Metabolic Muscle Disease Dr. Simon Olpin Consultant Clinical Scientist in Inherited Metabolic Disease Department of Clinical Chemistry Sheffield Children’s Hospital
Diagnosis of Muscle Disease A multi-disciplinary approach • Clinical • Physiology / Electrophysiology • Magnetic Resonance Imaging & Spectroscopy • Histopathology (histology & immunocytochemistry) • Biochemistry • Genetics • Haematology 06/05/11 Metabolic Muscle
Causes of Muscle disease • Structural/linkage proteins • Contractile proteins • Ion Channel proteins • Inflammatory & autoimmune myopathies • Endocrine disorders • Toxic myopathy e.g. statins • Defects of muscle energy metabolism 06/05/11 Metabolic Muscle
Causes of myoglobinuria Tein I (1996) Seminars in Ped Nur 3(2) 59 Age range - Childhood Age range 15-65 years diagnosis in 47% diagnosis in 23% (77) (100) CPT 2 17 16 GSD V McArdle’s 10 2 Phosphorylase “b” kinase 4 0 Phosphoglycerate kinase 1 1 Myoadenylate deaminase 3 0 (AMP) Phosphoglycerate mutase 0 3 Lactate dehydrogenase 0 1 AMP + CPT 2 1 0 NB. Absence of VLCAD Respiratory chain
Clinical presentations of defects of FAOD • Hypotonia /delayed development • Exercise intolerance/chronic weakness/muscle pain/stiffness/cramps/atrophy/contractures • Acute rhabdomyolysis / renal failure • Respiratory / neck muscle involvement, wheelchair dependency / episodic ketoacidosis • Ponto bulbar palsy, deafness • Peripheral neuropathy / polyneuropathy / abnormal gait 06/05/11 Metabolic Muscle
Synergistic heterozygosity • Combined defects – – myoadenylate deaminase deficiency • Found in ~ 2% of muscle biopsies • PLUS – partial deficiency/carrier status for another defect • e.g. partial CPTII deficiency plus myoadenylate deaminase deficiency • OR – carrier status for two other separate disorders Vladutiu G (2001) Mol Genet Metab 74:51-63 Vockley et al (2000) Mol Genet Metab 71:10-18 06/05/11 Metabolic Muscle
Presentation with pain / stiffness / weakness / rhabdomyolysis / bulbar palsy Family History of Disease Exclude non-metabolic causes: -inflammatory myopathies -toxicological, infection Full Clinical Examination Neurological, Gastrointestinal / liver, Cardiac, Opthalmology, Audiology 1 st line Biochemical Investigations Consider Additional Testing Urine Plasma (non invasive) Organic acids Lactate Forearm exercise testing Creatine kinase Exercise testing (e.g. treadmill studies) Acylcarnitines Nerve conduction studies Free carnitine P-MRS, EMG 06/05/11 Metabolic Muscle
Fatty Acid Oxidation defects can cause “mild/moderate” neuro / myopathic disease • Exercise intolerance - pain /stiffness/myoglobinuria – typically on prolonged sustained exercise – exacerbated by poor food intake / cold / heat – There may be myalgia with intercurrent infection • Peripheral polyneuropathy & episodic rhabdomyolysis • mild TFP deficiency / (?)LCHAD • Progressive myopathy/acute encephalopathy – rr-MADD • CK usually normal between episodes • Urine organic acids sometimes abnormal • DCA & (OH)DCA • Acylglycines • Plasma acylcarnitines MAY be abnormal
Detection of FAOD’s • Preliminary investigations – Urine OA’s – Plasma free carnitine /acylcarnitine profile – CK – NB. It is important to send samples to a recognised metabolic centre as many of the biochemical abnormalities are subtle! 06/05/11
Detection of FAOD’s • Second line investigations – skin biopsy for fatty acid oxidation studies – specific enzyme assay on fibroblasts e.g. CPTII – mutation studies • Generally NOT MUSCLE BIOPSY – CPT & VLCAD assays in muscle not usually reliable 06/05/11 Metabolic Muscle
What do we offer at Sheffield Children’s Hospital? A Fatty Acid Oxidation Service – UK and beyond e.g. Dublin & Toronto Children’s Hospitals • Investigate >400 patient fibroblast cell lines each year – measure the rate of fatty acid oxidation – ( using 3 fatty acids) • The results tell us if there is a defect that slows the rate of fatty acid oxidation in the patient – fibroblast acylcarnitine profiling • Pattern of abnormal by-products of fatty acid oxidation • Helps to pinpoint what step in fatty acid oxidation is affected Individual enzyme assays e.g. CPT I , CPT II, CAT, LCHAD, carnitine transporter activity Molecular Genetics – full mutation CPTII, CAT, GSDV & the rest 06/05/11 Metabolic Muscle
Fatty acid oxidation CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COOH • Sources of long chain fatty acids for use as fuel during fasting /sustained exercise • Diet • Adipose tissue • Long chain fatty acids are oxidised in the body to produce carbon dioxide (CO 2 ), water (H 2 O) & energy (ATP) 06/05/11 Metabolic Muscle
