genetics of mitochondrial respiratory chain disorders mitochondria nucleus lee-jun c. wong, ph.d....
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Genetics of Mitochondrial Respiratory Chain Disorders
mitochondria
nucleus
Lee-Jun C. Wong, Ph.D.Molecular and Human GeneticsBaylor College of Medicine
Dept Medical GeneticsZhong-Shang University, ChinaMay 28, 2007
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mitochondrion
The only animal cellular organelle that contains its own DNAHundreds to thousands of mitochondria per cellEgg cells: ~200,000, sperm cells: ~10
2-10 copies of mtDNA per mitochondrion
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Major Function of mitochondria:electron transport chainOxidative Phosphorylation producing energy, ATP
NADH
FADH2
O2
ADP
H2O
ATP
NAD
FAD
Proton gradient
Outer membrane
Inner membrane
Electron transport chain
Matrix
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Respiratory chain subunits encoded by two genomes: Nuclear and Mitochondria
7/43 0/4 1/11 3/13 2/13Mito/nuclear
complex I:NADH DH II: SDH III: cyt c red IV: COX V: ATPase
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Outer membrane
Inner membrane
Matrix
cytosol
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Unique Features ofMitochondrial 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
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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
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• Homoplasmy– 0 or 100%
• Heteroplasmy– Between 0-100%
Homoplasmy and Heteroplasmy
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Johns, D. New Eng J Med 1996;333:638-644
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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
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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
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Mutation hot spot
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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
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Mutation hot spot
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MRNA mutationsT8993G (Leu to Arg in ATPase6): Continuous phenotypic spectrum
nl > RP > NARP > Leigh syndromedepends on % mutant heteroplasmy
NA
RP
= Neuropathy/Neurogenic weakness= Ataxia= Retinitis pigmentosa
NARP:
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Leigh Syndrome
• Mitochondrial encephalopathy– Presents in infancy– Psychomotor regression– Signs of brainstem dysfunction– Ataxia– Often fatal
• Characteristic MRI findings
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Spectrum of Clinical Phenotypes for T8993GBased on Percentage of Mutant Mitochondria
0%
100%
75%
60%
90%
Retinitis Pigmentosa
NARP
Leigh Syndrome
“ Normal”
Carelli et al. (2002) Arch Neurol 59: 264-270.
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Percentage of mtDNA in Leucocytes Carrying the
T8993G Mutation
94%Leigh
80%NARP
31%carrier
82%Phenotypicallynormal Failure to thrive
Developmental delayhypotonia
Failure to thriveDevelopmental regressionHypotoniaSeizuresAbn MRI
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Tissue % mutant
Brother blood 94
(Leigh) buccal 97
hair 95
Proband blood 80
(NARP) buccal 81
hair 81
Sister blood 82
(?) buccal 92
hair 45
Mother blood 31
(carrier) buccal 36
hair 18
Tissue Variation of T8993G mtDNA
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• 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
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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
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I
II
d86y
d48y
III
IV
5
6 7
d2y d15y
1 2 3
1 2
4
3 4 5 11
7
12
6
8 9 10
8
13 14
G14459A mutation A72V in ND6Variable expression: LHON, dystonia
IV-2LimpHemiparesisMRS lactate peak
IV-8HemiparesisNF1Global delayDysarthriaspasticityMRS lactate peak
IV-10StrokeDystoniaDevelopmental delayshortSpasticityHearing lossMRS lactate peak
III-6NF1unaffected
All Homoplasmy
IV-9: unaffected
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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:377-382
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Gropman, chen, Perng, Krasnewich, Chernoff, Tifft, and Wong. AJMG 2004;124A:377-382
patient IV-2
bilateral symmertric increased T2 signal in the putamen
patient IV-8
unilateral increased T2 signal in the putamen
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mtDNA point mutationstRNA
• 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
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MitoDNA pedigree
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I
II
III
2
1 2 3 4 5 6 7 8
1 2 3 4
1
2.5%
4% 24% 3% 0% 27%
45% 29% 27% 0%
5
B:
H: 7.6%
B:
H: ND
B:
H: 20% 12% 0% 26%
B:
H: 59% 42% 23% NDM: 90%
A3243G Melas family
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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
DiabetesHearing lossMacular pattern retinal dystrophy
DiabetesHypertensionHeart disease
A3243G: diabetes, hearing loss, retinopathy
Am J Ophthalmol 1997;124:219
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B: 0% 16% 14%
B: 4% 30% ~0%
~0% 0% ~0%
B: 5% ~0% ~0% ~0%~0% ~0% ~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
1
1
2
2
9
543
43
10
8765
54321 131211
876 161514
~0%
A8344G MERRF family
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1 2 3 4
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6
76%
88% 82%83% 73%60%
84%90%94%98%87%91%
73%
I
II
III
Mitochondrial Cardiomyopathy and peripheral neuropathyMutation in tRNA lys (8363G>A)
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Single deletion Multiple deletion depletion
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F8389-R8529
F12093-R12170
F3212-R3319
F16498-R32mtDNA deletions
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Mitochondrial DNA Deletion Syndrome
• Kearns Sayre syndrome Ophthalmoplegia (inability to move eyes)
Ptosis (droopy eyes
Onset second decade
muscle
• Pearson syndromeSideroblastic anemia with pancytopenia
Exocrine pancreatic insufficiency
Onset: early infancy
Blood
• Multisystemic disease• PEO• Mitochondrial myopathy
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Muscle or Blood?
