Laboratory investigation of mitochondrial disease · 2014-04-21 · Laboratory investigation of...

Laboratory investigation of

mitochondrial disease

Johannes A. Mayr („Hans“)

Department of PaediatricsParacelsus Medical University, Salzburg

[email protected]

www.mito-center.org

Mitochondria in disease

• Inherited metabolic disorders

(1/5000)

• Neurodegenerative disorders

(Parkinson, Alzheimer, etc.)

• Tumours

• Aging

• …

Reactions in mitochondria leading to

ATP from pyruvate

Mitochondrial Energy Metabolismdefinition

>100 enzymes

Ou

ter M

em

bran

e

Cytosol

Lactate

NADHNAD+

Mitochondrial Matrix

Inner Membrane

Metabolite-

Protein-ADP/ATP-

Redox-Transport

NADH

NADH

NADH

Krebs-cycle

FADH2 FAD

ATP ADP+Pi

Oxidative phosphorylation

V

H+

H+

IIIH+

I

NADH NAD+

IIcyt.c

H+

IV

H2O O2

Q

PDHC NADHAcetyl-CoA

PyruvateGlucoseGlycolysis

PyruvateATP

Fatty acids,Ketone bodies

Glycolysis, anaerobic2 ATP/glucose

Oxidation in mitochondriaca. 30 ATP/glucose

Energy Metabolismenergetic yield: anaerobic versus aerobic

Ou

ter M

em

bran

e

Cytosol

Lactate

NADHNAD+


Inner Membrane

Metabolite-

Protein-ADP/ATP-

Redox-Transport

NADH

NADH

NADH

Krebs-cycle

FADH2 FAD

ATP ADP+Pi

Oxidative Phosphorylation

V

H+

H+

IIIH+

I

NADH NAD+

IIcyt.c

H+

IV

H2O O2

Q

PDHC NADHAcetyl-CoA


PyruvateATP

Fatty acids,Ketone bodiesPyruvate oxidation

Krebs-cycle

Respiratory chain ATP-synthesis

Transmembrane-transport

Lipide-pattern(e.g. cardiolipin)

Cofactors(e.g. coenzyme Q)

DEFECTSMotilityfission, fusion

Toxic substances(e.g. H2S in ETHE1)

Anaplerosis

EnzymesPyruvate oxidationKrebs cycleRespiratory chainATP synthesis

Biogenesis of mitochondriaMitochondrial replication, transcription, translationImport and assembly of proteinsTransport (import+export) of metabolitesFission and fusionLipid metabolismRegulation of the amount of mitochondria

Mitochondrial Energy Metabolismpossible different kinds of defects

CofactorsThiamine, biotin, FeS-Clusters, lipoic acid, etc.

neonatallethal

systemiclactate

nuclear genes

adultslowly progressiveoligosymptomatic

lactate -mtDNA

Mitochondrial Diseases -spectrum

juvenile

adult

infantile

neonatal frequency 1:5000

Transition juvenile/adult:e.g. KSS, CPEO, CPEO+,LHON,MELAS,MERRF, CoQ10-Def.

Johns DR. NEJM 1995,10: 638-44

Genetics of mitochondrial disorders

Nuclear genome (>98% of proteins)

Inheritance: Mendelian (autosomal

recessive, autosomal dominant), X-linked,

sporadic, somatic

Mitochondrial genome (13 proteins)

Inheritance: Maternal, sporadic, somatic

mitochondrium

I II III IV V

nucleus

endopl. reticulum

~1000 mitochondrial

proteins

nukl. DNA

mtDNAreplication

mtDNA

transcription

translationassembly

13 mitochondrial proteins22 tRNAs2 rRNAs

oute

r m

em

bra

ne

inner membrane

humanmtDNA16569 bp

22x tRNA

mitochondrial genescyt b

12S

16S

2x rRNA

D-loop

1x D-loop

H+H+

NADH NAD+

0/4 complex II

II

FADH2 FAD

cyt.c

Q

H+

H2O O2H+

ATP ADP+Pi

ATP6/8

2/16 complex V

V

COX1

COX2COX3

3/15 complex IV

IV

ND1

ND2

ND3

ND4/

4L

ND5

ND6

7/~50 complex I

I

cyt.b

1/11 complex III

III

Special features of mtDNAmutations

• Inherited from the oozyte

• Random segregation of mutations in

the germ line

• Variable copy number per cell

• Cell cycle independent replication

• Quantitative mutations (depletion)

• Heteroplasmy (wildtype and mutations)

• Threshold phenomenon

• Higher mutation rate

Middle Europe: H, V South Europe: J, K

Nord Europe: T, U, X Middle East: J, N

Africa: L, L1, L2, L3, L3* Asia: A, B, C, D, E, F, G (M is made up of C, D, E, and G) Native American: A, B, C, D and a small proportion of X

Haplogrupsof mitochondrial DNA

Mutations of the mtDNAheteroplasmy - gradual mutation load

100% normal 10% normal

0102030405060708090

100

0 20 40 60 80 100Mutation load [%]

