1. Introduction 2. Materials 2.1. Stock Solutions ( see Note 1) 2.2. Equipment and Buffer for Mitochondrial Isolation 2.3. Reagents for Polarography 2.4. Equipment for Polarography 3. Methods 3.1. Isolation of Mitochondria from Tissue 3.2. Preparation of Equipment for Polarography 3.3. Performing Mitochondrial Polarography 3.4. Data Analysis 4. Notes References Chapter 6: Mitochondrial Respiratory Chain: Biochemical Analysis and Criterion for Deficiency in Diagnosis Chapter 6: Mitochondrial Respiratory Chain: Biochemical Analysis and Criterion for Deficiency in Diagnosis 1. Introduction 1.1. Mitochondrial Respiratory Chain 1.2. Spectrophoto 1.3. Analytical Considerations 2. Materials 2.1. Solutions and Reagents 2.2. Instrumentation 2.3. Preparation of Biological Samples for MRC Activity Measurement 2.3.1. Blood Cells (see Note 2) 2.3.2. Skin Fibroblasts (see Note 3) 2.3.3. Fresh and Frozen Tissues (see Note 4) 2.3.4. Isolation of Mitochondria from Fresh Muscle Biopsy (see Note 5) 2.4. Determination of Protein Concentration by Bradford Method ( see Note 6) 3. Methods 3.1. Measurement of Complex I Activity 3.2. Measurement of Complex II Activity 3.3. Measurement of Complex II+III Activity 3.4. Measurement of Complex III Activity 3.5. Measurement of Complex IV Activity 3.6. Measurement of Complex V Activity 3.7. Measurement of Segment I+III Activity 3.8. Measurement of Citrate Synthase Activity 3.9. Calculation of Activity 3.10. Data Analysis: How to Define MRC Deficiency for Diagnosis 4. Notes References Chapter 7: Assays of Pyruvate Dehydrogenase Complex and Pyruvate Carboxylase Activity Chapter 7: Assays of Pyruvate Dehydrogenase Complex and Pyruvate Carboxylase Activity 1. Introduction 1.1. Pyruvate Dehydrogenase Complex 1.2. Dihydrolipoamide Dehydrogenase ( E 3) (and Other Components of PDC) 1.3. Pyruvate Carboxylase 1.4. Citrate Synthase 2. Materials 2.1. Pyruvate Dehydrogenase Complex 2.1.1. Reagents 2.1.2. Reaction Tubes and CO 2 Traps 2.1.3. Equipment 2.1.4. Preparation of Cells and Tissue Samples 2.2. E3 2.3. Pyruvate Carboxylase 2.3.1. Reagents 2.3.2. Preparation of Cells and Tissue Samples 2.4. Citrate Synthase 3. Methods 3.1. PDC Assay 3.2. E3 Assay 3.3. PC Assay ( see Table  4) 3.4. Citrate Synthase 4. Notes References Chapter 8: Assessment of Thymidine Phosphorylase Function Chapter 8: Assessment of Thymidine Phosphorylase Function: Measurement of Plasma Thymidine (and Deoxyuridine) and Thymidine Phosphorylase Activity 1. Introduction 2. Materials 2.1. Reagents 2.2. Standards for HPLC-UV Quantification 2.3. Other Materials and Equipment 3. Methods 3.1. Sample Collection and Preparation 3.2. dThd and dUrd Determination 3.3. TP Activity Assay 4. Notes References Chapter 9: Measurement of Mitochondrial dNTP Pools Chapter 9: Measurement of Mitochondrial dNTP Pools 1. Introduction 2. Materials 2.1. Reagents 2.2. Aqueous dNTP Standards 2.3. Other Materials and Equipment 3. Methods ( see Note 7) 3.1. Isolation of Mitochondria from Cultured Skin Fibroblasts (see Note 8) 3.2. Isolation of Mitochondria from Mouse Liver 3.3. dNTP Extraction from the Mitochondrial Pellets 3.4. DNA Polymerase Assay ( 20) 4. Notes References Chapter 10: Measurement of Oxidized and Reduced Coenzyme Q in Biological Fluids, Cells, and Tissues: An HPLC-EC Method Chapter 10: Measurement of Oxidized and Reduced Coenzyme Q in Biological Fluids, Cells, and Tissues: An HPLC-EC Method 1. Introduction 1.1. CoQ Function in the Electron Transport Chain 1.2. CoQ Redox State and Oxidative Stress 1.3. Clinical Considerations 1.4. Analytical Considerations 2. Materials 2.1. General Guidelines 2.2. Preparation of Standard Solutions 2.3. Instrumentation 3. Methods 3.1. Measurement of Ubiquinol-10 