Transcript Cytochrome oxidase (COX)

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
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
Major Function of mitochondria:
electron transport chain
Oxidative Phosphorylation
producing energy, ATP
NADH Proton gradient NAD
FAD
FADH2
O2
H2O
ADP
ATP
Outer membrane
Matrix
Inner membrane
Electron transport chain
Respiratory chain subunits encoded by
two genomes: Nuclear and Mitochondria
complexI:NADH DH
Mito/
nuclear
7/43
II: SDH
III: cyt c red
0/4
1/11
IV: COX
3/13
V: ATPase
2/13
cytosol
Outer membrane
Inner membrane
Matrix
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
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
Homoplasmy and Heteroplasmy
• Homoplasmy
– 0 or 100%
• Heteroplasmy
– Between 0-100%
Johns, D. New Eng J Med 1996;333:638-644
Mitochondrial DNA :
common point mutations
• MELAS: Mitochondrial Encephalopathy Lactic
Acidosis and Stroke-like episodes. A3243G
(80%), T3271C, in tRNA
• MERRF: Myoclonic epilepsy, Ragged Red
Fibers. A8344G (80%), T8356C, in tRNA
• 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
Leu(UUR)
Lys
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
Mutation
hot spot
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
Mutation hot spot
MRNA mutations
T8993G (Leu to Arg in ATPase6):
Continuous phenotypic spectrum
nl > RP > NARP > Leigh syndrome
depends on % mutant heteroplasmy
NARP:
N = Neuropathy/Neurogenic weakness
A = Ataxia
RP = Retinitis pigmentosa
Leigh Syndrome
• Mitochondrial encephalopathy
– Presents in infancy
– Psychomotor regression
– Signs of brainstem dysfunction
– Ataxia
– Often fatal
• Characteristic MRI findings
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: 264-270.
Leigh Syndrome
90%
100%
Percentage of mtDNA in
Leucocytes Carrying the
T8993G Mutation
31%
carrier
82%
Phenotypically
normal
80%
NARP
94%
Leigh
Failure to thrive
Failure to thrive
Developmental delay Developmental regression
hypotonia
Hypotonia
Seizures
Abn MRI
Tissue Variation of T8993G mtDNA
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
• 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
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
G14459A mutation A72V in ND6
Variable expression: LHON, dystonia
I
d86y
II
d48y
1
2
3
5
4
6
III-6
NF1
unaffected
III
d2y
d15y
1
2
3
4
5
6
7
8
9
7
10
11
8
12
13
14
IV
IV-2
Limp
Hemiparesis
MRS lactate peak
IV-9: unaffected
All Homoplasmy
IV-8
Hemiparesis
NF1
Global delay
Dysarthria
spasticity
MRS lactate peak
IV-10
Stroke
Dystonia
Developmental delay
short
Spasticity
Hearing loss
MRS lactate peak
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
patient IV-2
bilateral symmertric increased
T2 signal in the putamen
patient IV-8
unilateral increased
T2 signal in the putamen
Gropman, chen, Perng, Krasnewich, Chernoff, Tifft, and Wong. AJMG 2004;124A:377-382
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
MitoDNA pedigree
A3243G Melas family
1
2
I
B: 2.5%
H: 7.6%
1
II
2
4
3
B: 4%
H: ND
1
2
3
B: 24%
H: 20%
5
4
III
B:
H:
45%
59%
M: 90%
29%
42%
27%
23%
6
5
0%
ND
7
3%
12%
8
0%
0%
27%
26%
A3243G: diabetes, hearing loss, retinopathy
48 y o
47 y o
B: 12%
H: 33%
C: 30%
B: 8%
H: 6%
C: 18%
Diabetes
Hearing loss
Macular pattern
retinal dystrophy
Diabetes
Hypertension
Heart disease
28 y o
B: 23%
H: 15%
C: 16%
asymptomatic
Am J Ophthalmol 1997;124:219
A8344G MERRF family
I
1
2
3
4
5
II
B: 0%
1
2
16%
3
4
14%
6
1993, B:48%
1995, B:62%
1995, H:60%
5
III
B: 4%
~0
%
30%
~0%
9
1
2
3
4
5
6
7
10
11
7
8
0%
~0%
12
8
13
14
15
16
IV
B: 5% ~0% ~0% ~0% 10%
~0%
1993, B:75%
1995, B:nd
1995, H:nd
65%
65%
65%
43%
54%
43%
~0% ~0% ~0% 5%
Mitochondrial Cardiomyopathy and peripheral neuropathy
Mutation in tRNA lys (8363G>A)
1
2
3
4
I
76%
2
1
3
4
6
5
7
8
9
II
83%
1
III
91%
2
4
3
87%
98%
88% 82% 60%
5
94%
73%
6
90%
84%
73%
10
Single deletion
Multiple deletion
depletion
F16498-R32
mtDNA deletions
F3212R3319
F12093R12170
F8389-R8529
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
Muscle or Blood?
