Pathologies Mitochondriales De La Clinique À La Thérapie Jean-Marie Saudubray, Paris

Pathologies mitochondriales de la clinique à la thérapie Jean-Marie Saudubray, Paris

7e colloque "Meetochondrie » Evian 4-7 Mai 2014

1. Synthesis of bile acids 2. Synthesis of some steroid hormones 3. Synthesis of plasmalogens 4. Transmission of glyoxylate to glycine 5. Catalase Golgi complex 6. Oxidation of D-amino acids 7. α-oxidation of branched chain fatty acids, e.g., phytanic acid Post-translational modification of 8. β-oxidation of long-chain and very-long-chain fatty N-linked oligosaccharides of acids 9. Oxidation of pipecolic acid nascent glycoproteins, including 10. Spermine and spermidine oxidation glycosaminoglycan biosynthesis

Plasma membrane

1. Limiting loss of intracellular components of the cell, ensuring concentration and preservation Rough endoplasmic of optimum local milieu 2, Regulating uptake of amino acids reticulum (ER) 3. Neurotransmitter binding and uptake (nervous Translation (mRNA-directed synthesis tissue) of polypeptides) 4. Regulating uptake of metabolites, drugs, chemicals, hormones, etc. 5. Uptake (endocytosis) and intracellular trafficking of material suspended in extracellular milieu

Mitochondria Smooth endoplasmic reticulum (ER) 1. Oxidative phosphorylation 1. Post-translational modification of polypeptides, 2. Citric acid cycle 3. β-oxidation of fatty acids, with production of acetyl-CoA including N- and O-glycosylation 4. Oxidative degradation of some amino acids 2. Biosynthesis of cholesterol, 5. Proximal steps of urea biosynthesis phospholipids, triglycerides, 6. Biosynthesis of some amino acids 7. Proximal and distal steps of porphyrin biosynthesis Lysosome glycosphingolipids 3. Steroid hormone biosynthesis (endocrine tissues) Localization of hydrolytic enzymes involved in the 8. Mitochondrial transporters 4. Detoxifications (P450, liver) degradation of large, complex substrates, such as 5. Glucose-6-phosphate (liver) proteins, glycoconjugates, nucleic acids, complex lipids 6. Calcium sequestration (sarcoplasmic reticulum, Adapted from Dr. Joe Clarke Treatment: ERT;SRT,Chaperone muscle) 7. Porphyrin degradation © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org The mitochondrion

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. Energy production

• Electron transport chain (80% of cellular ATP)

• Fatty acid oxidation, ketogenesis, and ketolysis

• Krebs citric acid cycle . Urea cycle (partial) . Amino acid catabolism (branched chain, lysine, glycine, ornithine, proline)

. Pyrimidine biosynthesis . Heme and iron sulfur proteins synthesis . Cholesterol metabolism . Estrogen/testosterone synthesis . Calcium homeostasis at synapse  neurotransmission . Apoptosis

> 2001 On-line update continuous process….

. 1. Intoxication: IEM of intermediary metabolism (small molecules)

• Aminoacidopathies (catabolism, synthesis*)

• Organic acidurias

• Urea cycle defects

• Sugar intolerances (polyols)

• Metals, porphyrias

• Neurotransmitters (catabolism, synthesis)

In synthesis defects amino acid levels are DECREASED (*Serine,Glutamine,Proline) Intoxication common characteristics

. Accumulation of small diffusible water soluble molecules

. No embryonic or fetal consequences

. Neonate is typically normal and born at term

. Well interval between birth and first symptoms

. Easy to diagnose by plasma/urine chromatography

. Usually present emergently with acute recurrent crisis

. Most of them are treatable acutely and can be prevented

. Many of them can be detected by neonatal screening

free fay acids G6P glucose amino acids fay acyl-CoA pyruvate lactate organic acids acylcarnines PDH + NH4 β-oxidaon TCA cycle Urea cycle ketones reducing equivalents Urea respiratory chain