and Ubiquinone-10 in Human Plasma 3.1.1. Calibration of Ubiquinone-10 3.1.2. Calibration of Ubiquinol-10 and Ubiquinone-10 3.1.3. Specimen Collection, Processing, and Storage 3.1.4. CoQ10 Extraction 3.1.5. HPLC Conditions and Sample Analysis 3.2. Measurement of CoQ10 in Breast Milk 3.2.1. Calibration of Ubiquinone-10 3.2.2. Specimen Collection, Processing, and Storage 3.2.3. CoQ10 Extraction 3.2.4. HPLC Conditions and Sample Analysis 3.3. Measurement of CoQ10 in Blood Platelets 3.3.1. Calibration of Ubiquinone-10 3.3.2. Calibration of Ubiquinol-10 and Ubiquinone-10 3.3.3. Blood Platelet Preparation 3.3.4. CoQ10 Extraction 3.3.5. HPLC Conditions and Sample Analysis 3.4. Measurement of Ubiquinol-10 and Ubiquinone-10 in Muscle, Liver, and Brain 3.4.1. Calibration of Ubiquinone-10 3.4.2. Calibration of Ubiquinol-10 and Ubiquinone-10 3.4.3. Specimen Collection, Processing, and Storage 3.4.4. CoQ Extraction 3.4.5. HPLC Conditions and Sample Analysis 4. Notes References Chapter 11: Assay to Measure Oxidized and Reduced Forms of CoQ by LC–MS/MS Chapter 11: Assay to Measure Oxidized and Reduced Forms of CoQ by LC–MS/MS 1. Introduction 2. Materials 2.1. Chemicals and Reagents 2.2. Equipment 3. Methods 3.1. Calibration 3.2. Quality Control 3.3. Mononuclear WBC Pellet Preparation 3.4. Extraction of CoQ9 and CoQ10 3.5. LC–MS/MS Conditions 3.6. Protein Quantification 3.7. Quantification of CoQ9 and CoQ10 4. Notes References Chapter 12: Morphological Assessment of Mitochondrial Respiratory Chain Function on Tissue Sections Chapter 12: Morphological Assessment of Mitochondrial Respiratory Chain Function on Tissue Sections 1. Introduction 2. Materials 2.1. Succinate Dehydrogenase Histochemistry 2.2. Cytochrome c Oxidase Histochemistry 2.3. Immunohisto-chemical Preparations 3. Methods 3.1. Histochemical Staining ( see Note 2) 3.1.1. SDH Histochemical Staining ( see Note 3) 3.1.2. COX Histochemical Staining ( see Note 6) 3.2. Immunohisto-chemical Staining ( see Note 7) 3.2.1. Immunolocalization of Mitochondrial Proteins in Paraffin-Embedded Sections Using ABC Method ( see Note 8) 3.2.2. Simultaneous Visualization F (Double Labeling) of mtDNA- and nDNA-Encoded Subunits of the Respiratory Chain Using Different Fluorochromes ( see Note 9) 3.2.3. Localization of Mitochondrial DNA Using Immunofluorescent Probe ( see Note 11) 4. Notes References Chapter 13: Blue Native Polyacrylamide Gel Electrophoresis Chapter 13: Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Diagnostic Tool for the Detection of Assembly Defects in the Enzyme Complexes of Oxidative Phosphorylation 1. Introduction 2. Materials 2.1. Cell Culture and Sample Preparation 2.2. BN-PAGE 2.3. Western Blotting and Immunodetection 3. Methods 3.1. Cell Culture and Sample Preparation 3.2. BN-PAGE 3.3. Western Blotting and Immunodetection 4. Notes References Chapter 14: Radioactive Labeling of Mitochondrial Translation Products in Cultured Cells Chapter 14: Radioactive Labeling of Mitochondrial Translation Products in Cultured Cells 1. Introduction 2. Materials 2.1. Labeling of Mitochondrially Synthesized Proteins with Radioactive [ 35 S] Methionine and Cysteine 2.2. Sample Preparation 2.3. SDS–PAGE 2.4. Generation and Analysis of the Data 3. Methods 3.1. Labeling of Mitochondrially Synthesized Proteins with Radioactive [ 35 S] Methionine and Cysteine 3.2. Sample Preparation 3.3. SDS–PAGE 3.4. Generation and Analysis of the Data 4. Notes References Chapter 15: Transmitochondrial Cybrids: Tools for Functional Studies of Mutant Mitochondria Chapter 15: Transmitochondrial Cybrids: Tools for Functional Studies of Mutant Mitochondria 1. Introduction 2. Materials 2.1. Cell Culture 2.2. Generation of r 0 Cells 2.3. Transfection