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Genetics of Mitochondrial Respiratory Chain Disorders mitochondria nucleus Lee-Jun C. Wong, Ph.D. Molecular and Human Genetics Baylor College of Medicine.

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Presentation on theme: "Genetics of Mitochondrial Respiratory Chain Disorders mitochondria nucleus Lee-Jun C. Wong, Ph.D. Molecular and Human Genetics Baylor College of Medicine."— Presentation transcript:

1 Genetics of Mitochondrial Respiratory Chain Disorders mitochondria nucleus Lee-Jun C. Wong, Ph.D. Molecular and Human Genetics Baylor College of Medicine Dept Medical Genetics Zhong-Shang University, China May 28, 2007

2 mitochondrion The only animal cellular organelle that contains its own DNA Hundreds to thousands of mitochondria per cell Egg cells: ~200,000, sperm cells: ~ copies of mtDNA per mitochondrion

3 Major Function of mitochondria: electron transport chain Oxidative Phosphorylation producing energy, ATP NADH FADH 2 O2O2 ADP H2OH2O ATP NAD FAD Proton gradient Outer membrane Inner membrane Electron transport chain Matrix

4 Respiratory chain subunits encoded by two genomes: Nuclear and Mitochondria 7/43 0/4 1/11 3/13 2/13 Mito/ nuclear complex I: NADH DH II: SDH III: cyt c redIV: COXV: ATPase

5 Outer membrane Inner membrane Matrix cytosol

6 A3243G A8344G G8363A T8993G/C G11778A G3460A T14484C Eag I (2567) Hind III (12571) Hind III (11681) Hind III (6204) SnaBI (10737) BamHI (14258) PvuII (2650) Human Mitochondrial DNA Double stranded bp 13 mRNA genes 22 tRNA genes 2 rRNA genes Non- Universal Codon Light strand: 8 tRNA and 1 mRNA (ND6) Heavy strand: 14 tRNA, 2 rRNA, and 12 mRNA Polycistronic with posttranscriptional processing

7 Unique Features of Mitochondrial Genome No introns Except ~1.2kb (D-loop) at the origin of replication the remaining are coding regions Both strands are transcribed ND6 is encoded by light strand ATP6 and ATP8 are overlapped using different reading frame Mutations have been reported in all 13 mRNA, 2rRNA, and all tRNA (except tRNA Arg) Polycistronic

8 Characteristics of Mitochondrial Genetics Maternal inheritance High Mutation Rate limited proof reading & repair Lack of protective histone proteins close to the site of ROS production Heteroplasmy Threshold Effect Heterogeneous Expression Mitotic segregation

9 Homoplasmy –0 or 100% Heteroplasmy –Between 0-100% Homoplasmy and Heteroplasmy

10 Johns, D. New Eng J Med 1996;333:

11 Mitochondrial DNA : common point mutations MELAS: Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episodes. A3243G (80%), T3271C, in tRNA Leu(UUR) MERRF: Myoclonic epilepsy, Ragged Red Fibers. A8344G (80%), T8356C, in tRNA Lys NARP: Neuropathy, Ataxia, Retinitis Pigmentosa. Leigh disease. T8993G, T8993C, in ATPase 6 LHON: Leber Hereditary Optic Neuropathy. G11778A, G3460A, in ND4 and ND1 Diabetes/deafness: A3243G

12 MELAS: Mitochodrial Encephalopathy Lactic Acidosis and Stroke-like episodes The most common mtDNA point mutation: A3243G (80%) A severe mutation, usually heteroplasmy. Homoplasmy not seen Disease severity correlates with levels of mutant loads in affected tissues Sporadic or maternal inheritance Mechanism of pathogenesis –Abn RNA processing –tRNA post translation modification –Stability of tRNA –Aminoacylation –Protein translation

13 Mutation hot spot

14 MERRF: Myoclonic Epilepsy, Ragged Red Fibers Most common merrf mutation is A8344G (80%) usually heteroplasmy, not as severe as A3243G mutation, higher threshold Disease severity correlates with levels of mutant loads in affected tissues Require high level of mutant load (>60%) to show clinical symptoms Mitochondrial proliferation

15 Mutation hot spot

16 MRNA mutations T8993G (Leu to Arg in ATPase6): Continuous phenotypic spectrum nl > RP > NARP > Leigh syndrome depends on % mutant heteroplasmy N A RP = Neuropathy/Neurogenic weakness = Ataxia = Retinitis pigmentosa NARP:

