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