Fu

ncti

on

[%

]Mutations of the mtDNA

heteroplasmy - threshold

mtDNA depletion

Problems we face in the diagnosisof mitochondrial diseases

Family doctorPediatrician

Neuropediatrician

Laboratory 1, screening routine

Laboratory 2, Biochemistry

Metabolic specialist (second opinion)

(Rebiopsy)

Internet knowledge

PATIENT

Clinical geneticist

RadiologistFirst information

Genetic counselling

„definite“„probable“„possible“DIAGNOSIS

Final information

Laboratory 3, Genetics

Histochemistry, EM

Biopsy

Guidelines issued by the Working Group on Pediatric metabolic disorders (APS)

„Diagnostic and treatment approaches to mitochondriopathies

in children and adolescents“

*According to the prescription of the Guideline-Manual of AWMF and ÄZQ

http://www.uni-duesseldorf.de/WWW/AWMF/ll/index.html

Results of three conferences of experts (n=17)adopted by the APS and Society for Neuropediatrics

Current status of the guideline development:Development stage 2*

Suspicion of mitochondriopathy

Anamnesis

Bodily fluids - basal invest.

Physical examination including

neurological status

MITO?

MITO?

CNS?, PNS?

Muscle?

MRI (MRS), cerebrospinal fluid-lactate

Abandonment of

mito. diagnostics

CNS, PNS,

muscles Syndrome?

Encephalo-(myo)-neuropathies,

no defined syndrome

Syndrome-specific

Genetics diagnostics?

Genetically defined

syndrome?

Mutation

investigation

Blood?

Genetics in respect

of muscle biopsy

Genetically

defined

syndrome?

Specific diagnostics

Blood, urine?

1

Apparatus-based organ examinations,

stress tests, expanded laboratory

diagnostics

Muscle biopsy (+skin)

Biochemistry, histology

Unambiguous

diagnosis?

Specific genetics

appropriate?

Diagnosis

1

2

1

Resumé

MITO?

Re-enter further on


expanded laboratory diagnostics

Biopsy of the affected tissue

(+skin, muscles)

Non-neurological,

other organs

1

MITO?

MITO?Abandonment of

mito. diagnostics

Abandonment of

mito. diagnostics

Abandonment of

mito. diagnostics

Therapy

Abandonment of

mito. diagnostics

CNS-, PNS-, Muscle-involvement

MITO?Abandonment of

mito. diagnostics

Genetics, blood

Specific diagnostics, blood, urine

positive?

Specific genetics –

appropriate? Genetics, tissue

1

Biopsy of theaffected tissue? Note: We refer to the problems

associated with a biopsy

2

Syndrome with central nervous system,

peripheral nervous system, muscular involvement

2

Suspicion of mitochondriopathy

Anamnesis

Bodily fluids - basal invest.

Physical examination including

neurological status

MITO?

MITO?

CNS?, PNS?

Muscle?

MRI (MRS), cerebrospinal fluid-lactate

Abandonment of

mito. diagnostics

CNS, PNS,

muscles Syndrome?

Encephalo-(myo)-neuropathies,

no defined syndrome

Syndrome-specific

Genetics diagnostics?

Genetically defined

syndrome?

Mutation

investigation

Blood?

Genetics in respect

of muscle biopsy

Genetically

defined

syndrome?

Specific diagnostics

Blood, urine?

1


stress tests, expanded laboratory

diagnostics

Muscle biopsy (+skin)

Biochemistry, histology

Unambiguous

diagnosis?

Specific genetics

appropriate?

Diagnosis

1

2

1

Resumé

MITO?

Re-enter further on


expanded laboratory diagnostics

Biopsy of the affected tissue

(+skin, muscles)

Non-neurological,

other organs

1

MITO?

MITO?Abandonment of

mito. diagnostics

Abandonment of

mito. diagnostics

Abandonment of

mito. diagnostics

Therapy

Abandonment of

mito. diagnostics

CNS-, PNS-, Muscle-involvement

MITO?Abandonment of

mito. diagnostics

Genetics, blood

Specific diagnostics, blood, urine

positive?

Specific genetics –

appropriate? Genetics, tissue

1

Biopsy of theaffected tissue? Note: We refer to the problems

associated with a biopsy

2

Syndrome with central nervous system,

peripheral nervous system, muscular involvement

2

www.aps-med.de

www.mito-center.org

Guidelines for diagnostics and therapy

Diagnostic cascade

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites

Ergometry, loading tests

Investigation for organ involvement

Biopsy

Biochemistry Morphology

Molecular geneticsmtDNA, nuclear Genes

Diagnostic cascade

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites



Biopsy



SyndromesSuspicious symptoms

Organ involvement

Cardiomyopathies

(dilatative,

hypertrophic, Barth)

Myopathies

Hepatopathies

(mtDNA depletion)

Nephropathies

(Fanconi syndrom)

Peripheral

neuropathies

Endocrinopathies

Clinics of mitochondrial diseases

Leigh

Depletion

Pearson

Alpers

Barth

MELAS

MERRF

NARP

LHON

KSS

CPEO

MNGIE

Muscular hypotonia

Exercise intolerance

Convulsions

Ataxia and other

cerebellar

symptoms

Brainstem

involvement

Hearing loss

Short stature

Ptosis ...

