Mitochondria Flipped Lesson 4 PDF

Mitochondria MD210 – GGE – Genetics Flipped lesson 4 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Have a basic understanding of mitochondrial genetics and how this relates to inherited disorders of mitochondria • Understand the concept of heteroplasmy and how i...

Mitochondria MD210 – GGE – Genetics Flipped lesson 4 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Have a basic understanding of mitochondrial genetics and how this relates to inherited disorders of mitochondria • Understand the concept of heteroplasmy and how it relates to the phenotype of mitochondrial disease • Describe and recognise the matrilineal mitochondrial inheritance pattern 2 What is a mitochondrion? • A double-membrane bound organelle found in most eukaryotes • “Powerhouse” of the cell – generate ATP • Also involved in cell signalling, growth, death, cell cycle regulation • Has independent (of nuclear DNA) circular genome • Thought to have originated as free living bacteria which were taken up by eukaryotes to carry out oxidative phosphorylation (endosymbiotic theory) Mitochondria • Most nucleated cells contain 500 to 2000 mitochondria • In the cone cell photoreceptor of the eye, mitochondria make up 80% of the intracellular volume • In extra-ocular muscles like the lateral rectus, they account for 60% • In heart muscle they comprise 40% of the volume of the cell By Ivo Kruusamägi - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=54680466 4 Human Genome • Haploid nuclear genome ( c. 23,000 protein coding genes, 3200 Mb (million base pairs) Human Nuclear Genome 1.5 3 44 45 • 23 ds linear chromosomes (50-260 Mb each) • About 1.5% is “protein coding” sequences • Non-functional gene related sequences (pseudogenes and gene fragments) 6.6 Highly conserved (coding) Highly conserved (other) Transposon-based repeats Heterochromatin Other non-conserved • 2 copies per cell • Mitochondrial genome ( 37 genes, 16.6 kb) Mitochondrial genome 5 2 • Circular dsDNA (linear forms do exist) • 16 569 bp (93% coding mRNA or tRNA or rRNA) • Dozens of copies per mitochondrion and 1000’s of this genome / cell 93 5 mtDNA Sequence • The Cambridge Sequence • Determined from several different individuals • UK MRC 1981 • Fred Sanger (2nd Nobel Prize) Fred Sanger Public Domain, https://commons.wikimedia.org/w/ind ex.php?curid=1249932 6 Mitochondrial Genome • Total 37 genes – 24 encode for non mRNA • 22 mitochondrial tRNA (white) • 1 mitochondrial 23S rRNA (blue) • 1 mitochondrial 16S rRNA (blue) • 13 genes transcribed and translated to proteins on mt ribosomes (all related to oxidative phosphorylation) • • • • 7 NADH dehydrogenase subunits (ND – yellow) I 3 cytochrome C oxidase (COX – orange) IV 2 ATPase (ATP - red) V 1 cytochrome B (CYTB - peach) III By Emmanuel Douzery. CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=46726514 7 Energy Factory is an Important Function: Electron Transport Chain (Ox Phos) 8 ROS generation is Coupled to ATP Synthase • Reactive oxygen species (ROS) are generated during ATP synthesis • Rate of O2 consumption has to correspond to rate of ATP synthesis so that ROS are neutralized • Uncoupling of generation and consumption leads to accumulation of ROS: O2 ions and peroxides which are toxic 9 ROS Uncoupling & Accumulation • May occur in some mitochondrial diseases (especially if person has a fever) • ROS accumulation may result in oxidative damage (including to mtDNA) • May trigger apoptosis • CNS may be particularly vulnerable to this • Infection may trigger neurological problems in patients with mt disease Source: Murphy, MP. Biochem J. 2009 Jan 1; 417(Pt 1): 1–13. 