KSS vs
Multisystemic Disorder
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6 yo boy presented with Addison disease,Died of ARDS at 8 years of ageDeletion mutant in Autopsy tissues
5 kb common deletion in every autopsy tissue
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I
II
III
IV
1 2
1 2
1 2 3 4 5
1
2 3
39 3934 23
heart problems
wheelchair boundMRcleft lip
14
Clearly Kearns Sayre Syndrome, but deletion was not detected in blood.
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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
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DNA replicationTranscriptionTranslation Spinazzola and Zeviani, Gene 354 (2005) 162-168
MPV17
DNC
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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
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Autosomal dominant form of progressive external
ophthalmoplegia (adPEO)
• Twinkle gene: DNA helicase• ANT1 (Adenine Nucleotide
Translocase 1)• POLG
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Hepatocerebral form of mtDNA depletion syndrome
infantile hepatic failure
• DGUOK (deoxyguanosine kinase)• MPV17, a mitochondrial inner
membrane protein• POLG
Autosomal recessive
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P.W65X C.487ins4
P.W65X + c.487ins4 +
P.W65X C.487ins4
DGUOK mutations cause mtDNA depletion and respiratory chain enzyme deficienciesHepatocerebral form of mtDNA depletion syndrome
Both mutations are deleterious. Missense mutations in DGK appear to have similar clinical phenotype
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The liver biopsy showed portal fibrosis with extension into the lobule to surround hepatocytes. The hepatocytes are large with microvesicular steatosis and oncocytic change.
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Myopathic form of mtDNA depletion syndrome
• TK2 (thymidine kinase)
MNGIEMitochondrial
NeuroGastroIntestinal Encephlomyopathy
•TP (thymidine phosphorylase)
Both are Autosomal Recessive
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dGdA
dGKdGMPdAMP
dGDPdADP
dGTPdATP
NDPK
dCthymidine
dGMPdAMP
dGTPdATP
dGDPdADP
NDPKTK2
mtDNA
thymidine
thymine
TP
TK1dTMP
Thymidylatesynthase
dUMP
nDNA
mitochondrion
cytoplasm
POLG
POLG
dGdA
dGKdGMPdAMP
dGDPdADP
dGTPdATP
NDPK
dCthymidine
dCMPdTMP
dCTPdTTP
dCDPdTDP
NDPKTK2
mtDNA
thymidine
thymine
TP
TK1dTMP
Thymidylatesynthase
dUMP
nDNA
mitochondrion
cytoplasm
POLG
POLG
ANT1
ATP
ADP
DNCMPV17
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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;111:1913-1921
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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;111:1913-1921
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Genes encode for complex assembly factors
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Cytochrome cOxidase, (Complex IV)Assembly requires a series of factors:
SURF1SCO2SCO1COX10COX15LRPPRC
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Complex IV (cytochrome c oxidase)Assembly
Most common is mutations in SURF1 Gene, cause Leigh Disease
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normal
7567Mt7567G193S/G193S
SCO2 mutationG193S/G193S
SCO2 mutation analysis revealed homozygous G193S mutation
Isolated COX (cyt c oxidase, complex IV) deficiencyCardiomyopathy Lactic acidosis
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Mitochondrial Fission/fusion
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C. Ultrastructure of mitochondria from proband's muscle biopsy showing abnormal mitochondrial cristae forming concentric lamellae.(TEM,Mag.X30,000)
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Gomori trichrome stain
Muscle fibers have mild to moderate mitochondrial proliferation(Red rim & speckled sarcoplasm)
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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
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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.
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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
NormalMitochondrial disorder
Cytochrome oxidase (COX) stain
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Cytochrome oxidase (COX) stainSDH stain
Muscle fibers with excessive SDH staining (left) have reduced or absent COX (right) staining (arrows)