of Selection Marker 2.4. Enucleation of Cells 2.5. Generation of Cybrids 3. Methods 3.1. Regular Cell Passages 3.2. EtBr Treatment and Uridine Supplementation 3.3. Transfection of Selection Marker 3.4. Generation of Cybrids 4. Notes References Chapter 16: Fluorescence-Activated Cell Sorting Analysis of Mitochondrial Content, Membrane Potential, and Matrix Oxidant Burden Chapter 16: Fluorescence-Activated Cell Sorting Analysis of Mitochondrial Content, Membrane Potential, and Matrix Oxidant Burden in Human Lymphoblastoid Cell Lines 1. Introduction 2. Materials 2.1. Cell Culture and Treatment 2.2. Fluorescence-Activated Cell Sorting Flow Cytometry 3. Methods 3.1. LCL Culture Plate Preparation 3.2. LCL Incubation with Antimycin A and N -Acetyl-Cysteine 3.3. LCL Incubation with MitoTracker Green FM 3.4. LCL Incubation with TMRE 3.5. LCL Incubation with MitoSOX Red 3.6. LCL Imaging by Fluorescence Microscopy 3.7. FACS Analysis of LCL Fluorescence Intensity 4. Notes References Chapter 17: Molecular Profiling of Mitochondrial Dysfunction in Caenorhabditis elegans Chapter 17: Molecular Profiling of Mitochondrial Dysfunction in Caenorhabditis elegans 1. Introduction 2. Materials 2.1. Total RNA Isolation from Synchronous Worm Populations 2.2. DNA Isolation from Synchronous Worm Populations 2.3. qRT-PCR Analysis of Relative mtDNA Content in Synchronous Worm Populations 2.4. qRT-PCR of Nuclear Gene Relative Expression in Synchronous Worm Populations 2.4.1. DNase Treatment of Total RNA 2.4.2. RT-PCR to Generate cDNA 2.4.3. qRT-PCR to Assay Nuclear Gene Relative Expression 2.5. Affymetrix GeneChip C. elegans Genome Array Analysis 2.5.1. Microarray Performance 2.5.2. Microarray Platform Description 3. Methods 3.1. C. elegans Growth and Treatment 3.2. Pharmacologic Treatment of Synchronous Young Adult or Developing Worms 3.2.1. If Studying the Effects of a Study Drug, Add the Desired Drug or Appropriate Buffer Control to a 10-cm NGM Agar Plate After the Bacteria Lawn Has Dried and Prior to Plating Worms 3.2.2. Worms Can Be Treated with Study Drugs by Feeding Either (a) Young Adults for 24 h or (b) Developing Larvae from the L1-Arrested Stage Through the Young Adult Stage Pharmacologic Treatment of Young Adult Stage Worms Pharmacologic Treatment of Developing Worms 3.3. Total RNA Extraction from Adult C. elegans 3.4. DNA Isolation from Larval and Adult C. elegans 3.4.1. Preparation of Adult Stage Worms for DNA Isolation 3.4.2. Preparation of Larval Stage Worms for DNA Isolation 3.4.3. DNA Isolation from Adult Stage Worms 3.5. qRT-PCR Analysis of Relative mtDNA Content in C. elegans 3.6. qRT-PCR Analysis of Relative Nuclear Gene Expression in C. elegans 3.6.1. DNase Treatment of RNA 3.6.2. RT-PCR to Generate cDNA from DNase-Treated RNA 3.6.3. qRT-PCR 3.7. Affymetrix GeneChip C. elegans Genome Array Analysis 3.7.1. Probe Remapping 3.7.2. Data Normalization 3.7.3. Gene Expression Analysis Methods 4. Notes References Part III: Molecular Analysis of Mitochondrial Disorders Chapter 18: Analysis of Common Mitochondrial DNA Mutations by Allele-Specific Oligonucleotide and Southern Blot Hybridization Chapter 18: Analysis of Common Mitochondrial DNA Mutations by Allele-Specific Oligonucleotide and Southern Blot Hybridization 1. Introduction 2. Materials 2.1. General Guidelines 2.2. ASO Dot Blot for Point Mutation Analysis 2.2.1. PCR Reagents 2.2.2. Agarose Gel Electrophoresis 2.2.3. ASO Probe Labeling 2.2.4. Dot Blot and Hybridization 2.3. Southern Blot Hybridization for Large mtDNA Deletion Analysis 2.3.1. Restriction Digestion and Agarose Gel Electrophoresis 2.3.2. Gel Treatment and Transferring Reagents 2.3.3. Synthesis of DIG-Labeled Probes for Southern Blot by PCR 