. 2. Energetic disorders: IEM of intermediary metabolism Mitochondrial • Congenital lactic acidemias (PC, PDH,CAC) • Respiratory chain disorders • FAO and ketone bodies disorders Cytoplasmic • Gluconeogenesis, glycogenolysis, glycolysis defects • Cerebral creatine disorders • Pentose phosphate pathway

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Energetic disorders: common characteristics . Disturbances of physiologic parameters including pH, glucose, ketones, lactate, or carnitine . Diagnosis relies on functional tests or metabolic profiles . Requires specific enzyme and/or DNA assays . Can present with recurrent crisis triggered by catabolism/food . Progressive symptoms including antenatal/congenital presentations (liver, brain, heart, and muscle) . Presentation in any organ at any age . Only a few are treatable

free fay acids G6P glucose amino acids

fay acyl-CoA pyruvate lactate organic acids

acylcarnines PDH + NH4 β-oxidaon Urea cycle TCA cycle ketones

reducing equivalents Urea

respiratory chain

ATP

free fay acids G6P glucose amino acids fay acyl-CoA pyruvate lactate organic acids acylcarnines PDH + NH4 β-oxidaon Urea cycle TCA cycle ketones Urea reducing equivalents

respiratory chain

GLYCOGEN GALACTOSE G.1.P G.6.P GLUCOSE F.6.P F.16.dP FRUCTOSE NAD GA3P GLYCEROL NADH 1-3 DPG XTP XTP PEP

PYRUVATE LACTATE ASPARTATE NADH NAD PYRUVATE XTP ALANINE OXALO ACET ACETYL Co A FATTY ACIDS KREBS CITRATE NAD KETONE α KETOGLU BODIES NADH GLUTAMATE

LACTATE NAD

NADH PYRUVATE Pyruvate carrier PYRUVATE NADH NAD PDH Alanine PC AcetylCoA OAA TCA Citrate Aspartate

NAD

NADH + H

CO2

Pyruvate Acetyl-CoA CoA

New partners Mito FAS II Lipoic acid synthetase (J Mayr AJHG 2011) NAD Lipoic acid Lipoyltransferase (Soreze Orphanet J.2013) NADH + H

CO2

Pyruvate SLC25A19 (J Mayr AJHG 2007) Acetyl-CoA Mito carrier TPP (Science 2013) TPP kinase CoA (J Mayr AJHG 2011) Thiamine Cytoplasmic SLC19A3 Pyruvate Thiamine © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Branched chain ketoacid

CoA-SH

E3:Variant MSUD NAD

GDH

SCHAD MCT1

Adapted from P de Lonlay, Necker, Paris

LIAS LIPT1 GCS PDH KGDH BCKDH Keto adipate IBA57 NFU1 ISCU NFS1 NUBPL ISD11 GLRX2 BOLA3

FXN

After Lim S C et al. Hum. Mol. Genet. 2013;22:4460-4473

Complex I III IV thio-modiﬁed mitochondrial tRNAs subunits ( tRNA-Lys, tRNAGln,and tRNA-Glu) LIAS Acute Infantile LIPT1 GCS Liver Failure PDH and TRMU mut TRMU KGDH BCKDH Keto adipate IBA57 ISCU NFU1 Cystathionine NFS1 Metallothioneine ISD11 NUBPL GLRX2 Metabolic proﬁle Postnatal BOLA3 Development (1-4 months) FXN

Mitochondrial Fe-S Assembly System

. 3. Complex molecules: IEM of organelle metabolism • Lysosomes • Peroxisomes • CDG (ER and Golgi) • Cholesterol (mito and ER) • Phospholipids (ER and mito) • Glycosphingolipids (ER ,Golgi and lysosome)

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Pyruvate NAD+ PDH Co A Oxaloacetate NADH IDH ATP CoA synthase Acetyl-CoA Isocitrate Glucose 1-P phosphopantetheine citrate Glucose 6-P PANK2 - PFK G6PD 6-P- CTP gluconolactone