KSS
vs
Multisystemic Disorder
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
1
I
2
heart problems
2
1
II
1
3
2
4
5
III
39
39
34
wheelchair bound
MR
cleft lip
1
IV
2
23
3
14
Clearly Kearns Sayre Syndrome, but deletion was
not detected in blood.
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
MPV17
DNC
DNA replication
Transcription
Translation
Spinazzola and Zeviani, Gene 354 (2005) 162-168
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
Autosomal dominant form of
progressive external
ophthalmoplegia (adPEO)
• Twinkle gene: DNA helicase
• ANT1 (Adenine Nucleotide
Translocase 1)
• POLG
Hepatocerebral form of mtDNA
depletion syndrome
infantile hepatic failure
• DGUOK (deoxyguanosine kinase)
• MPV17, a mitochondrial inner
membrane protein
• POLG
Autosomal recessive
DGUOK mutations cause mtDNA depletion
and respiratory chain enzyme deficiencies
Hepatocerebral form of mtDNA depletion syndrome
P.W65X
+
P.W65X C.487ins4
c.487ins4
P.W65X
+
C.487ins4
Both mutations are deleterious.
Missense mutations in DGK appear to have similar clinical phenotype
The liver biopsy showed portal fibrosis with extension into the lobule to surround hepatocytes.
The hepatocytes are large with microvesicular steatosis and oncocytic change.
Myopathic form of mtDNA
depletion syndrome
• TK2 (thymidine kinase)
MNGIE
Mitochondrial
NeuroGastroIntestinal
Encephlomyopathy
•TP (thymidine phosphorylase)
Both are Autosomal Recessive
ADP
ANT1
dG
dA
dGK
dGMP
dAMP
dGDP
dADP
NDPK
ATP
dGTP
dATP
POLG
mtDNA
TK2
dC
thymidine
NDPK
dCMP
dGMP
dAMP
dTMP
dCDP
dGDP
dADP
dTDP
dCTP
dGTP
dATP
dTTP
POLG
mitochondrion
MPV17
TK1
thymidine
TP
thymine
cytoplasm
dTMP
Thymidylate
synthase
dUMP
nDNA
DNC
Mechanism leading to mtDNA
mutations
• Nucleotide imbalance cause misincorporation
• 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
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
Genes encode for complex
assembly factors
Cytochrome c
Oxidase,
(Complex IV)
Assembly
requires a
series of
factors:
SURF1
SCO2
SCO1
COX10
COX15
LRPPRC
Complex IV (cytochrome c oxidase)
Assembly
Most common is mutations in SURF1
Gene, cause Leigh Disease
SCO2 mutation analysis revealed homozygous G193S mutation
SCO2 mutation
G193S/G193S
normal
Mt7567
G193S/G193S
7567
Isolated COX (cyt c oxidase,
complex IV) deficiency
Cardiomyopathy
Lactic acidosis
Mitochondrial Fission/fusion
C. Ultrastructure of mitochondria from proband's muscle biopsy showing
abnormal mitochondrial cristae forming concentric lamellae.(TEM,Mag.X30,000)
Gomori trichrome stain
Muscle fibers have mild to moderate mitochondrial proliferation
(Red rim & speckled sarcoplasm)
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
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.
COX deficiency: Child
Mitochondrial disorder
Cytochrome oxidase (COX) stain
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
SDH stain
Cytochrome oxidase (COX) stain
Muscle fibers with excessive SDH staining (left) have
reduced or absent COX (right) staining (arrows)