Mitochondrial Fatty Acid Oxidation Deficiencies Prof. Niels Gregersen

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1 Mitochondrial Fatty Acid Oxidation Deficiencies Research Unit for Molecular Medicine Clinical Institute Aarhus University Hospital and Faculty of Health Sciences Aarhus University, Aarhus, Denmark 1 Menu Biochemistry of mitochondrial fatty acid metabolism Fatty acid β-oxidation deficiencies Diagnostic strategies Molecular genetics and pathogenesis Prevalent mutations and diagnosis Conclusion 2 Enzymes and metabolites in mitochondrial fatty acid oxidation Carnitine transporter VLCAD MTP Carnitine CACT CPTII CAT CPTI LCAD Fatty acid transport protein RCOSCoA RCOSCoA FATP ETF ACAS ETF DH Acyl-CoA synthetase Cytosol EH Cell membrane Mitochondria KT HAD Carnitine/acylcarnit palmitoyl-coa ine transferase translocase I 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Enzymes and metabolites in mitochondrial fatty acid oxidation Cell membrane VLCAD MTP Mitochondria Carnitine CACT CPTII CAT CPTI LCAD RCOSCoA KT RCOSCoA FATP ETF Acetyl-CoA ACAS ETF DH Cytosol EH HAD MTP: Acyl-CoA Long-chain dehydrogenases: 3-hydroxy 3-hydroxy acyl-coa acyl-coa dehydrogenases dehydrogenase Long-chain LCAD: long-chain-acyl-coa enoyl-coa 3-keto acyl-coa hydratase dehydrogenase thiolases VLCAD, Long-chain MTP, very-long-chain mitochondrial trifunctional acyl-coa dehydrogenase protein Carnitine : medium-chain palmitoyl-coa 3-keto Enoyl acyl-coa hydratases acyl-coa transferase thiolase dehydrogenase II : short-chain acyl-coa dehydrogenase 4 Enzyme Enzymes deficiencies and metabolites in mitochondrial in mitochondrial fatty acid fatty oxidation acid oxidation Cell membrane VLCAD MTP Mitochondria Carnitine CACT CPTII CAT CPTI LCAD RCOSCoA KT RCOSCoA FATP ETF FAD Acetyl-CoA NAD ACAS FADH2 ETF DH NADH Resp. chain Cytosol EH HAD Resp. chain ETF DH: ETF: electron electron transfer transfer flavoprotein flavoprotein dehydrogenase 5 25 Discovery rate of mitochondrial fatty acid oxidation deficiencies Number of deficiencies CPTII 1 ETF/ ETF DH CAT Year The screen versions of these slides have full details of copyright and acknowledgements 2

3 Elucidation sequence for deficiency Pt with recurrent hypoglycaemic attacks and lethargy Urine metabolites Biochemical investigation; Dicarboxylic acid in urine Enzyme investigation; Decreased activity in cultured fibroblasts ACAD activity Control Patient C6 C8 C10 gene sequencing A C G T A C G T Gene investigation; Mutation in the gene 1990 Patient Control 7 Number of deficiencies Discovery rate of mitochondrial fatty acid oxidation deficiencies ETF/ ETF DH 6 CPTI 7 8 LCHAD CACT LCKAT 9 VLCAD SCKAT FATP SCHAD See Gregersen, N., et al., Eur. J. Biochem., 271, 2004: Year 8 Strategy of investigation Clinical symptoms Hypoketotic hypoglycaemia, vomiting, drowsiness or coma are common symptoms Cardiomyopathy, hepatomegaly or muscle weakness may be indicative Development delay, failure to thrive and feeding difficulties are also seen Metabolic investigations Urine organic acids Blood acyl-carnitines 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Gas chromatographic mass spectrometric analysis of organic acid in urine Adipic acid Detector response Detector response Patient Control IS IS Suberic acid Sebasic acid Suberylglycine Hexanoylglycine IS: internal standard Minutes Picture from Ernst Christensen, Rigshospitalet, Copenhagen, Denmark Minutes 10 deficiency indicated LC-fatty acyl-coa VLCAD Decanoyl-CoA Octanoyl-CoA Hexanoyl-CoA Butyryl-CoA Acetyl-CoA Citric acid cycle Decanoylcarnitine Decenoylcarnitine Octanoylcarnitine Hexanoylcarnitine Hexanoylglycine Sebacinsyre Suberinsyre Adipinsyre Suberylglycine 11 Strategy of investigation Clinical symptoms Metabolic investigations Urine organic acids Blood acyl-carnitines Enzyme determination Pathway flux Specific enzyme activity Genotyping Neonatal screening 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Neonatal screening of fatty acid oxidation defects Sufficiently high frequency D: 1/10,000 50,000 in Caucasians Treatable disease Frequent meals and fasting avoidance Rapid and reliable analysis Tandem mass spectrometry 13 Strategy of investigation Clinical symptoms Metabolic investigations Urine organic acids Blood acyl-carnitines Enzyme determination Pathway flux Specific enzyme activity Genotyping Neonatal screening 14 Fatty acid oxidation genes with defects Gene Structure Carnitine transporter (CAT) OCTN2 10 exons Carnitine palmitoyltransferase I (CPT I) CPT1A 18 exons Carnitine/acyl-carnitine translocase (CACT) Carnitine palmitoyltransferase II (CPT II) Very-long-chain acyl-coa dehydrogenase (VLCAD) Mitochondrial trifunctional protein (MTP) (Long-chain 3-hydroxy acyl-coa dehydrogenase (LCHAD)) Medium-chain acyl-coa dehydrogenase () Short-chain acyl-coa dehydrogenase () Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) Electron transfer flavoprotein (ETF) CACT CPT2 ACADVL HADHA HADHB ACADM ACADS HADHSC ETFA ETFB 9 exons 5 exons 20 exons 20 exons 16 exons 12 exons 10 exons 8 exons 12 exons 6 exons Electron transferring flavoprotein: ubiquinone ETFDH 13 exons oxidoreductase (ETF-QO) See Gregersen, N., et al., Eur. J. Biochem., 271, 2004: The screen versions of these slides have full details of copyright and acknowledgements 5