Vitamine B5 F 1,6-BP NADP+ citrate NADPH Citrate Lyase Ribulose 5-P Aceto- Acetyl-CoA Acetyl-CoA Acetyl -CoA Carboxylase G3P DHase + TPI Glycerol 3-P DHAP GA3-P HMG-CoA Synthase Glyceraldehyde Malonyl-CoA 3-P-DHase NADPH 1,3-DPG FAS NADP+ P-Glycerate HMG-CoA Kinase Fatty acyl-CoA 3-PG HMG-CoA G3-P Acyltransferase DHAPAT Reductase Phosphatidate Elongases Acyl-DHAP AGK ELOV 1-6 Serine ADHAPS Mevalonate Cardiolipin Serine Palmitoyl NADPH Diacylglycerol transferase SERAC-1 NADP+ Alkyl-DHAP Ceramide

Cholesterol Ubiquinone LCFA Plasmalogens Sphingolipids Dolichol Triglycerides Phospholipids VLCFA

1 = Hydrophilic phosphate group head located at the surface of a bilayer 2 = Hydrophobic fatty acid chain tail located inside

Biosynthesis takes place mostly in the ER but also at the mitochondrial membrane and in the Golgi

Vlis & Daum, Cold Spring Harb Perspect Biol 2013 © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Overview of mitochondrial membrane phosphatidylethanolamine synthesis

Lysophosphatidic acid

Acyl CoA ABHD5 AG-AT CoA-SH AGK Phosphatidic acid Monoacylglycerol CTP CDP-DAG PAP Synthase PPi ADP ATP + DGKE PNPLA2 Phosphoglycero- Diacylglycerol CDP Diacylglycerol phosphate Triacylglycerol DGAT Phse

Phosphatidyl- CDP-PEth PC-Cytidylyl Glycerol CHK transferase CMP Phospho- choline choline CDP-PChol PE-N-methyl CMP CH2 CH2 ATP ADP transferase SAH SAM - O C O - O P O. P O . PG O. H H O PC PE SERAC1 O C16:0/C18:1 O CH CH2 Chol PS Ser 2 Ser CH CO Eth CO CH PSS1 CH2 CH2 TAZ O O CO CO Cardiolipin remodeling

Monolysocardiolipin Cardiolipin © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Mitochondrial cardiolipin synthesis

DAK PA AGPAT LPA GPAT G3P TAG DAG OMM

Ups1 (PRELID1) ADP Mdm35 (TRIAP1) Mitochondrial intermembrane space PL CDP- DAG PA PGP PG CRLS1 CL ANT IMM TAG X DAG AGK Tam41 PGS1 PTPMT1 (TAMM41) ATP

Protein [µg] 2.5 5.0 10 10 5.0 2.5

Sengers syndrome SDHA Acylglycerol kinase deﬁciency: ANT is absent

ANT

Control Sengers- patient From J Mayr AJHG 2012

1. Clinical/radiology phenotype (clinical algorhytms) 2. Basic screening tests at the bedside (pH,glucose, ketone,lactate,ammonia,) 3. Metabolic proﬁles (AA,OA….basal and function tests) 4. Therapeutic tests (Dopa,Vitamins ….) 5. Ex vivo tests (whole fatty acid oxidation,respiratory chain) 6. Targeted enzymatic or molecular tests (in case of suggestive phenotype) 7. DNA screening tests (targeted or general)

In every situations search ﬁrst for treatable disorders

. Urine tests • Organic acids, (Amino acids) • Orotic acid • Porphyrias • Glycosamine glycans • Sulfatides,Oligosaccharides,Sialic acid • Purines,Pyrimidines • Creatine metabolites • Polyols • Neurotransmitters (best in CSF)

. Blood tests • Amino acids • Total homocysteine • Acyl carnitine (OA,FAO) • VLCFA,Phytanic,Pristanic,Plasmalogens (PZO) • Free fatty acids profile • Copper,Ferritin,ceruloplasmin,transferrin • Fibroblast growth factor 21? • IEF and immunofixation of transferrin (CDG) • Thyroid hormones (MCT 8) • Cholesterol,bile acids

