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

47. dGK mtDNA NDPK TK2 TP TK1 dTMP dUMP nDNA POLG MPV17 dG dA dGMP dAMP
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)