10 Mitochondria are encoded mainly on nuclear genome • It takes about 3000 Gene products to make a mitochondrion • So most mitochondrial proteins are encoded on nuclear genome (transferred from mitochondrial genome over evolutionary time) and synthesised in cytoplasm • Therefore most inherited disorders of mitochondria are related to changes in nuclear DNA rather than mtDNA Courtesy Nature Education: https://www.nature.com/scitable/topicpage/mitoch ondria-14053590/ 11 Mitochondria - more than simple energy factories • About 90 mitochondrial gene products needed to make ATP • 13 encoded on mtDNA, 77 on nuclear genome • Remaining gene products serve other functions e.g. • • • • • • Signaling molecules involved in regulation of: Membrane potential Cell-cycle control Development Apoptosis Cellular metabolism 12 Mitochondrial inheritance • Mitochondrial DNA is inherited (almost*) exclusively along maternal line • Fertilised oocyte degrades mtDNA carried by sperm • Mothers transmit mtDNA to sons and daughters • Only daughters can transmit their mtDNA to the next generation • Sons can inherit mtDNA disease but can not transmit Typical pedigree of mitochondrial inheritance of a disorder mt DNA Sequence Variation • mtDNA of any individual shows variation from the Cambridge Consensus Sequence • Most variation is silent polymorphisms • One region (Control Region) is highly polymorphic - useful for forensic purposes (suspects in criminal investigation) 14 Heteroplasmy - mtDNA Sequence Variation • Remember – 100s-1000s of mtDNA copies per cell • Variable – replication not coordinated with cell cycle • Reduced stringency of “proofreading” and replication error correction + no mtDNA repair mechanism = many fold higher sequence variation than genomic DNA • Homoplasmy: all mtDNA sequences the same • Heteroplasmy: variation in mtDNA sequences (common) • Variation between cells (intercellular heteroplasmy) or within cells (intracellular heteroplasmy) 15 Mitochondrial heteroplasmy • Mitochondria do not undergo recombination during cell division – mutation represents only source of mtDNA genetic diversity (more frequent) • Individual may have a mtDNA mutation associated with a dysfunctional allele product • But the proportion of mtDNA sequences in each cell/tissue that carries the dysfunctional allele may vary • During cell division random segregation of mitochondria (and mitochondrial DNA) between 2 daughter cells occurs • However, mtDNA mutations in somatic cells are not heritable 16 Mitochondrial heteroplasmy and “threshold effect” Heteroplasmy & Phenotype • Different oocytes from one woman may vary in the extent of heteroplasmy • Some may get a high proportion of pathogenic mtDNA – termed a genetic bottleneck • Predicting phenotype from genotype and genetic counseling is especially difficult • Phenotype can depend on proportion of pathogenic mtDNA in tissues of each family member – highly variable By James B. Stewart & Nils-Göran Larsson - Keeping mtDNA in shape between generations (PLoS Genetics, 2014), CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=36894437 18 Same sequence – different disease phenotype • It is a hallmark of disease associated with mtDNA that identical mtDNA mutations may be associated with different manifestations • It can also happen that very similar manifestations arise from different mutations 19 Inherited Disease associated with Mitochondrial Genome • Mitochondrial Disease is often associated with multi-organ degenerative disease • Especially high energy organ systems • • • • CNS Muscle (skeletal & heart) Liver Kidney Source: DR Johns: Mitochondrial DNA and disease. N Engl J Med 333:638, 1995 20 Things to Remember 1. Mitochondria have their own genome (mtDNA) which has 16.6 kb and 37 genes (93% protein coding) – there are 1000s of copies per cell 2. Most mitochondrial proteins are synthesised in the cytoplasm from genes on the nuclear genome and transported to the mitochondria 3. Therefore most inherited disorders of mitochondria are related to changes in nuclear DNA rather than mtDNA 4. Mitochondrial DNA is inherited (almost) exclusively along the maternal line 5. Heteroplasmy is variation in mtDNA sequence and is common. The proportion of pathogenic mtDNA in tissues influences the phenotypic expression of mitochondrial diseases (threshold effect) 21