Metabolites

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites

Loading tests, ergometry Ergometry, loading tests


Biopsy



Plasma/Serum:lactate, pyruvate, alaninecreatinethymidine, deoxyuridine3-hydroxybutyrate/acetoacetateCSF:lactate, alanineUrine:methylmalonic acid3-methylglutaconic acidethylmalonic acidKrebs cycle metabolites

Ou

ter M

em

bran

e

Cytosol

Lactate

NADHNAD+


Inner Membrane

Metabolite-

Protein-ADP/ATP-

Redox-Transport

NADH

NADH

NADH

Krebs-cycle

FADH2 FAD

ATP ADP+Pi

Oxidative Phosphorylation

V

H+

H+

IIIH+

I

NADH NAD+

IIcyt.c

H+

IV

H2O O2

Q

PDHC NADHAcetyl-CoA


PyruvateATP


Lactate in physiologyCori cycle

Gerty and Carl CoriNobel Prize in Physiology or Medicine 1947

anaerobic ATP in muscle

Lactate in physiologyCori + alanine cycle

Cave: Plasma lactate and alanine are in correlation with liver function

Boumezbeur et al., J Neurosci. 2010;30:13983-91

Lactate in physiologyfuel in brain metabolism

10% under basal plasma lactate conditions60% at supraphysiological plasma lactate concentrations

Debray et al., Clin Chem 2007;53:916-21

Metabolites in mitochondrial diseaseslactate/pyruvate (L:P) ratio

Clear difference between PDHC deficient and other mitochondrial patients especially at high lactate concentrations.

Ou

ter M

em

bran

e

Cytosol

Lactate

NADHNAD+


Inner Membrane

Metabolite-

Protein-ADP/ATP-

Redox-Transport

NADH

NADH

NADH

Krebs-cycle

FADH2 FAD

ATP ADP+Pi

Oxidative phosphorylation

V

H+

H+

IIIH+

I

NADH NAD+

IIcyt.c

H+

IV

H2O O2

Q

PDHC NADHAcetyl-CoA


PyruvateATP


Shaham et al., Proc Natl Acad Sci U S A 2010;107:1571-5

Metabolites in mitochondrial diseases

H+

IIIH+

I

NADH NAD+

cyt.cH+

IV

H2O O2

Q

rotenone antimycin A


Metabolites in mitochondrial diseaseselevation of creatine

creatine elevation also found by: Boenzi et al., J Inherit Metab Dis 2011;34(Suppl. 3):S160

Metabolites in mitochondrial diseaseselevation of creatine


Metabolites in mitochondrial diseasesthymidine, deoxyuridine in MNGIE

MNGIE ... Mitochondrial NeuroGastroIntestinal Encephalomyopathy (TYMP)

Martí et al., Clin Chem 2004;50:120-4

Metabolites in mitochondrial diseasesMMA – Succinyl CoA ligase

Ostergaard, J Inherit Metab Dis (2008);31:226–229

Metabolites in mitochondrial diseases3-methylglutaconic acid

Type IV 3-MGA: TMEM70, ATP5EPOLG, SUCLA2, mtDNA deletions

Wortmann, J Inherit Metab Dis (2010)

Loading tests, ergometry

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites



Biopsy



Ergometry (spiroergometry)Exercise physiology (near infrared

spectroscopy, 31PMRS)

Fasting testPre, postprandial lactateGlucose-, alanine- etc. pyruvate-loading

Organ involvement

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites



Biopsy



CNS: MRI/MRS, PET?Heart: ECHO/ECG Eye: ERGMuscle Nerve: EMG/NLG etcKidney, endocrine system etc.

Lactate

6 a old boy with myopathy1H-magnetic resonance spectroscopy

A3243G mutation (MELAS)

4 years later: „stroke-like episode“

Feedback questions 1

1. Mitochondrial diseases have been described by the statement “... any disease, any organ, any age …”. Please comment this statement.

2. Which unique features of the mitochondrial genome do you remind?

3. Which metabolites are useful for the diagnosis of mitochondrial disease?

Please prepare answers together with your neighbour (10 min).

Biopsy

Suspicious symptoms

SyndromesOrgan

involvement

Metabolites



Biopsy



Muscle: Open – Needle ?Fresh – frozen ?

Skin - fibroblastsOther tissues (Liver, heart muscle...)