17 Leigh Syndrome Mitochondrial encephalopathy –Presents in infancy –Psychomotor regression –Signs of brainstem dysfunction –Ataxia –Often fatal Characteristic MRI findings

18 Spectrum of Clinical Phenotypes for T8993G Based on Percentage of Mutant Mitochondria 0% 100% 75% 60% 90% Retinitis Pigmentosa NARP Leigh Syndrome Normal Carelli et al. (2002) Arch Neurol 59:

19 Percentage of mtDNA in Leucocytes Carrying the T8993G Mutation 94% Leigh 80% NARP 31% carrier 82% Phenotypically normal Failure to thrive Developmental delay hypotonia Failure to thrive Developmental regression Hypotonia Seizures Abn MRI

20 Tissue% mutant Brotherblood94 (Leigh)buccal97 hair95 Probandblood80 (NARP)buccal81 hair81 Sisterblood82 (?)buccal92 hair45 Motherblood31 (carrier)buccal36 hair18 Tissue Variation of T8993G mtDNA

21 T8993G NARP/Leigh syndrome: a continuous phenotypical spectrum Roughly correlates with heteroplasmy Heteroplasmy variation important Known heteroplasmy may not fully explain all the variation in phenotype Prenatal testing: caution Age Tissue distribution Modifier gene Genetic background

22 Lebers Hereditary Optic Neuropathy (LHON) Mostly involve homoplasmic mutations: 80% G11778A in ND4, 15% G3460A in ND1 A degenerative eye disease Age of onset: mid 20s Variable penetrance, 20-80%, with male to female ratio of about 4 to 1 Missense mutation in conserved domain of complex 1 subunits Primary mutations and secondary mutations

23 G14459A mutation A72V in ND6 Variable expression: LHON, dystonia IV-2 Limp Hemiparesis MRS lactate peak IV-8 Hemiparesis NF1 Global delay Dysarthria spasticity MRS lactate peak IV-10 Stroke Dystonia Developmental delay short Spasticity Hearing loss MRS lactate peak III-6 NF1 unaffected All Homoplasmy IV-9: unaffected

24 Proband, patient IV-10: Bilateral increased T2 signal in the putamen MRS shows elevated lactate Gropman, chen, Perng, Krasnewich, Chernoff, Tifft, and Wong. AJMG 2004;124A:

25 patient IV-2 bilateral symmertric increased T2 signal in the putamen patient IV-8 unilateral increased T2 signal in the putamen

26 mtDNA point mutations tRNA pathogenic ones are usually heteroplasmic Affect overall mito protein translation, all subunits encoded by mtDNA mRNA Affect a specific protein subunit Homoplasmic missense mutations do occur Distinguish primary mutations and secondary mutations

27 MitoDNA pedigree

28 M: 90% A3243G Melas family

29 48 y o 47 y o 28 y o B: 8% H: 6% C: 18% B: 12% H: 33% C: 30% B: 23% H: 15% C: 16% asymptomatic Diabetes Hearing loss Macular pattern retinal dystrophy Diabetes Hypertension Heart disease A3243G: diabetes, hearing loss, retinopathy Am J Ophthalmol 1997;124:219

30 B: 0%16%14% B: 4%30% ~0 % 0%~0% B: 5%~0% 10% 5% 1993, B:48% 1995, B:62% 1995, H:60% 1993, B:75% 65% 43% 1995, B:nd 65% 54% 1995, H:nd 65% 43% II III I IV ~0% A8344G MERRF family

31 % 88%82% 83% 73% 60% 84% 90%94% 98% 87%91% 73% I II III Mitochondrial Cardiomyopathy and peripheral neuropathy Mutation in tRNA lys (8363G>A)

32 Single deletionMultiple deletion depletion

33 F8389-R8529 F R12170 F3212- R3319 F16498-R32 mtDNA deletions

34 Mitochondrial DNA Deletion Syndrome Kearns Sayre syndrome Ophthalmoplegia (inability to move eyes) Ptosis (droopy eyes Onset second decade muscle Pearson syndrome Sideroblastic anemia with pancytopenia Exocrine pancreatic insufficiency Onset: early infancy Blood Multisystemic disease PEO Mitochondrial myopathy

35 Muscle or Blood? KSS vs Multisystemic Disorder

36 6 yo boy presented with Addison disease, Died of ARDS at 8 years of age Deletion mutant in Autopsy tissues 5 kb common deletion in every autopsy tissue

37 I II III IV heart problems wheelchair bound MR cleft lip 14 Clearly Kearns Sayre Syndrome, but deletion was not detected in blood.