6 Disease-associated gene variations VLCAD Missense variation In-frame ins/del Out-of-frame ins/del Stop codon Splice error For details see: Gregersen, N., et al., Eur. J. Biochem., 271, 2004: Effect of gene variations pmrna Out-of-frame Splice error del/ins Stop & In-frame del/ins # Missense * mrna RNA surveillance PTC # * Non-sense mediated decay # * Polypeptide Degradation Protein quality control # * Mature protein 17 Mitochondrial protein quality control system Hsp70 Ribosome Protein mrna Nucleus Gene variation DNA CPTI VLCAD TP T CPTII LCAD Hsp70 Hsp60 Hsp10 Degradation Aggregation KT Mitochondria HAD EH 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Expression of variant proteins cdna containing plasmid E. coli cell The transformed E. coli cells were cultivated for 3 hr at 31 o C and activity determined in extracts In addition, the activity was determined after co-over-expression of GroESL (Hsp60/10) Andresen, B. S., et al., Hum. Mol. Genet., 6, 1997: GroESL (Hsp60/10) cdna containing plasmid Expression of activity in variant proteins without and with GroE 985A > G - GroESL + GroESL Folding mutations Andresen, B. S., et al., Hum. Mol. Genet., 6, 1997: Severe mutations 20 Modifiable Inactive Active site Picture done by Peter Bross, Research Unit for Molecular Medicine 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Expression of variant proteins Missense variant proteins may fold more or less efficiently, depending on the cellular conditions, such as chaperone efficiency It is not possible to predict with any certainty the effect of a given missense gene variation, because the folding pathway is not yet predictable 22 VLCAD Disease-associated gene variations 985A > G; Lys304Glu Missense variation In-frame ins/del Out-of-frame ins/del Stop codon Splice error For details see: Gregersen, N., et al., Eur. J. Biochem., 271, 2004: Prevalence of 985A>G Genotyping of 172 symptomatic patients for 985A>G G/G G/non-G Non-G/non-G Europe: 84: USA: 88: % 18% 2% Prevalence of 985A>G alleles: 90% 24 Coates, P. M., Chen, Y. T., Curtis, D., Gregersen, N., et al., Prog. Clin. Biol. Res., 1992, 375: The screen versions of these slides have full details of copyright and acknowledgements 8

9 Frequency of 985A>G Disease frequency: 1/29000 Tanaka, K., Gregersen, N., et al., Pediat. Res., 1997, 41: Diagnostic strategy for suspected deficiency Patient Clinical symptoms Urine/blood abnormalities 985A>G assay 985G/985G Heterozygous 985G or non 985G Family study gene sequencing ß-ox assay Diagnosis confirmed 985G/X or X/Y Family study Low Diagnosis confirmed 26 Fatty acid oxidation genes with defects Gene Prev. mut. Carnitine Transporter (CAT) OCTN2 Carnitine palmitoyltransferase I (CPT I) CPT1A Carnitine/acyl-carnitine translocase (CACT) Carnitine palmitoyltransferase II (CPT II) Very-long-chain acyl-coa dehydrogenase (VLCAD) Mitochondrial trifunctional protein (MTP) (Long-chain 3-hydroxy acyl-coa dehydrogenase (LCHAD)) Medium-chain acyl-coa dehydrogenase () Short-chain acyl-coa dehydrogenase () Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) Electron transfer flavoprotein (ETF) CACT CPT2 ACADVL HADHA HADHB ACADM ACADS HADHSC ETFA 439C>T 1528G>C 985A>G 625G>A 511C>T Electron transferring flavoprotein: ubiquinone oxidoreductase (ETF-QO) See Gregersen, N., et al., Eur. J. Pediatr., 159 (suppl. 3) 2000: S ETFB ETFDH 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Conclusion Fatty acid oxidation deficiencies (FAOD) are some of the most common metabolic disorders FAOD may be severe, early onset, with life-threatening liver/heart symptoms, or they may be milder, later onset, with neuromuscular symptoms FAOD are included in neonatal screening programs in many countries, especially because of the high frequency of deficiency Many different types of gene variations have been identified, some resulting in no protein, and many resulting in missense variant proteins, the amount of which are dependent on the cellular conditions Prevalent mutations in some of the genes make molecular genetic diagnosis easy The screen versions of these slides have full details of copyright and acknowledgements 10