. CSF tests • Amino acids (Glycine,Serine,Threonine…) • Neurotransmitters (GABA,Monoamines,Pterins,Folates) • Glucose (plasma/CSF ratio in GLUT 1 deficiency) • Lactate,Pyruvate,L/P ratio (when plasma lactate is < 2.5 mmol/l) (PDH,Pyruvate transporter)

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org . Although most identified by exome sequencing thus far, lipidomics likely to revolutionize discovery, treatment, and monitoring of complex lipid synthesis and recycling diseases and mitochondrial disorders

. Many new disorders likely to be defined

. Direct molecular analysis is deemed the most appropriate diagnostic approach if • There is a very specific phenotype to genotype correlation • The biochemical marker is unavailable (many tissue specific defects..) or unreliable (respiratory chain..) • The test requires an invasive procedure (CSF,stress test, biopsy) • The test is difficult to access (complex lipids..) • The molecular testing is easy compared to the enzyme assay • The cost:effectiveness ratio is low

• Glucose:GLUT1(SLC6A19),GLUTII(SLC2A2),GLUT10 (SLC2A10) • Monocarboxylic acids:MCD8(AllanDudley),MCD1:EI/HI(SLC16A1) (Otontoski T Am J Hum Genet 2007) • Creatine cerebral transporter(SLC6A8) • Carnitine:Carnitine uptake(OTCN 2)

• Thiamine:BRBGD(SLC19A3)(Zeng Am J Hum Genet 2005) • Acetyl CoA:Cataract,hearing loss,low copper(SLC33A) (Huppke P, Am J Hum Genet 2012)

• Glutamate (SLC25A22) :Neonatal Epilepsy (F Molinari AJHG 2005)

• Aspartate (SLC25A13):Citrullinemia type II (T Saheki MGM 2010)

• Pyruvate (MPC1) :Lactic acidosis(DK Bricker Science 2012)

• Citrate (SLC25A1):D-L-2-Hydroxyglutaric aciduria (B Nota AJHG 2013)

• Adenine nucleotide translocator:mtDNA deletions (L Palmieri HMG 2005)

• Phosphate (SLC25A3) :Neonatal cardiomyopathy (J Mayr AJHG 2007 ) • Thiamine pyrophosphate (SLC25A19):Amisch microcephaly,Striatal necrosis (J Mayr AJHG 2007)

. Some keys to interpret metabolic markers • Interorgan metabolism (brain versus others) • Production versus consumption (organs,organelles) • Physiological state (fed,fasted,exercise,rest) • Development(foetus to adult) • Other (moon lighting proteins, transcription factors…) • Conformational versus metabolic disease with characteristic aggregation-prone protein (Ex:TPI) . Most common markers • Lactate, Ammonia • Organic acids,amino acids …… • Profiles: Metabolic signature (Metabolomics)

Benarroch E. Neurology. 74:919-923 (2010)

% Basal metabolic rate

LACTATE NAD

NADH PYRUVATE Pyruvate carrier PYRUVATE NADH NAD PDH Alanine PC AcetylCoA OAA TCA Citrate Aspartate

O LDH OH || | CH3 — C — COOH CH3 — CH — COOH NADH NAD+ | Pyruvate H+ Lactate

. Increased NADH/NAD+ Most common • Increased lactate/pyruvate • Anoxia/ischemia Mostly not metabolic + } . Increased H (acidosis) . Increased pyruvate • Normal or low lactate/pyruvate • Mostly inborn errors © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Pyruvate metabolism:L/P ratio

. Timing of lactate elevation

• Fasting

• Postprandial

• Permanent . Hypoglycemia . Hepatomegaly . Neurologic signs

. Hypoglycemia

• Gluconeogenesis – Primary (FBPase, G6Pase) – Secondary (FAOD,respiratory chain)

• Glycogen storage disorder type I (G6Pase defect) . Probably not an oxidative disorder (but RC…)

. In fasting ketotic hypoglycemia context: Glycogenesis and glycogenolysis defects:Liver size ? . In neurologic disease context: Oxydative defect : L/P ratio?

