MS523.L17.Mitochondrial Genetics.Q3.23.pptx

Lecture: Mitochondrial Genetics Presenter: Dr. Darl Swartz Course: Human Genetics MS523 Date: 2/17/23 12/12/ Dr. Darl Ray. Swartz 1 Objectives: 1. Describe the features of the mitochondrial genome relative to the nuclear genome both quantitatively (number per cell) and qualitatively (genomic structure). 2. Describe the types of coding genes within the mitochondrial genome. 3. Explain the inheritance pattern of mtDNA variants and compare this to nDNA variant inheritance. 4. Define homoplasmy and heteroplasmy and how this influences mitochondrial disease genetics and phenotype. 5. Describe the features of mitochondrial biogenesis considering fusion, fission, and regulation of this process via the environment as mediated by master transcription factors (PGC-1 ). 6. Explain why every type of inheritance pattern is observed in genetic mitochondrial diseases and thus why genetic counseling is a challenge. 7. Explain why many genetic mitochondrial diseases have age-dependent penetrance and threshold effects as well as unique phenomic features ranging from cell-type specific to syndromic. 8. Justify using genomic (mt and nDNA) as a first-line tool in diagnosis of primary mitochondrial diseases. 9. Explain how and why exercise (both mental and physical) is important to 2 12/12/2 Dr. Darl Ray. Swartz Outline: I. II. III. IV. V. 12/12/2 Mitochondrial Genome Mitochondrial Inheritance Mitochondrial Function and Biogenesis Genetic Diseases of Mitochondria Multifactorial Diseases Associated with Mitochondria Dr. Darl Ray. Swartz 3 Mitochondrial Genome Kivisild Investigative Genetics (2015) 6:3 12/12/2 Dr. Darl Ray. Swartz 4 Mitochondrial Genome A) Circular dsDNA with so-called heavy and light strands 1) Heavy has more G and designated outer strand 2) As “nucleoid” in terms of chromatin structure B) Called nucleoid when associated with proteins and in tertiary structure 1) Major protein component is TFAM (transcription factor of mitochondria) (a)Major component likely involved in compacting the genome 2) Other proteins include proteins involved in mtDNA replication and transcription factors (a)Single stranded binding proteins (mtSSBP) 12/12/2 Dr. Darl Ray. Swartz Cell 172, January 11, 2018 5 Mitochondrial Genome C) Can have multiple copies per “mitochondria” and multiple mitochondria per cell 1) 1-5 mt genomes/mitochondria and 1 – 100 mitochondria/cell (a) 1 – 500 mt genomes/nuclear genomes 2) Cell type dependent with higher levels in metabolically active cells (a) Cardiac and some types of skeletal muscle (b) Kidney epithelial cells (c) Neurons 12/12/2 Dr. Darl Ray. Swartz JOURNAL OF CELLULAR PHYSIOLOGY 143:160-164 (1990) 6 Mitochondrial Genome D) Genome is 16.6 kbp containing prokaryote-like organization 1) 93% is coding and 7% regulatory 2) No introns 3) Cistronic (one-gene after another) 4) Minimal replications/transcriptional regulatory sites (DNA elements, 7% of genome) (a) Major one at control region of H-stand (so-called D-loop) 5) Genes code for inner membrane proteins, tRNAs, and rRNA 12/12/2 Dr. Darl Ray. Swartz (a) Gene names contain prefix Experimental Gerontology Volume 56, August 2014, Pages 175- 7 Mitochondrial Genome D) 16.6 kbp containing prokaryote-like organization 6) 13 protein-coding genes with most part of the electron transport chain (a)6 different NADH-dehydrogenase genes (MT-ND1 – MT-ND6) (b)Cytochrome B (c) Cytochrome C oxidase (d)2 ATP synthase gene subunits (e)Other 1,500 + mitochondrial proteins coded for by nuclear genes 7) 2 rRNA genes for mitochondrion-specific large (16S) and small (12S) ribosomal subunits (a)Involved in protein synthesis within the matrix 8) 22 tRNA genes involved in mitochondrial protein synthesis (a)Uses fewer than the 64 for cytosolic protein synthesis (b)Only 1 base at wobble position instead of 2-4 in 12/12/2 Dr. Darl Ray. Swartz NATURE REVIEWS | DISEASE PRIMERS VOLUME 2 | 2016 | 1 8 Mitochondrial Genome E) Genome replicated by DNA Pol gamma that is