Download Presentation - MetBio.Net

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 metabolism06/05/11 Metabolic Muscle

Causes of myoglobinuriaTein I (1996) Seminars in Ped Nur 3(2) 59Age range 15-65 yearsAge range - Childhooddiagnosis in 47%(77)CPT 2 17 16GSD V McArdle’s 10 2Phosphorylase “b” kinase 4 0Phosphoglycerate kinase 1 1Myoadenylate deaminase(AMP)3 0Phosphoglycerate mutase 0 3Lactate dehydrogenase 0 1AMP + CPT 2 1 0diagnosis in 23%(100)NB. Absence of VLCADRespiratory chain

Clinical presentations of defects ofFAOD• Hypotonia /delayed development• Exercise intolerance/chronic weakness/musclepain/stiffness/cramps/atrophy/contractures• Acute rhabdomyolysis / renal failure• Respiratory / neck muscle involvement,wheelchair dependency / episodic ketoacidosis• Ponto bulbar palsy, deafness• Peripheral neuropathy / polyneuropathy /abnormal gait06/05/11 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 disordersVladutiu G (2001) Mol Genet Metab 74:51-63Vockley et al (2000) Mol Genet Metab 71:10-1806/05/11 Metabolic Muscle

Presentation with pain / stiffness / weakness / rhabdomyolysis / bulbar palsyFamily History of DiseaseExclude non-metabolic causes:-inflammatory myopathies-toxicological, infectionFull Clinical ExaminationNeurological, Gastrointestinal / liver, Cardiac, Opthalmology,Audiology1 st line Biochemical InvestigationsPlasmaUrineLactateOrganic acidsCreatine kinaseAcylcarnitinesFree carnitineConsider Additional Testing(non invasive)Forearm exercise testingExercise testing (e.g. treadmill studies)Nerve conduction studiesP-MRS, EMG06/05/11 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 & episodicrhabdomyolysis• 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 arecognised metabolic centre as many of thebiochemical 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 reliable06/05/11 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 acidoxidation 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 affectedIndividual enzyme assayse.g. CPT I , CPT II, CAT, LCHAD, carnitine transporter activityMolecular Genetics – full mutation CPTII, CAT, GSDV & the rest06/05/11 Metabolic Muscle

Fatty acid oxidationCH 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 asfuel during fasting /sustained exercise• Diet• Adipose tissue• Long chain fatty acids are oxidised in the body to producecarbon dioxide (CO 2 ), water (H 2 O) & energy (ATP)06/05/11 Metabolic Muscle

How we measure fatty acidoxidationMUSCLE CELLSfatty acid oxidation enzymes (+ Kreb’s cycle + RES)• Fatty acid CO 2 + H 2 O + energyOUR ASSAY in fibroblastsfatty acid oxidation enzymes• Labelled fatty acid 3 H CO 2 + 3 H 2 O + energy06/05/11 Metabolic Muscle

3H 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 2CH 2 C 3 H C 3 H CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COOH

% OleateDetection of VLCAD using[9,10- 3 H]substrates in fibroblasts1401201008060CONTROLSmild VLCAD40severe VLCAD20LCHAD00.5 0.6 0.7 0.8 0.9 1 1.1 1.2Ratio Palmitate/Myristate

Diagnosis FAOD that can cause muscle disease(Sheffield)1995-2011All 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• Primary Carnitine Deficiency 35• 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• Muscle 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-CoAdehydrogenase deficiency (VLCAD)Three phenotypes– neonatal / infantile with/without cardiomyopathy– mild - late onsetMILD (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 MADDKetone bodiesAcetyl-CoATCASEFADEFADH 2ETFoxSH 2Very-long-chain acyl-CoA DHMedium-chain acyl-CoA DHShort-chain acyl-CoA DHLong-chain acyl-CoA DHAcyl-CoA DH-9Short/branched-chain acyl-CoA DHIsobutyryl-CoA DHIsovaleryl-CoA DHGlutaryl-CoA DHDimethylglycine DHSarcosine DHADPETFredH+ H+ H+Fatty acid metabolismAmino acid metabolismCholine metabolismATPETFQOI IIQ III Cytc IVVH+06/05/11 Metabolic MuscleBackground