38 mtDNA multiple deletion and depletion syndrome Caused by nuclear genes responsible for the maintenance of mtDNA integrity, genes involved in mtDNA replication and balance of dNTP pools Disorders of intergenomic signaling

39 DNA replication Transcription Translation Spinazzola and Zeviani, Gene 354 (2005) MPV17 DNC

40 DNA polymerase gamma mutations Cause mtDNA multiple deletions and depletion Autosomal recessive: eg, Alpers synd (infantile CNS and liver disease) Autosomal dominant: Progressive external ophthalmoplegia

41 Autosomal dominant form of progressive external ophthalmoplegia (adPEO) Twinkle gene: DNA helicase ANT1 (Adenine Nucleotide Translocase 1) POLG

42 Hepatocerebral form of mtDNA depletion syndrome infantile hepatic failure DGUOK (deoxyguanosine kinase) MPV17, a mitochondrial inner membrane protein POLG Autosomal recessive

43 P.W65X C.487ins4 P.W65X +c.487ins4 + P.W65X C.487ins4 DGUOK mutations cause mtDNA depletion and respiratory chain enzyme deficiencies Hepatocerebral form of mtDNA depletion syndrome Both mutations are deleterious. Missense mutations in DGK appear to have similar clinical phenotype

44 The liver biopsy showed portal fibrosis with extension into the lobule to surround hepatocytes. The hepatocytes are large with microvesicular steatosis and oncocytic change. The liver biopsy showed portal fibrosis with extension into the lobule to surround hepatocytes. The hepatocytes are large with microvesicular steatosis and oncocytic change.


46 Myopathic form of mtDNA depletion syndrome TK2 (thymidine kinase) MNGIE Mitochondrial NeuroGastroIntestinal Encephlomyopathy TP (thymidine phosphorylase) Both are Autosomal Recessive


48 Mechanism leading to mtDNA mutations Nucleotide imbalance cause mis- incorporation Lack of DNA repair Acceleration of DNA polymerase activity by increased conc of dTTP Nishigaki Y et al. J Clin Invest. 2003;111:

49 Why Mitochondrial DNA ? Mito dNTP pools are physically separate and are regulated independently More vulnerable to toxic effects of excessive dT because mtDNA is more dependent on dT SALVAGE pathway Lack of an efficient mismatch repair system Nishigaki Y et al. J Clin Invest. 2003;111:

50 Genes encode for complex assembly factors

51 Cytochrome c Oxidase, (Complex IV) Assembly requires a series of factors: SURF1 SCO2 SCO1 COX10 COX15 LRPPRC

52 Complex IV (cytochrome c oxidase) Assembly Most common is mutations in SURF1 Gene, cause Leigh Disease

53 normal 7567 Mt7567 G193S/G193S SCO2 mutation G193S/G193S SCO2 mutation analysis revealed homozygous G193S mutation Isolated COX (cyt c oxidase, complex IV) deficiency Cardiomyopathy Lactic acidosis


55 Mitochondrial Fission/fusion

56 C. Ultrastructure of mitochondria from proband's muscle biopsy showing abnormal mitochondrial cristae forming concentric lamellae.(TEM,Mag.X30,000)



59 Gomori trichrome stain Muscle fibers have mild to moderate mitochondrial proliferation (Red rim & speckled sarcoplasm)

60 Muscle fibers with mitochondrial proliferation stain darkly for succinic dehydrogenase (SDH). SDH is the most sensitive stain for detecting mitochondrial proliferation. Increased SDH staining in muscle fibers with mitochondrial proliferation Adult Child Mitochondrial disorder normal

61 Cytochrome oxidase (COX) stain Type I fibers stain more darkly than type II. Several fibers have no staining for cytochrome oxidase (COX). On SDH, COX- muscle fibers may be normal or have increased staining In normal biopsies virtually all fibers have staining for COX.

62 COX deficiency: Child Cytochrome oxidase (COX) levels are reduced in all muscle fibers (Left) Normal muscle (Right) has COX staining in all muscle fibers: More in type I than type II Normal Mitochondrial disorder Cytochrome oxidase (COX) stain

63 SDH stain Muscle fibers with excessive SDH staining (left) have reduced or absent COX (right) staining (arrows)

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