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Genetics of Mitochondrial Respiratory Chain Disorders

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

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: ~10 2-10 copies of mtDNA per mitochondrion

3 Major Function of mitochondria: electron transport chain
Oxidative Phosphorylation producing energy, ATP NADH FADH2 O2 ADP H2O ATP NAD FAD Proton gradient Outer membrane Matrix Inner membrane Electron transport chain

4 Respiratory chain subunits encoded by
two genomes: Nuclear and Mitochondria 7/ / / / /13 Mito/ nuclear complex I:NADH DH II: SDH III: cyt c red IV: COX V: ATPase

5 cytosol Outer membrane Inner membrane Matrix

6 Human Mitochondrial DNA Double stranded 16569 bp 13 mRNA genes
22 tRNA genes 2 rRNA genes Eag I (2567) PvuII (2650) T14484C BamHI (14258) A3243G G3460A Hind III (12571) G11778A Hind III (11681) SnaBI (10737) Hind III (6204) A8344G Non- Universal Codon G8363A T8993G/C 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 Mitochondrial Genetics
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 and Heteroplasmy
0 or 100% Heteroplasmy Between 0-100%

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 tRNALeu(UUR) MERRF: Myoclonic epilepsy, Ragged Red Fibers. A8344G (80%), T8356C, in tRNALys 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 0% Spectrum of Clinical Phenotypes for T8993G Based on Percentage of Mutant Mitochondria “ Normal” 60% Retinitis Pigmentosa 75% NARP Carelli et al. (2002) Arch Neurol 59: 90% Leigh Syndrome 100%

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

20 Tissue Variation of T8993G mtDNA
% mutant Brother blood 94 (Leigh) buccal 97 hair 95 Proband 80 (NARP) 81 Sister 82 (?) 92 45 Mother 31 (carrier) 36 18

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 Leber’s Hereditary Optic Neuropathy (LHON)
Mostly involve homoplasmic mutations: 80% G11778A in ND4, 15% G3460A in ND1 A degenerative eye disease Age of onset: mid 20’s 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 Variable expression: LHON, dystonia
G14459A mutation A72V in ND6 Variable expression: LHON, dystonia I II d86y d48y III IV 5 6 7 d2y d15y 1 2 3 4 11 12 8 9 10 13 14 III-6 NF1 unaffected IV-8 Hemiparesis NF1 Global delay Dysarthria spasticity MRS lactate peak IV-10 Stroke Dystonia Developmental delay short Spasticity Hearing loss MRS lactate peak IV-2 Limp Hemiparesis MRS lactate peak IV-9: unaffected All Homoplasmy

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 patient IV-8 bilateral symmertric increased
T2 signal in the putamen unilateral increased T2 signal in the putamen Gropman, chen, Perng, Krasnewich, Chernoff, Tifft, and Wong. AJMG 2004;124A:

26 mtDNA point mutations tRNA mRNA
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 A3243G Melas family M: 90%

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

30 A8344G MERRF family I 1 2 3 4 5 II B: 0% 16% 14% 1 2 3 6 7 8 4 5
1995, H:60% III ~0% B: 4% 30% ~0% 0% ~0% 9 10 11 12 1 2 3 4 5 6 7 8 13 14 15 16 IV 1993, B:75% 65% % 1995, B:nd 65% % 1995, H:nd 65% % B: 5% ~0% ~0% ~0% 10% ~0% ~0% ~0% ~0% 5%

31 Mitochondrial Cardiomyopathy and peripheral neuropathy
Mutation in tRNA lys (8363G>A) 1 2 3 4 I 76% 5 6 7 8 9 10 2 3 4 1 II 83% 88% 82% 60% 73% 4 5 1 2 3 6 III 94% 90% 84% 91% 87% 98% 73%

32 Single deletion Multiple deletion depletion

33 F16498-R32 mtDNA deletions F3212-R3319 F12093-R12170 F8389-R8529

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 Multisystemic Disorder
Muscle or Blood? KSS vs Multisystemic Disorder

36 5 kb common deletion in every autopsy tissue
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 Clearly Kearns Sayre Syndrome, but deletion was not detected in blood.
1 2 I heart problems 1 2 II 4 3 5 1 2 III 39 39 34 23 wheelchair bound MR cleft lip 1 IV 2 3 14 Clearly Kearns Sayre Syndrome, but deletion was not detected in blood.

38 mtDNA multiple deletion and depletion syndrome
Disorders of intergenomic signaling 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

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

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 DGUOK mutations cause mtDNA depletion
and respiratory chain enzyme deficiencies Hepatocerebral form of mtDNA depletion syndrome P.W65X + c.487ins P.W65X C.487ins4 P.W65X C.487ins4 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

ANT1 ATP ADP dG dA dGK dGMP dAMP dGDP dADP dGTP dATP NDPK dC thymidine TK2 mtDNA thymine TP TK1 dTMP Thymidylate synthase dUMP nDNA mitochondrion cytoplasm POLG dCMP dCTP dTTP dCDP dTDP DNC MPV17

48 Mechanism leading to mtDNA mutations
Nucleotide imbalance cause mis-incorporation Lack of DNA repair Acceleration of DNA polymerase g 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 SURF1 SCO2 SCO1 COX10 COX15 LRPPRC 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 SCO2 mutation analysis revealed homozygous G193S mutation
G193S/G193S normal Mt7567 G193S/G193S 7567 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 Mitochondrial disorder normal
Adult Increased SDH staining in muscle fibers with mitochondrial proliferation Child Muscle fibers with mitochondrial proliferation stain darkly for succinic dehydrogenase (SDH). SDH is the most sensitive stain for detecting mitochondrial proliferation. 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) stain Mitochondrial disorder Normal 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

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

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