DARIA DIODATO Matr. N°. 745002 Identification and functional validation of new mtDNA and nuclear gene variants responsible for mitochondrial disorders Coordinator: Prof. Andrea Biondi Tutor: Dr. Massimo Zeviani I II A mia madre Francesca e mio padre Ernesto, che stimo immensamente e senza i quali tutto questo non sarebbe stato possibile. A Luca, che ha meravigliosamente cambiato la mia vita. III IV Table of Contents Chapter 1: General Introduction 1.1 Mitochondria page 3 1.1.1 Mitochondrial structure page 4 1.1.2 Mitochondrial genetics page 6 1.1.3 Oxidative phosphorylation system (OXPHOS) page 12 1.1.4 Other mitochondrial functions: ROS production and apoptosis page 17 1.2 Mitochondrial disorders page 23 1.2.1 Diagnosis of mitochondrial disorders page 31 1.2.2 Mitochondrial disorders due to mtDNA mutations page 41 1.2.3 Nuclear gene mutations page 55 V 1.2.3.1 Defects of genes encoding structural or assembly factors of respiratory chain complexes page 56 1.2.3.2 Defects of genes altering the stability of mtDNA page 66 1.2.3.3 Defects of genes encoding factors involved in metabolic pathways influencing the biogenesis of mitochondria including OXPHOS page 80 1.2.3.4 Defects of genes encoding factors involved in the biosynthesis of lipids and cofactors page 100 1.3 Identification of the genetic defect in mitochondrial disorders page 103 1.3.1 Cybrids and mtDNA mutations page 105 1.3.2 Exome sequencing and mitochondrial disorders page 111 1.4 Treatment of the mitochondrial disorders page 119 Scope of the thesis page 125 VI References page 129 Chapter 2: MELAS-like encephalomyopathy caused by a new pathogenic mutation in the mitochondrial DNA encoded cytochrome c oxidase subunit I page 161 Chapter 3: Novel (ovario)leukodystrophy related to AARS2 mutations page 195 Chapter 4: VARS2 and TARS2 mutations in patients with mitochondrial encephalomyopathies page 283 Chapter 5: Summary, conclusions and future perspectives page 351 Summary page 353 VII Discussion and Conclusions page 369 Future Perspectives page 386 References page 397 Abbreviations and WEB resources page 401 VIII CHAPTER 1 General introduction: Mitochondria and mitochondrial medicine 1 2 1.1 Mitochondria Mitochondria are double-membrane organelles ubiquitous in eukaryotes and essential for survival. They are responsible for cell energy supply and have also been shown to play an important role in cell signaling, particularly in the apoptotic process (Letai et al, 2006). Mitochondria host different metabolic pathways, including β-oxidation, the Krebs cycle, and lipid and cholesterol synthesis. Their primary function is to produce adenosine triphosphate (ATP), the energy currency of the cell, through the Oxidative Phosphorylation System (OXPHOS). Given their essential role in the human body, dysfunctions eventually occurring in different aspects of their functioning can result in a multitude of clinical conditions, often 3 configuring very severe disease phenotypes (Schapira, 2006). 1.1.1 Mitochondrial structure Origin of mitochondria date back to primitive bacteria-like organism, which developed an endosymbiotic relationship with eukaryotic cells. Their capacity to generate ATP through OXPHOS made them the main intracellular energy source (Margulis et al. 1976). Mitochondria density varies between different tissues depending on their energy demand. Thus, neurons, cardiac and skeletal muscle cells have a high density of mitochondria, and this explains their sensitivity to energy- dependent defects caused by mitochondrial dysfunctions. Mitochondria are surrounded by a double-membrane; the outer membrane is porous and allows diffusion of low molecular 4 weight substances between the cytosol and the intermembrane space (IMS). They have always been considered as non-communicative organelles, but recently an opposite true is emerging: they are involved in frequent fission and fusion events and in exchange of mtDNA (Margineantu et al, 2002). The inner mitochondrial membrane (IM) separates the IMS from the mitochondrial matrix and contains the complexes of the mitochondrial respiratory chain (MRC); it functions also as a barrier to ionic diffusion, thus allowing the formation of the proton gradient necessary to produce ATP. The mitochondrial matrix hosts several enzymes involved in different pathways (i.e. tricarboxylic acid-TCA- cycle and oxidation) and the mitochondrial deoxyribonucleic acid (mtDNA) (Fig.1). 