How we measure fatty acid oxidation MUSCLE CELLS fatty acid oxidation enzymes (+ Kreb’s cycle + RES) • Fatty acid CO 2 + H 2 O + energy OUR ASSAY in fibroblasts fatty acid oxidation enzymes • Labelled fatty acid 3 H CO 2 + 3 H 2 O + energy 06/05/11 Metabolic Muscle
3 H release from labelled [9,10- 3 H] fatty acids [9,10- 3 H] Myristic acid (C14:0) CH 3 CH 2 CH 2 CH 2 C 3 H 2 C 3 H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COOH [9,10- 3 H]Palmitic acid (C16:0) CH 3 CH 2 (CH 2 ) 3 CH 2 C 3 H 2 C 3 H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COOH [9,10- 3 H]Oleic acid (C18:1) CH 3 (CH 2 ) 5 CH 2 CH 2 C 3 H C 3 H CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COOH
Detection of VLCAD using [9,10- 3 H]substrates in fibroblasts 140 120 100 CONTROLS % Oleate 80 mild VLCAD 60 40 severe VLCAD 20 LCHAD 0 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 Ratio Palmitate/Myristate
Diagnosis FAOD that can cause muscle disease (Sheffield)1995-2011 All fatty acid oxidation defects ~ 400 • Myopathic CPTII 31 • Myopathic CPTII carrier only 5 • Myopathic VLCAD 16 • rr-MADD (myopathic) 20 • Brown-Vialetto Van Laere 3 • Mild TFP 3 35 • Primary Carnitine Deficiency • SCAD (?) 9
Carnitine Palmitoyl Transferase type II Deficiency • Three phenotypes of CPTII deficiency • neonatal / infantile with/without cardiomyopathy – Late onset (mild) - myopathic with rhabdomyolysis • Prolonged exercise related • exacerbated by heat/cold/stress/poor food intake • MYOPATHIC - “most common inherited cause of myoglobinuria in young adults” 06/05/11 Metabolic Muscle
Diagnosis of mild CPTII • Clinical suspicion – myalgia in young children – rhabdomyolysis in adolescence / adults • All biochemical parameters may be normal between episodes but:- – raised plasma C 18:1 + C 16 /C 2 ratio • M uscle biopsy may be abnormal (~20% lipid) – muscle CPTII assays are usually unreliable!! • Fibroblasts – fatty acid oxidation @41 o C – acylcarnitine profiling C 16 , C 18 • Specific CPT II assay in fibroblasts (will detect carriers!!) • Mutation analysis • Common S113L mutation – (accounts for ~50% of disease)
Very long chain acyl-CoA dehydrogenase deficiency (VLCAD) Three phenotypes – neonatal / infantile with/without cardiomyopathy – mild - late onset MILD (onset usually >10 years) – exercise intolerance (prolonged) – rhabdomyolysis – may be exacerbated by missing meals / cold / heat – raised C14:1 acylcarnitine in plasma – NOT ALWAYS!!
The Biochemical defect in MADD Very-long-chain acyl-CoA DH Ketone bodies Medium-chain acyl-CoA DH Short-chain acyl-CoA DH Fatty acid metabolism Long-chain acyl-CoA DH Acetyl-CoA S SH 2 Acyl-CoA DH-9 EFAD Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Amino acid metabolism Isovaleryl-CoA DH EFADH 2 Glutaryl-CoA DH ETFox Dimethylglycine DH Choline metabolism TCA Sarcosine DH ADP ATP ETFred H+ H+ H+ ETFQO I II III IV V Cytc Q H+ 06/05/11 Metabolic Muscle Background
The defect in riboflavin-responsive MADD? Very-long-chain acyl-CoA DH Medium-chain acyl-CoA DH Fatty acid metabolism Short-chain acyl-CoA DH Long-chain acyl-CoA EFAD Acyl-CoA DH-9 Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Amino acid metabolism Isovaleryl-CoA DH EFADH 2 Glutaryl-CoA DH ETFox Dimethylglycine DH Choline metabolism TCA Sarcosine DH ADP ATP ETFred H+ H+ H+ ETFQO I II III IV V Cytc Q H+ 06/05/11 Metabolic Muscle Background
The defect in riboflavin-responsive MADD? Very-long-chain acyl-CoA DH Medium-chain acyl-CoA DH Riboflavin FMN FAD Short-chain acyl-CoA DH Fatty acid metabolism Long-chain acyl-CoA DH Acyl-CoA DH-9 EFAD Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Amino acid metabolism Isovaleryl-CoA DH EFADH 2 Glutaryl-CoA DH ETFox Dimethylglycine DH Choline metabolism TCA Sarcosine DH ADP ATP ETFred H+ H+ H+ ETFQO I II III IV V Cytc Q H+ 06/05/11 Metabolic Muscle Background
How should these ETFDH mutations give rise to a riboflavin-responsive phenotype? • For several flavoproteins, FAD-binding has been demonstrated to promote assembly and/or stabilization of holoenzyme N agao and Tanaka 1992; Saijo and Tanaka 1995; Sato et al., 1996 Hypothesis We hypothesise that the ETFDH mutations cause impaired FAD- binding /-stabilization thereby increasing intra-cellular degradation of mutant ETFQO protein. The vulnerability to degradation could be modulated by the FAD content of the cell resulting in a riboflavin-responsive phenotype 06/05/11 Metabolic Muscle
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