Tissuefor the analysis of mitochondrial energy metabolism

1. Clinically affected tissue

2. If not possible, other tissue (muscle, fibroblasts)

Amount: Depends on investigations planned (biochemistry 20-100 mg, histology 10-50 mg, electron microscopy 10-50 mg, myoblast cultivation 10 mg, back-up 50 mg), 200 mg muscle

Muscle biopsyNeedle-biopsy under vacuum

Ø = 5 mm

Typical yield: 70-200 mg muscle

approx. 40 mg muscle

~2 rice grains

Needle biopsy:•Higher acceptance for a biopsy

•No anaesthesia in severely affected children•Miniaturized biochemical procedure necessary

Methodsanalysis of mitochondrial energy metabolism

Histological & histochemical staining, electron microscopy

Functional investigations (unfrozen cells)

Enzyme investigations

Blue Native electrophoresis

Immunological methods (western blot, immunohistochemistry, dip sticks)

Membrane potential

Histochemical staining of muscleSuccinate dehydrogenase/Cytochrome c oxidase

COX-negative COX-positive

Methods:

Measurement of

O2 (polarography, fluorimetry)

ATP (endpoint, luciferase-mediated)

CO2 (14C-labelled substrates)

Functional investigationsunfrozen biopsy tissue

ou

ter m

em

bran

e

Cytosol

mitochondrial matrix

inner membrane

NADH

NADH

NADH

Krebs-cycle

FADH2 FAD

ATP

oxidative phosphorylation

V

H+

H+

IIIH+

I

NADH NAD+

IIcyt.c

H+

IV

H2O O2

Q

PDHC NADHacetyl-CoA

pyruvatepyruvate

malate

14CO2

ADP+PiADP+Pi

[1-14C]-pyruvate

malate

ADP, Pi

Functional investigationsunfrozen biopsy tissue

Advantages:

• Measurement of transport processes

• Total activity of all enzymes of the mitochondrial energy metabolism

• Investigation of the Krebs cycle

• First results within few hours

Functional analysis – patientreport substrate oxidation

Activity in relation toprotein

Activity in relation tomarker enzyme

Clinical synopsis of patient 1:Neonatal onset hypertrophic cardiomyopathy, muscular hypotonia. Elevation of lactate up to 10 mmol/L.Excretion of 3-methylglutaconic acid in urine.Muscle biopsy at the age of 2 months.

II. Substrate oxidation analysis form 600g supernatant of fresh muscle homogenate[nmol/h/mg protein] normal range [nmol/h/mUnit CS] normal range

[1-14C]pyruvate+malate 127 6 263 - 900 P+M/CS 0,56 6 1,54 - 3,55

[1-14C]pyruvate+carnitine 298 302 - 856 P+C/CS 1,32 6 1,65 - 3,66

[1-14C]pyruvate+malate-ADP 48 32 - 102 P+M-ADP/CS 0,21 0,21 - 0,41

[1-14C]pyruvate+malate+CCCP 565 304 - 889 P+M+C/CS 2,50 1,31 - 3,11

[1-14C]pyruvate+malate+atractyloside 110 5 19 - 90 P+M+A/CS 0,49 0,16 - 0,55

[U-14C]malate+pyruvate+malonate 123 6 282 - 874 M+P+Mal/CS 0,54 6 1,56 - 3,87

[U-14C]malate+acetylcarn.+malonate 176 6 273 - 678 M+AC+Mal/CS 0,78 6 1,16 - 2,82

[U-14C]malate+acetylcarn.+arsenite 159 156 - 378 M+AC+As/CS 0,70 0,57 - 1,52

[1,4-14C]succinate+acetylcarnitine 47 6 167 - 488 G+AC/CS 0,21 6 1,36 - 2,53

Functional analysis – ATP synthesissubstrate oxidation of intact muscle-mitochondria

Patients with defective ATP-synthesis

pyruvate + malate + CCCP

pyruvate + malate + ADP

Respiratory chain(w/o ATP-synthesis)

Oxidative phosphorylation(incl. ATP-synthesis)

CCCP/ADP-activation

rati

o0

1

2

3

4

5

6

7

8

9

P1

Synthesis of ATPvia oxidative phosphorylation

Fo

H+

PiC

H+ Pi

ANT

ADP ATP

F1

Pi ADP+ ATP

ATP synthesisapparatus

1. F1Fo ATP synthase

2. Adenine-nucleotide-translocator, ANT

3. Mitochondrial phosphate-carrier, PiC

340 nm

ATP ADP + P

ATPase

pyruvate PEP

pyruvate kinase

lactate dehydrogenase

lactateNAD

NADH

Enzyme investigationse.g. oligomycin-sensitive ATPase (complex V)

Tris pH 8.0 40 mM

MgCl2 5 mM

KCl 10 mM

lactate dehydrogenase 4 U/ml

pyruvate kinase 4 U/ml

NADH 0.2 mM

PEP 2 mM

ATP 0.5 mM

BSA 0.1 %

antimycin A 1 µM

FCCP 3 µM

Conc.Substance

Oligomycin-sensitive ATPaseassay mixture

• 10 s sonification to open the mitochondrial membranes

• Follow NADH decrease

at 340 nm 3-5 min.

• Inhibition with

oligomycin.