• Normal lactate/pyruvate: Hyperpyruvicemia – PDH,Pyruvate transporter

• High lactate/pyruvate: High NADH/NAD ration – Pyruvate carboxylase ,Krebs cycle – OXPHOS (primary and secondary)

Respiratory Show All High chain, PC Permanent L/P with neuro signs Normal PDH, Pyr carrier Or Low

Fasting ketotic GSD III Hypoglycemia GSD 0 Only in post- Hyperlactatemia prandial phase High RC, PC Neuro signs L/P Low PDH

Gluconeogenic G6Pase FBPase Only at fast enzyme defects with hypoglycemia Energetic defects FAO, RC

Courtesy of JM Saudubray

ORNITHINE

AKG

ACETYL Orotic GLUTAMATE acid

NH3

Pyruvate Urea PC 2KG Cycle GLU Aspartate Oxaloacetate Citrate

PCC MMM PP-CoA MM-CoA Succinyl-CoA Lys 2-Ketoglutarate Saccharopine Urea AA NH3 ASAT Cycle GDH 2KA Glutamine Glutamate

CO2 X CO3H X CO3H

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org C van Karnebeeke Am J Hum Genet 2014 94:453-461 © 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Metabolic markers III organic acid proﬁle

L-VALINE L-ISOLEUCINE THREONINE METHIONINE

2-OXO-BUTYRYL-CoA ACETO-ACETATE LACTATE 3-OH-BUTYRATE C15-C17 FA CHOLESTEROL 3-OH-ISOVALERATE

PYRUVATE PROPIONYL-CoA ETHYLMALONYL-CoA ACETYL-CoA MALONYL-CoA

ODD-NUMBERED FA CITRATE Propionic acidemia OXALOACETATE D-METHYLMALONYL-CoA

CH3-CITRATE L-METHYLMALONYL-CoA PROPIONYLGLYCINE

PROPIONYLCARNITINE

3-OH-PROPIONATE SUCCINYL-CoA

Characteristic compounds accumulated in propionic acidemia and visible on urinary GCMS Characteristic compounds accumulated in propionic acidemia and NOT visible on urinary GCMS

3-CH3-GLUTACONIC

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org « Mitochondrial » disorders associated with secondary 3-CH3-Glutaconic aciduria 1.Due to defective phospholipids remodeling • Tafazzin (Barth syndrome) (type 2) • Serac 1 (Megdhel syndrome) • Acylglycerol kinase (Sengers syndrome) 2. Due to mitochondrial membrane associated disorders • OPA3 (Costeff syndrome) (type 3) • DNJAC19 ( DCMA syndrome) (type 5) • TMEM70 ( with complex V deficiency)(type 4) 3. Due to ATP synthesis and ATP synthase assembly factors • ATP 5 E,ATP 12 4. Due to mitochondrial DNA dysfunction • mtDNA deletions and depletions • POLG1 mtDNA replication • tRNA leucine mtDNA translation

1. At any age from foetus to late adulthood 2. Any symptoms (specific ,systemic) 3. Any scenarios (acute,chronic,intermittent) 4. Any mode of inheritance 5. Neonatal screening (at-risk screening) 6. Presymptomatic medicine

© 2008-2013 Society for Inherited Metabolic Disorders www.simd.org Take-home clinical messages from a bedside clinical physician (retired….) . Be very careful to the clinical phenotype . START ALWAYS WITH SCREENING TESTS . LOOK CAREFULLY AT THE METABOLIC PROFILES . DO NOT MISS A TREATABLE DISORDER . DON’T BE SNOB (and/or limited minded!) nor NAIVE . RARE DISEASES are often described first on only one or a very few patients . Understanding pathophysiology is crucial . Molecular investigations are not the alpha and omega