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 FADH2 O2 ADP H2O ATP NAD FAD Proton gradient Outer membrane Matrix Inner membrane Electron transport chain

Respiratory chain subunits encoded by two genomes: Nuclear and Mitochondria 7/43 0/4 1/11 3/13 2/13 Mito/ nuclear complex I:NADH DH II: SDH III: cyt c red IV: COX V: ATPase

cytosol Outer membrane Inner membrane Matrix

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

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

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

Homoplasmy and Heteroplasmy 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 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 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:

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. 90% Leigh Syndrome 100%

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

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

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

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

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 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:377-382

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

MitoDNA pedigree

A3243G Melas family M: 90%

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

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% 43% 1995, B:nd 65% 54% 1995, H:nd 65% 43% B: 5% ~0% ~0% ~0% 10% ~0% ~0% ~0% ~0% 5%

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%

Single deletion Multiple deletion depletion

F16498-R32 mtDNA deletions F3212-R3319 F12093-R12170 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

Multisystemic Disorder Muscle or Blood? KSS vs Multisystemic Disorder

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

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.

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

DNA replication Transcription Translation MPV17 DNC 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 + c.487ins4 + P.W65X 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. 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

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

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

SURF1 SCO2 SCO1 COX10 COX15 LRPPRC 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 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)

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

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) 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

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