a heterotrimeric protein 1) POLg is the catalytic gamma subunit 2) POLg2 dimer associates with POLg and stimulates activity and processivity 3) DNA Polg has a 10-20X higher error rate than nuclear DNA polymerases (a) Higher rate of spontaneous mutations in mitochondrial genome 4) Twinkle helicase upstream of POL 5) Strands replicated in opposite directions (no lagging strand) 6) Replicated continuously independent of the cell cycle 7) Half life of about 7 days Cell 172, January 11, 2018 12/12/2 Dr. Darl Ray. Swartz 9 Mitochondrial Genome F) Transcription involves nucleus-encoded proteins that are imported into the matrix 1) Few transcription factors since less complex nucleoid (chromatin) 2) TFAM, TFB2M, and POLRMT forms the active transcription site Cell 172, January 11, 2018 12/12/2 Dr. Darl Ray. Swartz 10 Mitochondrial Genome G) Genomes can be all the same (homologous?) or a mixture (heterozygous?) 1) Homoplasmy = all the same 2) Heteroplasmy = different (a) A mixture of potentially disease variants and non-disease variants (i) Inherited (ii)Spontaneous from errors in replication and/or repair (b) Can occur via certain in vitro fertilization methods (i) Buy design (“3-parent” zygotes) (ii)IVF via whole sperm injection (c)Proportion of disease-causing variants can result in variable penetrance and expressivity (i) Mostly threshold-dependent penetrance 12/12/2 Dr. Darl Ray. Swartz http://hihg.med.miami.edu/code/ http/modules/education/Design/ Print.asp? CourseNum=2&LessonNum=4 11 Mitochondrial Genome NaTure revIeWS | GeNeTICs G) Genomes can be all the same (homologous?) or a mixture (heterozygous?) volume 22 | February 2021 | 109 12/12/2 Dr. Darl Ray. Swartz 12 Mitochondrial Inheritance 12/12/2 Dr. Darl Ray. Swartz 13 Mitochondrial Inheritance A) Evolved from phagocytosis of alpha proteobacteria (likely lacking a cell wall) 1) Endosymbiotic theory 2) Biology of mitochondria is somewhat similar to bacteria (a) Circular DNA (b) Reproduction by fission 3) Over evolutionary time, most proteobacterial genes were incorporated into the host genome 12/12/2 Dr. Darl Ray. Swartz 14 Mitochondrial Inheritance B) Eve’s mitochondria and her peopling of the world (mitochondrial haplotype phylogeny) started about 200,000 ya 1) Variants develop via DNA replication/repair errors and subsequent selection (a) Single nucleotide variants (b) Deletions and duplications (c) Mutation rate at 1 – 1000 X 10-9 bp/y (i) Slower in coding regions than non-coding (hypervariable regions) 2) Since haploid, does not have reductive division (meiosis/recombination) so genome sequence related to spontaneous mutations and selection (a) Some evidence of recombination with heteroplasmic cells/individuals 3) Early methods of mitochondrial molecular phylogeny used restriction 12/12/2 Dr. Darl Ray. Swartz length polymorphism/gel electrophoresis to name the haplotypes 15 Mitochondrial Inheritance B) Eve’s mitochondria and her peopling of the world (mitochondrial haplotype phylogeny) started about 200,000 ya 4) Eve’s mitochondria appeared about 200,000 YA giving the L0 haplogroup in Africa 5) Main haplogroups and “progeny” are: (a) L (L0, L1, and L3) with the most genetic diversity (similar to nuclear genomes) (b) Some individuals with L3 migrated out of Africa about 75,000 ya (i) Bottleneck/founder effect decreasing the diversity of the other haplogroups (ii)Diverged into M and N haplogroups (c)M populated mostly South Asia (d) N populated mostly Eurasia > reduced electron transfer efficiency resulting in less efficient/more heat generation of mitochondria (e) M and N divergents (A, B, C, D, and X) populated the Americas about 15,000 ya (f) Some regions with few haplogroups resulting from founder effects 16 12/12/2 Dr. Darl Ray. Swartz Mitochondrial Inheritance B) Eve’s mitochondria and her peopling of the world (mitochondrial haplotype phylogeny) started about 200,000 ya 12/12/2 Dr. Darl Ray. Swartz Kivisild Investigative Genetics (2015) 6:3 17 Mitochondrial Inheritance C) Nearly