Oligomycin-sensitive ATPaseassay mixture

Oligomycin-sensitive ATPasedecrease at 340 nm

start measurement after sonification

inhibition with oligomycin

• 1st derivative:

Total activity

Oliogomycin inhibited

*specific enzyme activity:oligomycin-sensitive ATPase

*

Oligomycin-sensitive ATPasedecrease at 340 nm

Enzyme activitiesreport

I. Enzyme activities form 600g supernatant of fresh muscle homogenate[mUnit/mg protein] normal range [mUnit/mUnit CS] normal range

Citrate synthase (CS) 226 150 - 325

Complex I (CI) 18 6 28 - 76 CI/CS 0,08 6 0,17 - 0,31

Complex I+III (C13) 60 64 - 218 C13/CS 0,27 0,24 - 0,81

Complex II (CII) 78 39 - 102 CII/CS 0,35 0,23 - 0,41

Complex II+III (C23) 119 93 - 180 C23/CS 0,52 0,55 - 0,67

Complex III (CIII) 564 426 - 762 CIII/CS 2,49 2,12 - 3,09

Cytochrome c oxidase (COX) 583 452 - 889 COX/CS 2,58 2,59 - 3,12

Complex V (CV) 5 6 70 - 397 CV/CS 0,02 6 0,47 - 1,47

Pyruvate dehydrogenase (PDHC) 10,5 6,1 - 19,8 PDHC/CS 0,046 0,034 - 0,079

1. Decrease of complex V (oligomycin-sensitive ATPase)2. Less severe: defect of complex I

Activity in relation toprotein

Activity in relation tomarker enzyme

Blue Native PAGE muscle tissue

Complex I -

F1Fo-ATP synthase -

Complex III -

Cytochrome c Oxidase -

C P1

1. Decrease of complex V (oligomycin-sensitive ATPase)2. Less severe: defect of complex I

Genetic cause of the diseasehomozygosity mapping

control

patient

INTRON 2 EXON 3

Cízková et al., Nat Genet 2008;40:1288-90

TMEM70, mitochondrial transmembrane protein

Activitie

s a

t 25°C

-37°C

[U

/g N

CP

])

1

10

100

1000Lab 1 (37°C)

Lab 2 (30°C)

Lab 3 (37°C)

Lab 4 (30°C)

Lab 5 (25°C)

Lab 6 (30°C)

Lab 7 (37°C)

Lab 8 (25°C)

Lab 9 (30°C)

Lab 10 (37°C)

Lab 11 (25°C)

Lab 12 (30°C)

C I

C I+

III

C II+

III

CO

X

CS

AT

Pase

NC

P

C III

C II

PD

HC

6

2

Cave: comparison of enzyme activitiesinternational multicenter trial of 12 diagnostic laboratories

Gellerich et al., Mitochondrion 2004;4:427-39

Feedback questions 2

1. Which tissue should be analysed to identify a mitochondrial disease?

2. Which biochemical methods are used for the identification of mitochondrial disorders?

3. What are advantages of the investigation of intact mitochondria?

Please prepare answers together with your neighbour (10 min).

• Girl, normal birth after uneventful pregnancy.• Neonatal hearing test revealed congenital deafness• 8 weeks, lack of head control, truncal hypotonia.• Plasma lactate 1.3 to 8.9 mmol/L (normal 0.5-2.2 mmol/L).

• Aminoaciduria, glucosuria, renal loss of phosphate and urate indicated proximal tubulopathy.

• MRI at the age of 10 weeks was normal.• 3 months, poor sucking and recurrent vomiting necessitated continuous nasogastric tube feeding

• She died under palliative care at the age of 4 month.

Case Aclinical synopsis

Case Ainvestigation of a muscle biopsy

• Histological: ragged red fibers and fat accumulation, severe decrease of cytochrome c oxidase staining

-

% o

f n

orm

al

16%22%

101%

31%23%

11%

34%

0%

100%

com

plex

I/C

S

com

plex

I+III/CS

com

plex

II/CS

com

plex

II+

III/CS

com

plex

III/C

S

cyto

chro

me c ox

idas

e/CS

oligom

ycin-s

ens. A

TPas

e/CS

Case Aenzymes in relation to citrate synthase

ou

ter

mem

bran

einner membrane

humanmtDNA16569 bp

22x tRNA

mitochondrial genecyt b

12S

16S

2x rRNA

D-loop

1x D-loop

H+H+

NADH NAD+

0/4 complex II

II

FADH2FAD

cyt.c

Q

H+

H2O O2

H+

ATP ADP+Pi

ATP6/8

2/16 complex V

V

COX1

COX2COX3

3/15 complex IV

IV

ND1

ND2

ND3

ND4/

4L

ND5

ND6

7/~50 complex I

I

cyt.b

1/11 complex III

III

control

mitochondrial DNA nuclear DNA

patientmtDNA-content:

4% of control

Case Aquantitation of mtDNA content

Case Amutation in RRM2B

Acham-Roschitz et al., Mol Genet Metab 2009;98:300-4

Yen et al. Clin Cancer Res 2003; 9:4304-8

NDP dNDP

Ribonucleotide reductase(cytosolic enzyme)

ribonucleotides

deoxyribonucleotides

MNGIE

Copeland WC. Annu Rev Med 2008,59:309-24

Encephalomyopathykidney

Encephalo-myopathy,mild MMA

Myopathy Hepato-cerebral

symptoms

SUCLG1

Mitochondrial DNA depletion

• A girl was born after normal pregnancy.• Free walking at the age of 17 months.• At 7 years referred to gastroenterologist due to loss of appetite, weakness and slight growth retardation.