The defect in riboflavin-responsive MADD?TCAEFADEFADH 2ETFoxVery-long-chain acyl-CoA DHMedium-chain acyl-CoA DHShort-chain acyl-CoA DHLong-chain acyl-CoAAcyl-CoA DH-9Short/branched-chain acyl-CoA DHIsobutyryl-CoA DHIsovaleryl-CoA DHGlutaryl-CoA DHDimethylglycine DHSarcosine DHADPETFredH+ H+ H+Fatty acid metabolismAmino acid metabolismCholine metabolismATPIIIETFQOQIIICytcIVVH+06/05/11 Metabolic MuscleBackground

The defect in riboflavin-responsive MADD?Riboflavin FMN FADTCAEFADEFADH 2ETFoxVery-long-chain acyl-CoA DHMedium-chain acyl-CoA DHShort-chain acyl-CoA DHLong-chain acyl-CoA DHAcyl-CoA DH-9Short/branched-chain acyl-CoA DHIsobutyryl-CoA DHIsovaleryl-CoA DHGlutaryl-CoA DHDimethylglycine DHSarcosine DHADPETFredH+ H+ H+Fatty acid metabolismAmino acid metabolismCholine metabolismATPETFQOI IIQ III Cytc IVVH+06/05/11 Metabolic MuscleBackground

How should these ETFDH mutations give rise to ariboflavin-responsive phenotype?• For several flavoproteins, FAD-binding has been demonstrated to promoteassembly and/or stabilization of holoenzymeNagao and Tanaka 1992; Saijo and Tanaka 1995; Sato et al., 1996HypothesisWe hypothesise that the ETFDH mutations cause impaired FADbinding/-stabilization thereby increasing intra-cellular degradationof mutant ETFQO protein. The vulnerability to degradation couldbe modulated by the FAD content of the cell resulting in ariboflavin-responsive phenotype06/05/11 Metabolic Muscle

Poor riboflavin statusa disease triggering factor?• Symptoms may have been precipitated by a deterioration inriboflavin status during adolescence.– The national Diet and Nutrition Survey of young peopleaged 4-18 y has reported a high prevalence (95%) of poorriboflavin status among adolescent girls in the UK Gregory2000.– Interestingly, 12 of our 16 patients are adolescent girls.06/05/11 Metabolic Muscle

Brown-Vialetto-Van Laere Syndrome BVVLor Fazio Londe syndrome FL• BVVL Sensorineural deafness plus variety of cranial nerve palsies• FL – same disease without the deafness• Usually presents in second decade of life (progessive ponto-bulbarpalsy)– Limb weakness– Difficulty breathing– Slurred speech & difficulty swallowing– Facial weakness– Neck & shoulder weakness– May include mental retardation & seizures• 30% patients survive >10 years after diagnosis06/05/11 Metabolic Muscle

Brown-Vialetto-Van Laere Syndrome• RFT2 riboflavin transporter 2 (rats)• riboflavin transporter – small intestine• mutations in C20orf54 gene• Bosch et al. 2011 J Inher Metab Dis 34(1), 159-64– Brown-Vialetto-Van Laere and Fazio Londe syndrome isassociated with a riboflavin transporter defect mimicking mildMADD: a new inborn error of metabolism with potentialtreatment.06/05/11 Metabolic Muscle

Brown-Vialetto-Van Laere Syndrome BVVLor Fazio Londe syndrome FL• OR presents in infants• often without deafness• Neurological deterioration• Hypotonia• Respiratory insufficiency• Early death06/05/11 Metabolic Muscle

Diagnostic clues• Urine organic acids• Often subtle but variable metabolites asseen in MADD• i.e. isobutyrylglycine, isovalerylglycine,hexanoylglycine, suberylglycine,ethylmalonate, 2-hydroxyglutarate06/05/11 Metabolic Muscle

Diagnostic clues• Plasma acylcarnitines• Often subtle but variable metabolites asseen in MADD• i.e. generally mild increases in C4-C12acylcarnitines• Low plasma FAD, FMN or EGRAC06/05/11 Metabolic Muscle

Treatment• Usually responds well to riboflavin• 100 mg /day in two doses• Early treatment to prevent irreversibleparalysis of diaphragm06/05/11 Metabolic Muscle

Muscle disease• “Could this be metabolic?”– CK– Urine organic acids– Plasma acylcarnitines– Fibroblasts/ (?)mutation analysis• Riboflavin responsive disorders are underdiagnosed!06/05/11 Metabolic Muscle

AcknowledgementsShirley ClarkHelen HindCamilla ScottNigel ManningMelanie DowningRodney PollittMark SharrardJim BonhamJoanne CroftJane Dalley06/05/11 Metabolic MuscleRikke Olsen