all mitochondria are inherited maternally via egg 1) Mitochondrial number increases as oocytes develop (a)Primordial germ cell division dilutes mitochondria in oogonia giving 10 - 100 copies (i) If heteroplasmic can have variable distribution of disease-causing variants in resultant mature eggs (ii) Selection for eggs with less mitochondrial disease burden can occur (b)Oogonia mature and replicate mitochondria giving 100,000 – 600,000 in a mature human egg (i) Can have variable heteroplasmy in mature eggs resulting in variable penetrance/expressivity in offspring from 12/12/2 Dr. Darl Ray. Swartz 18 Mitochondrial Inheritance D) During cleavage of embryonic development, no new mitochondria are synthesized resulting in dilution of mitochondria again 1) Can do 8-cell stage or blastocyst testing for disease-causing mtDNA variants in assisted reproductive technologies (a) Proportion of heteroplasmic variants in cells 2) Can do embryo/fetal testing at different stages 12/12/2 NATure GeNeTiCs | VOL 50 | DECEMBER 2018 | 1642–1649 Dr. Darl Ray. Swartz 19 Mitochondrial Function and Biogenesis Liver JOURNAL OF CELLULAR PHYSIOLOGY 143:160-164 (1990) Brain 12/12 Kidney Heart 20 Mitochondrial Function and Biogenesis A) Mitochondria are involved in energy metabolism and other processes essential for most cell types 1) Integrate intermediary metabolism International journal of biological sciences (a) Oxidative phosphorylation > efficient synthesis of ATP 15(12):2707-2718 (b) Fatty acid oxidation (c)Urea cycle (d) Krebs cycle (e) Gluconeogenesis and ketogenesis 2) Non-shivering thermogenesis (beige and brown fat) 3) Amino acid and lipid metabolism 4) Synthesis of haem and iron-sulfur clusters 5) Calcium homeostasis 6) Apoptosis 12/12/20 Dr. Darl Ray. Swartz 21 Mitochondrial Function and Biogenesis B) Mitochondria have different morphologies involving fusion and fission 1) Not always football-shaped in live cells (classical view of football mostly a tissue processing artifact for electron microscopy) (a) Reticular networks formed by fusion are common in metabolically active cells (b) Small spheres and short rods by fission in quiescent cells 12/12/20 Dr. Darl Ray. Swartz NATuRe ReVIews | Molecular cell Biolog VoluMe 11 | deceMBeR 2010 | 873 22 Mitochondrial Function and Biogenesis B) Mitochondria have different morphologies involving fusion and fission 2) Fission like bacterial fission but uses cytoplasmic proteins for pinching of membranes to break (a) Dynamin-like proteins that constrict down the membranes (b) Fission involved in increasing mitochondrial number for cell division (c)Excessive fission can result in loss of nucleoid from mitochondria 3) Fusion also uses cytoplasmic proteins like those involved in vesicle fusion with the membrane (during neurochemical release – SNAPs and SNAREs) (a) Allows for mixing of mtDNA copies for cells with heteroplasmic mitochondria (i) Can complement (repair) mitochondrial defects if good mtDNA genes available 12/12/20 Dr. Darl Ray. Swartz NATuRe ReVIews | Molecular cell Biology VoluMe 11 | deceMBeR 2010 | 873 23 Mitochondrial Function and Biogenesis C) Biogenesis involves replication of mtDNA, synthesis of membrane lipids, synthesis of membrane and matrix proteins 1) mtDNA replication, mtRNA, and mt protein synthesis in genome section 2) Most proteins synthesized in the cytoplasm and imported into mitochondrion (a) Have N-terminal sequence as import signal (b) Transported through outer membrane and inner membrane transporters (i) TOM (ii)TIM (c)Energy dependent process using chaperones to facilitate pulling peptides through and formation of tertiary structure 3) Biogenesis increases during G1/S and upon 12/12/20 Dr. Darl Ray. Swartz upstream signaling associated with increased J. Dudek et al. / Biochimica et Biophysica Acta 1833 (2013) 274– 285 24 Mitochondrial Function and Biogenesis D) In Go/terminally differentiated cells mitochondrial number is increased by increased energy demand 1) Readily occurs in