• Discrete muscular atrophy.• Slightly elevated transaminases, lactate dehydrogenase and creatine kinase.

• Bicycle ergometry showed an endurance performance of 70% of age-matched controls, increase in lactate from 4.0 to 8.1 mmol/l

• Myopathic pattern in electromyographic investigation.

Case Bclinical synopsis

Case Bmuscle biopsy investigations

Gömöri staining:ragged-red fibres

Cytochrome c oxidase (brown)

succinate dehydrogenase

(blue)

Electron microscopy:large mitochondria with tubular cristae

Case Bmuscle biopsy investigations

Gömöri staining:ragged-red fibres

Cytochrome c oxidase (brown)

succinatedehydrogenase

(blue)

Electron microscopy:large mitochondria with tubular cristae

ou

ter

mem

bran

einner membrane

humanmtDNA16569 bp

22x tRNA

mitochondrial genecyt b

12S

16S

2x rRNA

D-loop

1x D-loop

H+H+

NADH NAD+

0/4 complex II

II

FADH2FAD

cyt.c

Q

H+

H2O O2

H+

ATP ADP+Pi

ATP6/8

2/16 complex V

V

COX1

COX2COX3

3/15 complex IV

IV

ND1

ND2

ND3

ND4/

4L

ND5

ND6

7/~50 complex I

I

cyt.b

1/11 complex III

III

Case B - Single fibre PCRmutation in a mitochondrial tRNA gene

Mayr et al., Neuromuscul Disorders 2006;16:874-77

DHPLC

mitochondrialtRNAGlu

sequenceanalysis

single muscle fibre

dissection+

mutation quantification

Patient C – Case reportfamiliy of Turkish origin, non consanguineous

Findings

Sex

Birth weight [g]

Hypertrophic CMP

Muscular hypotonia

Hyperlactatemia [mmol/l]

Poor feeding

Death at the age of

Organic and amino acids,

carnitine, acylcarnitine,

Enzymes: OXPHOS, PDHC

patient C2

female

2870

+

+

4-17

+

9 months

normal

normal

patient C

female

2600

+

+

4-15

+

6 months

normal

normal

Patient Cresults of biochemical investigations



Complex I (CI) 63 28 - 76 CI/CS 0,32 0,17 - 0,31






Complex V (CV) 137 70 - 397 CV/CS 0,70 0,47 - 1,47




[1-14C]pyruvate+carnitine 98 6 302 - 856 P+C/CS 0,50 6 1,65 - 3,66

[1-14C]pyruvate+malate-ADP 35 32 - 102 P+M-ADP/CS 0,18 6 0,21 - 0,41

[1-14C]pyruvate+malate+CCCP 383 304 - 889 P+M+C/CS 1,96 1,31 - 3,11

[1-14C]pyruvate+malate+atractyloside 41 19 - 90 P+M+A/CS 0,21 0,16 - 0,55


[U-14C]malate+acetylcarn.+malonate 73 6 273 - 678 M+AC+Mal/CS 0,38 6 2,06 - 3,52

[U-14C]malate+acetylcarn.+arsenite 52 6 156 - 378 M+AC+As/CS 0,27 6 1,22 - 1,96

[1,4-14C]succinate+acetylcarnitine 24 6 167 - 488 S+AC/CS 0,12 6 1,36 - 2,53

III. Respirometric investigations of native permeabilised muscle fibres[nmol O2/min/mg muscle fibre] normal range [pmol O2/min/mUnit CS] normal range

Pyruvate+malate+ADP 0,54 6 1,19 - 2,20 P+M/CS 34 6 107 - 204

Succinate+rotenon+ADP 1,12 1,17 - 2,25 S+Rot/CS 71 6 106 - 212

TMPD ascorbate*+FCCP 3,97 2,41 - 5,49 TAsc+F/CS 251 229 - 517*N,N,N',N'-Te trame thyl-1,4 -phe nyle nd iamid + A sc orba t (subs tra te fo r c ytoc hrome c oxidase )

Functional analysis muscle vs. fibroblasts

ATP synthesis in patient C

Patient C: Tissue specific defect of thesynthesis of ATP

CC

CP

-vs.