5 1.1.2 Mitochondrial genetics Mitochondria contain their own DNA, a circular double-strand molecule of 16569 base pairs found in multiple copies within the mitochondrial matrix. MtDNA is much smaller than most nuclear genes and extremely compact, containing no introns and small non- coding regions; it codes for 13 protein of the OXPHOS system and for 2 ribosomal and 22 transfer RNAs, necessary for the mitochondrial protein synthesis (Fig.1). Although human mtDNA encodes the rRNAs and tRNAs for protein synthesis, it completely depends upon the nucleus for the provision of the majority of structural MRC proteins and the enzymes for replication, repair, transcription and translation. Recently has been shown that mtDNA co- localizes with various proteins responsible for 6 their maintenance in structures called nucleoids, thought to provide the perfect microenviroment to protect and repair the mtDNA molecules from insults (Garrido et al, 2003). Fig.1 Mitochondrial structure (from Encyclopaedia Britannica, 1998) 7 Fig.2 Mitochondrial DNA (from www.ganfyd.org) MtDNA replicates continuously, in an independent manner from the nucleus, in dividing as well as in non-dividing cells (Chinnery and Samuels, 1999). It is transcribed polycistronically and translated by mitochondrial ribosomes. 8 MtDNA replication requires several nuclear encoded factors such as DNA polymerase gamma (POLG), mitochondrial transcription factor A (mtTFA), mt single-strand binding protein (mtSSB) and enzymes important for the supply of deoxynucleotides, such as thymidine kinase 2 (TK2) and deoxy- guanosine kinase (dGUOK). After fertilization, sperm mtDNA is degraded; therefore mtDNA is inherited exclusively from the mother. Only in one case has been reported a paternal inheritance of a microdeletion in a mtDNA complex I gene (Schwartz and Vissing, 2002) but this possibility has been studied and excluded in several further studies (Filosto et al, 2003; Taylor et al, 2003). There are thousands of mtDNA molecules per cell; these copies can be identical (a condition called homoplasmy) or differ for the presence 9 of one or more variants, inherited or accumulated during the ageing process (heteroplasmy). The percentage of a mutant variant (level of heteroplasmy) can vary between different cells and tissues, because mitochondria are randomly segregated at every cell division. A bottleneck effect is active in primordial female germinal cells (primary oogonia) when wild type and mutant mtDNA molecules are randomly partitioned between oocytes and drastically reduced. This means that in the final oocyte population some cells would carry a high percentage of mutation, others intermediate or low percentages, with important implications for disease severity in the offspring. Neutral mtDNA variants (polymorphisms) are usually homoplasmic, whereas pathogenic changes are usually, but no invariably, 10 heteroplasmic. The percentage of mutated mtDNA will result in a pathogenic phenotype after exceeding a certain threshold, related to the energy demand of different tissues and organs (Schapira 2006, DiMauro and Schon, 2001) and variable amongst different mutations (Fig.3). Leber hereditary optic neuropathy (LHON), the first disorder associated to mitochondrial DNA mutation (Wallace et al. 1988), is caused by homoplasmic mtDNA mutations. Fig. 3 Mitochondrial mtDNA transmission (from www.mitoresearch.org) 11 1.1.3 Oxidative Phosphorylation System Mitochondria main role is to generate ATP trough the oxidative phosphorylation system (OXPHOS). This is carried out by the MRC composed of five multiheteromeric complexes located in the IMM (complex I, CI; complex II, CII; complex III, CIII; complex IV, CIV or cytochrome c oxidase, COX; complex V, CV, or ATP synthase), and two mobile electron shuttles, ubiquinone (Coenzyme Q, CoQ), a lipoidal quinone, and cytochrome c (cyt c), a heme-containing small polypeptide (Fig.4). The electron flow through these components generates an electrical gradient (ΔΨ) and a pH gradient across the IMM. These gradients constitute the driving proton motive force for the phosphorylation of ADP to ATP, operated by CV, and for several other processes, such as 12 Ca++ import inside mitochondria, heat production and protein translocation. Complex I (NADH-ubiquinone oxidoreductase) is the largest complex in the OXPHOS system, composed of ≈45 subunits with a total molecular mass of 1000kDa (Carroll et al. 2006). Seven subunits (ND1- ND6, ND4L) are encoded by mtDNA, the others by nuclear genes. It catalyzes the oxidation of NADH, derived from the oxidation of pyruvate, fatty acids, and amino acids, by ubiquinone (CoQ). Complex II (succinate dehydrogenase ubiquinone–ubiquinol reductase) is the smallest MRC complex and the only membrane-bound member of the tricarboxylic acid (TCA) cycle. It is composed of four subunits all encoded by nuclear genes (SDHA, SDHB, SDHC, and SDHD). CII acts as a 13 succinate dehydrogenase (SDH) and catalyzes the oxidation and dehydration of succinate to fumarate; it takes part in the MRC by coupling this reaction to the reduction of ubiquinone to ubiquinol thus transfer electrons to