skeletal muscle upon an increase in muscle activity, fat upon cold exposure, and other tissues upon starvation 2) Involves multiple signaling pathways that activate transcription of a master transcriptional regulator of genes involved in energy metabolism (a)Peroxisome proliferator-activated receptor (G) co-activator-1 (PGC-1  (i) Upstream of the transcriptional activator of peroxisome proliferation (ii)A nuclear co-receptor with 12/12/20 Dr. Darl Ray. Swartz Essays Biochem. (2010) 47, 69–84 25 Mitochondrial Function and Biogenesis D) In Go/terminally differentiated cells mitochondrial number is stimulated by increased energy demand 3) Multiple signaling pathways increase the amount or activity of PGC-1that in turn increase transcription of nuclear genes and mt genes involved in mitochondrial biogenesis (a) Numerous growth factor, cytokines, specific adrenergic receptors and high cytosolic calcium can activate transcription (b) Post-translational modification can alter activity 12/12/20 Dr. Darl Ray. Swartz (both up and down) Am J Clin Nutr 2011;93(suppl):884S–90 26 Mitochondrial Function and Biogenesis D) In Go/terminally differentiated cells mitochondrial number is stimulated by increased energy demand 4) PGC-1  downstream targets are genes that up-regulate expression of mitochondrial transcription factors for mitochondrial proteins and nuclear genes for cytoplasmic proteins imported into the mitochondria or involved in mitochondrial fusion 12/12/20 J Cell Mol Med. 2020;24:4892– Dr. Darl Ray. Swartz 27 Mitochondrial Function and Biogenesis E) Loss of mitochondria occur through mitophagy 1) Mostly occurs in damaged/low functionality mitochondria 2) Fission allows for selective removal of defunct mitochondria 3) Involves autophagosome > lysosome pathway 12/12/20 Dr. Darl Ray. Swartz NATuRe ReVIews | Molecular cell Biology 28 VoluMe 11 | deceMBeR 2010 | 873 Genetic Diseases of Mitochondria 12/12 Dr. Darl Ray. Swartz NATURE REVIEWS | DISEASE PRIMERS VOLUME 2 | 2016 | 29 1 Genetic Diseases of Mitochondria A) Defined as a disease caused by mitochondria dysfunction resulting from mtDNA and nDNA variants coding for proteins intrinsic to or involved in formation of mitochondria 1) Current term is primary mitochondrial diseases 2) First genetic link was demonstrated in 1988 for Leber Hereditary Optic Neuropathy (LHON) B) Prevalence in population is age dependent related to age-dependent penetrance 1) About 6/100,000 in children 2) About 23/100,000 in adults 12/12/ Dr. Darl Ray. Swartz 30 Genetic Diseases of Mitochondria C) Complicated inheritance pattern because of variants in nuclear genes and mitochondrial genes involved in forming functional mitochondria 1) Mitochondrial genes variants from mtDNA via maternal inheritance (a) Heteroplasmic proportion (i) Can differ wildly between different cell types (b)De-novo mutations in gametes (c) De-novo mutations in somatic cells 2) Nuclear genes on autosome and X-chromosome (a)Autosomal recessive, autosomal dominant, X-linked dominant, X-linked recessive 3) Variable penetrance and expressivity at the organism, organ, and cell type level (a)Mostly related to % heteroplasmic proportion 4) Many have threshold effect related to heteroplasmic % and age > agedependent penetrance (a)Can be secondary to compensatory mechanisms involved in up-regulating mitochondrial biogenesis to compensate for reduced functionality component that accelerates disease onset 12/12/5) Multifactorial with environmental Dr. Darl Ray. Swartz 31 Genetic Diseases of Mitochondria E) mtDNA variants are either de-novo or maternally inherited 1) De-novo mutations mostly result from deletions of several genes with some examples of: (a) Kearns-Sayre syndrome > progressive myopathy, ophthalmoplegia (affecting ocular extrinsic and intrinsic eye muscles), cardiomyopathy, death usually < 2oyo (b) CPEO (chronic progressive external ophthalmoplegia) > ophthalmoplegia, eyes mostly impacted 12/12/ Dr. Darl Ray. Swartz (c)Pearson syndrome > pancytopenia