2.3.4. Hybridization and Wash Reagents 2.3.5. DIG Luminescent Detection Materials 3. Methods 3.1. ASO Dot Blot Hybridization for Point Mutation Analysis 3.1.1. Multiplex PCR to Amplify the Five mtDNA Fragments Containing the 13 Common Point Mutations 3.1.2. Dot Blot 3.1.3. Synthesis of Probes ( Note 6) 3.1.4. Pre-hybridization and Hybridization 3.1.5. Wash and X-ray Autoradiography 3.2. Southern Blot Hybridization for the Detection of Large mtDNA Deletions 3.2.1. Restriction Enzyme Digestion 3.2.2. Agarose Gel Electrophoresis 3.2.3. Agarose Gel Transfer and DNA Fixation 3.2.4. Synthesis of DIG-Labeled Probes 3.2.5. Hybridization and Wash 3.2.6. DIG Illuminescence Detection 4. Notes References Chapter 19: Sequence Analysis of the Whole Mitochondrial Genome and Nuclear Genes Causing Mitochondrial Disorders Chapter 19: Sequence Analysis of the Whole Mitochondrial Genome and Nuclear Genes Causing Mitochondrial Disorders 1. Introduction 2. Materials 2.1. PCR 2.2. Agarose Gel 2.3. Big Dye 2.4. PCR Product and Big Dye Reaction Purification 2.5. Sequencing and Analysis 3. Methods 3.1. Primer Design for Nuclear Genes 3.2. PCR Setup 3.3. PCR Amplification 3.4. Analysis of PCR Product on Agarose Gel 3.5. Purification of PCR Products for Sequencing Analysis 3.6. Big Dye Setup 3.7. Big Dye Purification 3.8. Sample Sequence Analysis 4. Notes References Chapter 20: Utility of Array CGH in Molecular Diagnosis of Mitochondrial Disorders Chapter 20: Utility of Array CGH in Molecular Diagnosis of Mitochondrial Disorders 1. Introduction 2. Materials 2.1. Equipment 2.2. Reagents and Materials ( see Note 2) 3. Methods 3.1. Restriction Digestion of Genomic DNA 3.2. Genomic DNA Labeling 3.3. Removal of Excess Unreacted Cy3-dCTP and Cy5-dCTP ( see Note 7) 3.4. Preparation of Hybridization Samples 3.5. Assembly of Hybridization Chamber 3.6. Washing the Slides 3.7. Scan 3.8. Data Extraction 4. Notes References Chapter 21: Quantification of mtDNA Mutation Heteroplasmy (ARMS qPCR) Chapter 21: Quantification of mtDNA Mutation Heteroplasmy (ARMS qPCR) 1. Introduction 2. Materials 2.1. Pre-PCR 2.2. Post-PCR 3. Methods 3.1. ARMs qPCR Primer Design 3.2. Generation of Positive Control and Normal Control Plasmid DNA 3.3. Pre-qPCR 3.4. PCR and Post-qPCR 3.5. Analysis of Data 3.6. Assessment of PCR efficiency 3.7. Quality Assessment of the ARMS qPCR Quantification of mtDNA Point Mutations 4. Notes References Chapter 22: Measurement of Mitochondrial DNA Copy Number Chapter 22: Measurement of Mitochondrial DNA Copy Number 1. Introduction 2. Materials 2.1. Pre-PCR 2.2. Post-PCR 3. Methods 3.1. qPCR Primer Design 3.2. qPCR Setup 3.3. Analysis of Data 4. Notes References Chapter 23: Determination of the Clinical Significance of an Unclassified Variant Chapter 23: Determination of the Clinical Significance of an Unclassified Variant 1. Introduction 2. Materials 2.1. Internet Access and General Databases 2.2. Disease-Specific Databases for Nuclear Genes-Related Mitochondrial Disorders 2.3. Databases Specific for Mitochondrial DNA (mtDNA) Variants 2.4. Computational Algorithms for the Prediction of Pathogenicity of Missense Variants 2.5. Computational Algorithms for the Prediction of Pathogenicity of Splice Site Alterations 3. Methods 3.1. Interpretation of Missense Variants ( see Note 2) 3.1.1. Human Genome Mutation Database 3.1.2. Single-Nucleotide Polymorphisms 3.1.3. Other Databases or Resources in the Public Domain 3.1.4. Novel Variants of Unknown Significance 3.2. Interpretation of Deletions/Duplications ( see Note 4) 3.3. Interpretation of Intronic and Close-to-Exon–Intron-Boundary Variants 3.4. Interpretation of Mitochondrial DNA (mtDNA) Variants ( see Note 5) 4. Notes References BackMatter_INDEX