Mitochondrial Diseases
Simon Heales
SWIM Conference – Taunton, November 29th 2018
Diabetes Thyroid
Disease Myopathy
Peripheral
Neuropathy
Deafness
Seizures /
Developmental delay
Respiratory Failure Optic Atrophy / Retinitis
Pigmentosa
Cardiomyopathy
Short Stature
Marrow Failure
Liver
Failure
Red Flags
1 in 5,000
Heteroplasmy & oogenesis
Gorman, G. S. et al. (2016) Mitochondrial diseases
Nat. Rev. Dis. Primers doi:10.1038/nrdp.2016.80
Adapted from Taylor, R. W. & Turnbull, D. M. Mitochondrial DNA
mutations in human disease. Nat. Rev. Genet. 6, 389–402 (2005), Nature Publishing Group
Clinical Picture
Biochemistry
Histochemistry
Genetics
Neuroimaging
Phenotypic Syndromes • Leigh syndrome
• Alpers syndrome
• Mitochondrial recessive ataxia syndrome
• Mitochondrial encephalomyopathy, lactic acidosis & stroke like episodes
(MELAS)
• Myoclonic epilepsy with ragged red fibres (MERFF)
• Pearson syndrome
• Kearns-Sayre syndrome (KSS)
• Progressive external ophthalmoplegia (PEO)
• Leber hereditary optic neuropathy (LHON)
• Neuropathy, ataxia and retinitis pigmentosa (NARP)
• Mitochondrial neurogastrointestinal encephalopathy (MNGIE)
• Myopathy lactic acidosis and sideroblastic anaemia (MLASA)
Metabolic Biochemistry
• Lactate – normal levels do not exclude. Secondary factors.
• Plasma amino acids, e.g. alanine
• Plasma acylcarnitines – differential diagnosis.
• Urine organic acids – lactate, TCA cycle intermediates,
ethylmalonic acid, 3-methylglutaconic acid, methylmalonic
acid (SUCLA2).
• FGF21
• GDF15
Diagnostic algorithm for suspected mitochondrial
diseases
Gorman, G. S. et al. (2016) Mitochondrial diseases
Nat. Rev. Dis. Primers doi:10.1038/nrdp.2016.80
Muscle Biopsy
• Skeletal muscle (50 - 100mg).
• Flash frozen at bedside.
• Store at -70oC.
• Transported on dry ice.
• Store at -70oC.
• Homogenise (1:9 w/v)
• Freeze Thaw (x3).
• Assay.
Citrate Synthase a Marker of Mitochondrial Enrichment
Mitochondrial Complex
Assays
I III II
IV
NADH
NAD+
Q
QH2
Succinate
Fumarate
Cyt c (Ox)
Cyt c (Red)
Cyt c (Red)
Cyt c (Ox)
O2
H2O
Rotenone Anitmycin
Cyanide
Q
Case
• 7 days; history of poor feeding and lethargy.
Elevated serum lactate and hypoglycaemia.
• 3 weeks; right-sided twitching episodes. EEG
revealed asymmetric discharges. MRI normal.
TCA cycle intermediates present in urine.
• 7 weeks: Skin, liver and muscle obtained
Muscle Respiratory Chain
Enzymes
Complex I 0.042 (0.104 - 0.268)
Complex II/III 0.156 (0.040 - 0.204) Complex IV 0.023 (0.014 - 0.034)
Liver Respiratory Chain
Enzymes
Complex I 0.017 (0.043 - 0.127)
Complex II/III 0.033 (0.037 - 0.099) Complex IV 0.018 (0.004 - 0.023)
Fatty Acid Oxidation Studies
• Fibroblasts - Sheffield
• All chain length activities depressed
• Can support diagnosis of mitochondrial defect
V (F0+ F1)
F1
Blue Native Gel Electrophoresis
Complex V Activity
Pooled control Patient
I
V III
IV
II Absent complex III
Blue Native Gel Electrophoresis
• Acute regression of motor skills. MRI brain demonstrates bilateral, symmetrical high signal in the basal ganglia and brain stem suggestive of Leigh's syndrome. Also noted to be some thinning of optic chiasm.