Gametic or somatic De novo mutation Nucleic Acids Research 47(14) · May 2019 32 Genetic Diseases of Mitochondria E) mtDNA variants are either de-novo or maternally inherited 2) Maternally inherited are SNVs that alter the function of genes (proteins, tRNAs, and rRNAs) with most having heteroplasmy (a)MELAS (myopathy, encephalopathy, acidosis, stroke-like episodes) > symptoms as per abbreviation > tRNA gene variants (b)MERRF (myoclonic epilepsy w/ragged-red fibers) > symptoms as per abbreviation > tRNA gene variants (c) MIDD (maternally inherited diabetes and deafness) > diabetes development and sensorineural hearing loss in adulthood > tRNA gene (d) Myopathy and diabetes > as per name with progressive proximal to distal myopathy and insulin dependent diabetes > tRNA gene (e) NARP (neuropathy, ataxia, retinitis pigmentosa) > symptoms as per abbreviation > ATPase subunit variant (f) MILS (maternally inherited Leigh syndrome) > progressive brainstem disorder > ATP synthase gene (same locus as NARP but different base change) (g)LHON > optic neuropathy > complex I protein variants (h)Leigh-like syndrome > as per + many peripheral nervous system 33 12/12/ Dr. MILS Darl Ray. Swartz Genetic Diseases of Mitochondria F) nDNA variants > 300 with various types of variants (SNVs, copy #, deletions) 1) About 20 newly discovered each year for the last decade 2) Can have mtDNA bits incorporated into the nDNA complicating analysis (need to isolate mt then NGS sequence longprimary read/single molecule Genes interrogated in Centogene’s Panel for using nDNA-linked mitochondrial diseases techniques) 12/12/ Dr. Darl Ray. Swartz 34 Genetic Diseases of Mitochondria G) Phenotypes of mitochondrial diseases are clinically heterogenous 1) Age dependent (a)Specific childhood and adult-onset disease variants (b) Have age-dependent mortality 2) Cell-type specific to syndromic 3) Variant-specific sex bias for some 4) Mostly affect cells with high metabolic activity (a)Neuromuscular system (i) CNS (ii)PNS (iii) Sensory structures (mostly ocular and otic) (iv) Skeletal muscles (b) Heart (c)Kidney 12/12/ Dr. Darl Ray. Swartz NATURE REVIEWS | DISEASE PRIMERS VOLUME 2 | 2016 | 1 35 Genetic Diseases of Mitochondria H) Clinical diagnosis involves numerous phenomic and metabolomic markers, biopsy/histology of skeletal muscle, and genetic testing 1) Phenomic include physical features described for many of the diseases, neurological exams, cardiovascular physiology, and neuroimaging 2) Metabolomic mostly focus on plasma or cerebrospinal fluid markers of mitochondrial insufficiency 3) Biopsy focuses on skeletal muscle and measurement of mitochondrial enzyme activity in muscle fibers 4) Genetic testing can focus on mtDNA or mtDNA + nDNA (a) mt DNA mostly complete genome sequence with the ability to detect > 5% heteroplasmy (b) nDNA mostly via targeted exomics/NGS for known or suspected disease-causing variants (i) So many that usually do not enrich in known genes, just do whole genome and focus on known/suspect genes (c)Trend towards early genetic testing because of the complexity of the 36 12/12/ Dr. Darl Ray. Swartz phenotypic features Genetic Diseases of Mitochondria H) Clinical diagnosis involves numerous phenomic and metabolomic markers, biopsy/histology of skeletal muscle NATURE REVIEWS | DISEASE PRIMERS VOLUME 2 | 2016 | 1 12/12/ Dr. Darl Ray. Swartz 37 Genetic Diseases of Mitochondria I) Treatment focuses on slowing progression of disease and supportive care 1) Numerous compounds tried/used in a disease-specific manner (a) Antioxidants (b) Enzyme supplementation for nDNA variants (c)Vitamin/co-factor therapy (B2, Coenzyme Q) J) Genetic therapy cure has potential for nDNA causing variants as per other nDNA disease causing variants K) Genetic therapy of existing patients for mtDNA complicated by inaccessibility of 12/12/ mtDNA by current technologiesDr. Darl Ray. Swartz 38 Genetic Diseases of Mitochondria L) Current clinical trials are using nDNA gene transfer (virally