AD

P-s

tim

ula

tio

n

Muscle Fibroblasts

Patient C

0

1

2

3

4

5

6

7

8

9

PC PC-2

Synthesis of ATPvia oxidative phosphorylation

Fo

H+

PiC

H+ Pi

ANT

ADP ATP

F1

Pi ADP+ ATP

ATP synthesisapparatus

1. F1Fo ATP synthase

2. Adenine-nucleotide-translocator, ANT

3. Mitochondrial phosphate-carrier, PiC

Mitochondrial Phosphate CarrierSLC25A3 gene on chromosome 12

3AIsoform A

3BIsoform B

“mutually exclusivealternative splicing”

Heart, skeletal muscle

Fibroblasts and other tissues

http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000075415

Patient 4: Mutation in the Exon 3Amitochondrial phosphate carrier, SLC25A3

A highly conserved glycine is replaced by glutamic acid.

Mayr et al., Am J Hum Genet 2007;80:478-84

Patient C Exon-3A E

Human PiC Exon-3A YSCDYGSGRFFILCGLGGIISCGTTHTALVPLDLVKCRMQ 94

Human PiC Exon-3B YSCEFGSAKYYALCGFGGVLSCGLTHTAVVPLDLVKCRMQ 93

Xenopus trop. FSCEYGSGTFYAYCGFGGILSCGLTHTAVVPLDLVKCRLQ 92

Drosphila mela. DSCEFGSTKYFALCGIGGILSCGTTHTFVVPLDLVKCRLQ 91

S.pombe KTLQLYTPQYYGLCTLGGLLACGTTHSAITPLDLIKCRKQ 52

S.cerevisiae-PIC2 RKIQLYTKEFYATCTLGGIIACGPTHSSITPLDLVKCRLQ 47

Arabidopsis thal. KGIEMYSPAFYAACTFGGILSCGLTHMTVTPLDLVKCNMQ 107

S.cerevisiae-MIR1 -AIPQYSVSDYMKFALAGAIGCGSTHSSMVPIDVVKTRIQ 46

homology : : :.* :.** ** :.*:*::* . *

16

543

2

Growth on YP glycerol (3%)1: wild type2: mp (mir1pic2)3: mp hPICA-E (mutant)4: mp hPICA-G (wild type)5: mp yMIR1-E (mutant)6: mp yMIR1-G (wild type)

• A boy, born after uneventful pregnancy at term.• At 2 years he became dizzy and vertiginous and developed a gait disturbance but recovered.

• Several similar episodes during the next years with spontaneous remission.

• 10 ½ years admitted to hospital with loss of speech• Seizure with clonic jerks of his arms.• Cerebellar and bulbar affection with dysarthria, intention tremor, confusion and episodic ataxia.

• Ophthalmoplegia and nystagmus were noted and there was spasticity right accented.

• MRI which was normal before showed now changes in the white matter of the cerebellum.

Case Dclinical synopsis

• MR-Spectroscopy of the cerebellum revealed a soft decrease of N-acetylaspartate und an increase of choline, lactate was normal.

• Respiratory insufficiency needing artificial ventilation for 6 days.

• He recovered and is mentally normal, attending high school, present age 17 years.

• Lactate was elevated up to 4.4 mmol/L in plasma and 3.3 mmol/L in the cerebrospinal fluid during the crisis otherwise normal.

• Histologically normal muscle biopsy.

Case Dclinical synopsis (continued)

Case Dmuscle biopsy investigations



Complex I (CI) 40 28 - 76 CI/CS 0,16 0,14 - 0,35






Complex V (CV) 170 86 - 257 CV/CS 0,70 0,42 - 1,26




[1-14C]pyruvate+carnitine 195 6 302 - 856 P+C/CS 0,80 6 1,65 - 3,66

[1-14C]pyruvate+malate-ADP 54 32 - 102 P+M-ADP/CS 0,22 0,21 - 0,41


[U-14C]malate+acetylcarn.+malonate 414 273 - 678 M+AC+Mal/CS 1,70 1,16 - 2,82

[U-14C]malate+acetylcarn.+arsenite 225 156 - 378 M+AC+As/CS 0,92 0,57 - 1,52

[U-14C]glutamate+acetylcarnitine 337 167 - 488 G+AC/CS 1,38 0,90 - 2,06

1. [1-14C]pyruvate+malate2. [1-14C]pyruvate+carnitine3. [U-14C]malate+pyruvate+malonate4. [U-14C]malate+acetylcarn.+malonate5. [U-14C]malate+acetylcarn.+arsenite6. [1,4-14C]succinate+acetylcarnitine

Diagnosis: Disturbed pyruvate oxidation

15% 14%24%

112%

97%103%

0%

100%

1. 2. 3. 4. 5. 6.

patient X

38% 37% 41%

89%99%

106%

0%

100%

1. 2. 3. 4. 5. 6.

patient D

Intact mitochondria – Pyruvate oxidation

patients X+D (muscle tissue)

15% 14%24%

112%

97%103%

0%

100%

1. 2. 3. 4. 5. 6.

patient X

38% 37% 41%

89%99%

106%

0%

100%

1. 2. 3. 4. 5. 6.

