Metabolic Muscle Disease Dr. Simon Olpin Consultant Clinical - - PowerPoint PPT Presentation

Metabolic Muscle Disease

• Dr. Simon Olpin

Consultant Clinical Scientist in Inherited Metabolic Disease Department of Clinical Chemistry Sheffield Children’s Hospital

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Diagnosis of Muscle Disease

A multi-disciplinary approach

• Clinical
• Physiology / Electrophysiology
• Magnetic Resonance Imaging & Spectroscopy
• Histopathology (histology & immunocytochemistry)
• Biochemistry
• Genetics
• Haematology

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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

Causes of myoglobinuria

Tein I (1996) Seminars in Ped Nur 3(2) 59 Age range 15-65 years Age range - Childhood

diagnosis in 47% (77) diagnosis in 23% (100) CPT 2 17 16 GSD V McArdle’s 10 2

Phosphorylase “b” kinase

4 Phosphoglycerate kinase 1 1 Myoadenylate deaminase (AMP) 3

Phosphoglycerate mutase

3 Lactate dehydrogenase 1 AMP + CPT 2 1

• NB. Absence of VLCAD

Respiratory chain

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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

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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

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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

1st line Biochemical Investigations Consider Additional Testing (non invasive) Forearm exercise testing Exercise testing (e.g. treadmill studies) Nerve conduction studies P-MRS, EMG

Plasma Lactate Creatine kinase Acylcarnitines Free carnitine Urine Organic acids

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

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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!

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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

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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
• xidation 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

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Fatty acid oxidation

CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 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 (CO2), water (H2O) & energy (ATP)

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How we measure fatty acid

• xidation

MUSCLE CELLS

fatty acid oxidation enzymes (+ Kreb’s cycle + RES)

• Fatty acid

CO2 + H2O + energy OUR ASSAY in fibroblasts

fatty acid oxidation enzymes

• Labelled fatty acid 3H

CO2 + 3H2O + energy

3H release from labelled [9,10-3H] fatty acids

[9,10-3H]Myristic acid (C14:0)

CH3 CH2 CH2 CH2 C3H2 C3H2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH

[9,10-3H]Palmitic acid (C16:0)

CH3 CH2 (CH2) 3 CH2 C3H2 C3H2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH

[9,10-3H]Oleic acid (C18:1)

CH3 (CH2)5 CH2 CH2 C3H C3H CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH

Detection of VLCAD using [9,10-3H]substrates in fibroblasts

20 40 60 80 100 120 140 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 Ratio Palmitate/Myristate % Oleate mild VLCAD severe VLCAD CONTROLS LCHAD

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

• Primary Carnitine Deficiency

35

• SCAD (?)

9

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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”

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 C18:1 + C16/C2 ratio

• Muscle biopsy may be abnormal (~20% lipid)

– muscle CPTII assays are usually unreliable!!

• Fibroblasts

– fatty acid oxidation @41oC – acylcarnitine profiling C16, C18

• 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!!

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The Biochemical defect in MADD

Background

Very-long-chain acyl-CoA DH Medium-chain acyl-CoA DH Short-chain acyl-CoA DH Long-chain acyl-CoA DH Acyl-CoA DH-9

Fatty acid metabolism

EFAD EFADH2

ETFox ETFred

SH2 S

Acetyl-CoA

II

ETFQO

Q

Cytc TCA

I III IV

H+ H+ H+

V

H+ ADP ATP

Dimethylglycine DH Sarcosine DH

Choline metabolism

Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Isovaleryl-CoA DH Glutaryl-CoA DH

Amino acid metabolism Ketone bodies

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The defect in riboflavin-responsive MADD?

Background

EFAD EFADH2

ETFox ETFred ETFQO

Q

Cytc TCA

III IV

H+ H+ H+

V

H+ ADP ATP

Dimethylglycine DH Sarcosine DH

Choline metabolism

Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Isovaleryl-CoA DH Glutaryl-CoA DH

Amino acid metabolism

II I

Very-long-chain acyl-CoA DH Medium-chain acyl-CoA DH Short-chain acyl-CoA DH Long-chain acyl-CoA Acyl-CoA DH-9

Fatty acid metabolism

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The defect in riboflavin-responsive MADD?

Background

Very-long-chain acyl-CoA DH Medium-chain acyl-CoA DH Short-chain acyl-CoA DH Long-chain acyl-CoA DH Acyl-CoA DH-9

Fatty acid metabolism

EFAD EFADH2

ETFox ETFred

II

ETFQO

Q

Cytc TCA

I III IV

H+ H+ H+

V

H+ ADP ATP

Dimethylglycine DH Sarcosine DH

Choline metabolism

Short/branched-chain acyl-CoA DH Isobutyryl-CoA DH Isovaleryl-CoA DH Glutaryl-CoA DH

Amino acid metabolism

Riboflavin FMN FAD

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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 Nagao 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

• f mutant ETFQO protein. The vulnerability to degradation could

be modulated by the FAD content of the cell resulting in a riboflavin-responsive phenotype

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Poor riboflavin status a disease triggering factor?

– The national Diet and Nutrition Survey of young people aged 4-18 y has reported a high prevalence (95%) of poor riboflavin status among adolescent girls in the UK Gregory 2000. – Interestingly, 12 of our 16 patients are adolescent girls.

• Symptoms may have been precipitated by a deterioration in

riboflavin status during adolescence.

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Brown-Vialetto-Van Laere Syndrome BVVL

• r 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-bulbar

palsy)

– Limb weakness – Difficulty breathing – Slurred speech & difficulty swallowing – Facial weakness – Neck & shoulder weakness – May include mental retardation & seizures

• 30% patients survive >10 years after diagnosis

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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 is associated with a riboflavin transporter defect mimicking mild MADD: a new inborn error of metabolism with potential treatment.

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Brown-Vialetto-Van Laere Syndrome BVVL

• r Fazio Londe syndrome FL
• OR presents in infants
• often without deafness
• Neurological deterioration
• Hypotonia
• Respiratory insufficiency
• Early death

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Diagnostic clues

• Urine organic acids
• Often subtle but variable metabolites as

seen in MADD

• i.e. isobutyrylglycine, isovalerylglycine,

hexanoylglycine, suberylglycine, ethylmalonate, 2-hydroxyglutarate

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Diagnostic clues

• Plasma acylcarnitines
• Often subtle but variable metabolites as

seen in MADD

• i.e. generally mild increases in C4-C12

acylcarnitines

• Low plasma FAD, FMN or EGRAC

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Treatment

• Usually responds well to riboflavin
• 100 mg /day in two doses
• Early treatment to prevent irreversible

paralysis of diaphragm

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Muscle disease

• “Could this be metabolic?”

– CK – Urine organic acids – Plasma acylcarnitines – Fibroblasts/ (?)mutation analysis

• Riboflavin responsive disorders are under

diagnosed!

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Acknowledgements

Shirley Clark Helen Hind Camilla Scott Nigel Manning Melanie Downing Rodney Pollitt Mark Sharrard Jim Bonham Joanne Croft Jane Dalley Rikke Olsen