• CSF lactate = 3.2 and serum lactate = 4 and 2.51 on repeat. • Current medication: Biotin 30mg bd, Thiamine 100mg tds and Ubiquinone 25mg bd.
Case
NDUFV1 mutation.
Oxidative Phosphorylation – CoQ and Cytochrome c
Gorman, G. S. et al. (2016) Mitochondrial diseases
Nat. Rev. Dis. Primers doi:10.1038/nrdp.2016.80
Neonatal presentation of CoQ10
Deficiency. J. Pediatr; 139: 456-458
• Presented at 6 hrs with poor feeding, hypothermia and shaking of both arms. Generalised increase in peripheral tone with reduced truncal tone.
• Initial investigations; lactic acidosis (plasma lactate 19.4 mmol/L). Renal tubulopathy and mild left ventricular hypertrophy with global hypokinesia. Brain MRI demonstrated generalised atrophy and cerebellar atrophy.
Muscle Complex II+III
III II
Succinate
Fumarate
Cyt c (Ox)
Cyt c (Red)
Anitmycin
Q
Complex Activity Ref Range
II+III <0.001 0.040-0.204
II 0.151 0.052-0.258
III 0.026 0.008-0.028
CoQ10 37* (140-580 pmol/mg)
•Oral CoQ10 therapy
60 to 300 mg/day
•Decreased plasma lactate
7.6 to 2.5 mmol/L after 4 months
•Not accompanied by clinical improvement
• Secondary muscle
CoQ10 Deficiency
associated with
mitochondrial
respiratory chain
defects.
• Reported benefits from
supplementation.
Groups Cytosolic
Cytochrome C
ng/mg protein
ETC Defects
(n=22)
63.7 ± 15.5*
Disease
Controls
(n=26)
27.7 ± 2.5
Methionine
Homocysteine
Adomet
Cystathionine
Cysteine
B12 Methyl-cbl MS
Methyl-THF
THF
Serine Diet
B6 CBS
MAT
Glycine
Betaine
DMG
X
Methyl-X
Cbl(II)
Methyl-cbl
Methyl transferases
SH
MT
Ado.hcy
Gly
Sar
GNMT
N
N
N
NH
NH2
NH
O
NH
O OH
OH
O
OH CH3
5-Methyltetrahydrofolate
•CSF deficiency documented in mitochondrial disorders
(Allen et al., 1983, Dougados et al., 1983)
•Responsive to folinic acid
•No apparent correlation with magnitude of defect
CSF 5-MTHF Deficiency & Mitochondrial
Respiratory Chain Defects
Sex Age (years) 5-MTHF Ref Range (nmol/L)
F 15 29 46 - 160
M 9 5 72 - 172
M 8 44 72 - 172
F 2 17 52 - 178
F 6 7 72 - 172
Associated in some (25%) but not all patients
5MTHF Endocytosis
Folate Polyglutamate
Pool
5MTHF FR1
RFC
sFR1
-ve
PLASMA CSF
1 2
O2
._
5MTHF ???
FGF21
FGF21
Al Shahrani et al. Unpublished
Decanoic Acid & Function
0
0.2
0.4
0.6
0.8
1
1.2
1.4
ControlC10
Re
lati
ve m
tDN
A c
on
ten
t
Control
C10 Treated
Specimen
Reception Biobank Trials, Research
Combined Omics
Platforms
Personalised Testing
Integrated
Comprehensive Report
?
Flow Chart Showing Gene-Discovery Approach with the Use of Collaborative Phenomics and Semiautomated Genomics.
Tarailo-Graovac M et al. N Engl J Med 2016;374:2246-2255
Amanda Lam
Stuart Bennett
Iain Hargreaves
Viruna Neergheen
Simon Pope
Acknowledgements
Simon Eaton
Michael Orford
Aziza Khabbush
Tom Baldwin
Sean Hughes
Marta Kanabus
Shamima Rahman