mediated) for mtDNA genes for LHON Yu-Wai-Man et al., Sci. Transl. Med. 12, eaaz7423 (2020) 9 December 1) Mt DNA gene has mitochondrial protein targeting sequence fused to the repaired protein 2) Eye (internally) is immunoprotected (from) making immune response to virus carrier less of a problem 3) Initial clinical trials used unilateral injection of the gene-containing virus 4) Observed improvement in bestcorrected visual acuity in both eyes with animal studies suggesting a transfer of virus via the optic nerve 12/12/ Dr. Darl Ray. Swartz between the eyes 39 Genetic Diseases of Mitochondria M) Assisted reproductive technologies can be used for future offspring for mtDNA variants 1) Testing of blastocyst for variant and degree of heteroplasmy to select which to implant 2) Three parent zygote via transfer of egg nucleus of diseased mother into non-diseased enucleated egg then fertilization (or derivatives thereof) NATure GeNeTiCs | VOL 50 | DECEMBER 2018 | 1642–1649 12/12/ Dr. Darl Ray. Swartz 40 tifactorial Diseases Associated with Mitochondria 12/12/ Dr. Darl Ray. Swartz https://www.hopeforfatigue.org 41 tifactorial Diseases Associated with Mitochondria A) Numerous environmental chemicals, drugs, and antibiotics can affect mitochondrial function 1) Mitochondrial toxins 2) DNA polymerase inhibitors used for HIV/HBV treatment > inhibit DNA POL 3) Some antibiotics (aminoglycosides) can cause organ of Corti hair cell loss and deafness for specific mtDNA variants (a)Variants in 12s rRNA gene make rRNA more like bacterial rRNA (b)Antibiotics selectively concentrated by hair cells and inhibit mt protein synthesis eventually leading to hair cell death (c) Specific variants at 5 – 20% in various populations studies (i) Pharmacogenomics should be 12/12/ Dr. Darl Ray. Swartz Journal of Otology 12 (2017) 1e8 42 tifactorial Diseases Associated with Mitochondria B) Gradual mitochondrial decline with aging 1) Potential for links to neurological disorders Correlates of Peripheral Blood Mitochondrial DNA Content in a General Population, Am J Epidemiol. 2016;183(2):138–146 12/12/ Dr. Darl Ray. Swartz 43 tifactorial Diseases Associated with Mitochondria C) Diet effects associated with obesity and adult-onset diabetes 1) Mitochondria are directly involved in  -cell signaling (a) Selective metabolism of glucose into G-6-P then shunting glycolysis products to mitochondria to increase ATP (b) ATP inactivates/closes K+ channel allowing depolarize the membrane (c)Depolarization activates voltage-gated Ca2+ channel (d) Calcium stimulates release of insulin from vesicles much like occurs at the axon terminal 12/12/ Dr. Darl Front. Ray. Swartz Cell Dev. Biol., 23 May 2017 44 tifactorial Diseases Associated with Mitochondria C) Diet effects associated with obesity and adult-onset diabetes 2) Several GWAS variants associated with type II diabetes involved in mitochondrial biogenesis (a) PPAR (b) PGC-1 (c)And a few more (d) Diet induces changes (reversibly) in methylation of promoter region for PGC-1 3) Some diabetes drugs target transcription factors that stimulate mitochondrial biogenesis (a) PPAR-agonists (thaizolidendiones/TZDs) to address 12/12/ Dr. Darl Ray. Swartz insulin resistance CMAJ January 18, 2005 172 (2) 213-226 45 tifactorial Diseases Associated with Mitochondria D) Exercise (mental and physical) stimulates mitochondrial biogenesis ameliorating many diseases associated with environmental effects 1) Signaling to PGC-1via multiple pathways 12/12/ Dr. Darl Ray. Swartz Am J Clin Nutr 2011;93(suppl):884S–90S. 46 Copyright Notice All materials found on Geisinger Commonwealth School of Medicine’s course and project sites may be subject to copyright protection, and may be restricted from further dissemination, retention or copying. Disclosure I have no financial relationship with a commercial entity producing health-care related products and/or services.

MS523.L17.Mitochondrial Genetics.Q3.23.pptx

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