patient D

142%

162%

119%

172%

82%

107% 108%

143%

16%

0%

100%

CS C I C I+III C II C II+III C III COX ATPase PDHC

PDHC: 16% residual activityE1 Gen: CGA->GGA, Arg263Gly

105%

83%

101%

115%

143%

70%

92% 96%

84%

0%

100%

CS C I C I+III C II C II+III C III COX ATPase PDHC

PDHC-activity is normal (84%)E1 ,E1β,E2,E3BP,PDP1: normal

Intact mitochondria – Pyruvate oxidation

patients X+D (muscle tissue)

C1 C1 P3 P3

E2 -E3BP -

E1α -E1β -

su OSCP -

- 132

- 78

- 45

- 32

- 18

Protein [µg]

C2 PD P2 C3

5 510 10 10 10 10 10 kDa

Western blot analysis

subunits of pyruvate dehydrogenase

Normal pattern of PDHC subunits in patient D.

E2

E3BP

Smolle et al., J Biol Chem 2006;281:19772-80

30x E1α subunit30x E1β60x E2 12x E3BP12x E3PD-Kinase (PDK)PD-Phosphatase (PDP)PDP regulatory protein

total size: ~10 MDalton!

PDHC - structure

[TPP]

CO2

CH3-C-COOH

=O

[CH3-C=TPP]-

OH

[CH3-C-S-LipSH]

=O

CH3-C-S-CoA

=O

CoA-SH

[Lip(SH)2]

[LipS2]

FAD

FADH2

NAD+

NADH + H+

pyruvate acetyl-CoA

E1E2

E3

Pyruvate dehydrogenase

reaction mechanism

PDHC reaction depends on cofactors

0

1

2

3

PD

HC

ac

t. -

TP

P

[mU

nit

s/g

pro

tein

]

0.00

0.05

0.10

0.15

0.20

rati

o:

PD

HC

ac

tivit

y -

/+T

PP

Patients ControlsPatients Controls


thiamine pyrophosphate dependency

TPP TMP

Thiamine

Control

Patient D

Standard

TPP

TMP Thiamine

TPPThiamine


thiamine pyrophosphate dependency

Muscle 600g sup. [nmol/g prot.]

TPP TMP Thiamine

P2 8.8 0.1 1.5P3 9.5 0.1 3.0Patient D 9.3 0.2 1.9Controls (n=9)Mean ± SD 58.7 ± 12.6 1.1 ± 0.4 0.9 ± 1.4

range 41.6 - 81.6 0.4 - 1.7 0.2 - 4.4

Blood [nmol/L] TPP TMP Thiamine

P3 68.0 2.2 21.4P4 50.4 1.2 5.3Patient D 96.9 18.1 67.5Controls (n=10)Mean ± SD 190.9 ± 41.5 6.2 ± 1.3 10.7 ± 6.1

range 132.2 - 271.2 4.1 - 8.8 5.0 - 26.4

Deficiency of TPP in muscle and blood.

Thiamine(TMP)

Thiamine

TPK1

SLC19A2Pla

sm

a m

em

bra

ne

SLC19A3

TPP

TPP

TransketolaseTPP

TPP

BCKDHTPP

PDHCTPP

α-KGDHTPP

SLC25A19

ATP AMP

Mitochondrion

Affected Individuals P3+P4 P1+P2 Patient D

Missense mutation P H S

H. sapiens 33 LWNKALLRACADGGANRLYDI .. LVSTSNTYDGS 224

M. musculus 33 LWKKALLRACADGGANHLYDL .. LVSTSNTYDGS 224

D. rerio 33 LWSKAQIRACADGGANHLYRL .. LVSTSNTYEDH 224

D. melanogaster 116 LWKNAAVRCAVDGGSNHWRDF .. MVSTSNTYATE 316

A. thaliana 40 LWEHAKLRLCADGGANRIYDE .. LISTSNLVKEE 239

S. cerevisiae 56 IWKLHDLKVCADGAANRLYDY .. RVSSSNRFVGD 293

Clostr. tetani 20 ELKDSDIIIAADKGAEALYKC .. GLGVSNEIKEN 195

Staph. aureus 18 AKSNEGKWGGVDRGALILLKH .. TLTISNEIESL 193

Consensus . .* .: : **

Mutation analysisthiamine pyrophosphokinase, TPK1 gene


GAPDH

TPKIsoform aIsoform b

Prot. [µg] 5 10 5 10 10 510 510 5

P2 C1 P3 C2 Patient D

Western blot analysisthiamine pyrophosphokinase


• “... any disease, any organ, any age ...”

• enzymes – biogenesis – cofactors

• mitochondrial DNA – nuclear DNA

• systematic clinical and laboratory analyses

• functional investigations: enzymes, proteins, intact mitochondrial membranes

Summarydefects in the mitochondrial energy metabolism

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Laboratory investigation of mitochondrial disease · 2014-04-21 · Laboratory investigation of...

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