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

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

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

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

Outer membrane

Inner membrane

Matrix

cytosol

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

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– 0 or 100%

• Heteroplasmy– Between 0-100%

Homoplasmy and Heteroplasmy

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 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

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 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:

Leigh Syndrome

• Mitochondrial encephalopathy– Presents in infancy– Psychomotor regression– Signs of brainstem dysfunction– Ataxia– Often fatal

• Characteristic MRI findings

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.

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

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

• 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

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

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

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

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

MitoDNA pedigree

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

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

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

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)

Single deletion Multiple deletion depletion

F8389-R8529

F12093-R12170

F3212-R3319

F16498-R32mtDNA deletions

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

Muscle or Blood?

KSS vs

Multisystemic Disorder

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

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.

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

DNA replicationTranscriptionTranslation Spinazzola and Zeviani, Gene 354 (2005) 162-168

MPV17

DNC

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

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

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)

MNGIEMitochondrial

NeuroGastroIntestinal Encephlomyopathy

•TP (thymidine phosphorylase)

Both are Autosomal Recessive

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

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

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 cOxidase, (Complex IV)Assembly requires a series of factors:

SURF1SCO2SCO1COX10COX15LRPPRC

Complex IV (cytochrome c oxidase)Assembly

Most common is mutations in SURF1 Gene, cause Leigh Disease

normal

7567Mt7567G193S/G193S

SCO2 mutationG193S/G193S

SCO2 mutation analysis revealed homozygous G193S mutation

Isolated COX (cyt c oxidase, complex IV) deficiencyCardiomyopathy 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)

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

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

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

Cytochrome oxidase (COX) stainSDH stain

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