mitokondriesykdom Muskelbiopsiog

Muskelbiopsi og mitokondriesykdom

Laurence Bindoff

Overview

• Indications for biopsy (for investigating mitochondrial disease!)

• Clinical features

• Indications/Are there quicker ways to the diagnosis

• New genetic methods are reducing the need for biopsy

• There are still definite indications for biopsy

• How muscle biopsy should be handled

• What can we get out of a biopsy

• Morphology, histochemistry, genetics

• Biochemical analyses (Yngve Thomas Bliksrud)

Clinical diagnostic decision map

Molecular diagnosis

Clinical

Genotype

The problem is that everything can be

“mitochondrial”

• Neurology:

• Encephalopathy, Epilepsy, Ataxia, Mental retardation, Deafness;

• Endocrine:

• Diabetes

• Gastro;

• Liver disease, bowel stasis

• Haematology:

• anaemia, pancytopenia

• etc. etc.

Inner mito membrane OUT

IN I

II

III

IV

V

Q pool

Q Q

Q

Q

c c c

mtDNA

• Assembly

• Transport

• Uptake

Respiratory chain

mtDNA

homeostasis

And there are A lot of genes

We need to recognise the classical ones!

• mtDNA

• CPEO/KSS/Pearson’s

• MELAS/MERRF

• LHON etc.

• nDNA

• Leigh

• Alpers/POLG

• etc.

Diagnostic decision map

Molecular diagnosis

Clinical

Next generation sequencing

Recognisable phenotype

Genetics

(mtDNA/nDNA)

mtDNA

nDNA

Single gene

When it is not straightforward and there are many differentials

• We look for supportive features

• Blood tests

• Lactate

• Organic acids etc

• Electrophyisology

• Imaging

• MRI

• MRS

• Tissue for biochemical confirmation

Diagnostic decision map

Molecular diagnosis

Clinical

Next generation sequencing

Suspect phenotype

Supportive tests:

Bl. Lactate; Metabolites incl.

organic acids ; FGF21/GDF15;

Imaging/MRS etc.

Recognisable phenotype

Genetics

(mtDNA/nDNA)

mtDNA

nDNA

Single gene

Tissue Biopsy Biochemical/

protein diagnosis

Choice of tissue

• Remember

• Nuclear gene defect are usually (but not 100%) identifiable in blood cells!

• The most common reasons for needing tissue are:

• A baby/child with a complex syndromic phenotype

• AND genetics have not helped

• You suspect a tissue restricted mtDNA mutation

• mtDNA mutations can segregate to ONE or FEW tissues

• Single mtDNA deletions (Kearns-Sayre, CPEO etc.)

• Several point mutations

• Incl. common ones e.g. m.3243A>G

• Usually the disease involves skeletal muscle so biopsy relevant

• Remember urine can be a good alternative

• The most common tissue is skeletal muscle

• Other tissues are problematical

• We often do not have a control range

Muscle biopsy - how?

How is the muscle biopsy handled?

Muscle Biopsy Procedure and Processing, Myopathology; Springer

Fixative Biochemical & molecular

studies

Ultrastructural studies

Routine histochemical studies

Snap frozen in isopentane cooled in liquid nitrogen

Fresh

Routine histochemical studies

• Standard stains performed on «frozen» muscle

• H&E, ATPase (pH 9,4; 4,3; 4,6), NADH, SDH, COX, Gomori, PAS, Oil red O

pH 4.3

I II

https://neuromuscular.wustl.edu/

Histochemical stains relevant for mitochondria

• Standard stains

• NADH, SDH, COX, Gomori, PAS, Oil red O

NADH SDH Gomori COX

PAS

Oil Red O

https://neuromuscular.wustl.edu/

Inner mito membrane OUT

IN I

II

III

IV

V

Q pool

Q Q

Q

Q

c c c

Cytochrome c oxidase

Succinate

dehydrogenase

Specific mitochondrial stains

Electron microscopy

Bindoff

https://neuromuscular.wustl.edu/

Mitochondrial pathology

H&E

Mitochondrial pathology

Inner mito membrane

OUT

IN I

II

III

IV

V

Q pool

Q Q

Q

Q

c c c

Cytochrome c oxidase

Succinate dehydrogenase

SDH/COX

Ragged blue fibre COX neg fibres

Mitochondrial pathology

Bruschigliaro & Zeviani

https://doi.org/10.1016/j.bbabio.2020.148335

Downhan E, et al.

https://doi.org/10.1016/j.bbabio.2020.148335

Total COX deficiency Ragged Brown fibres

Some traps - myositis

Mitochondrial abnormalities in inclusion-body myositis

Oldfors, A. R. et al. doi.org/10.1212/01.wnl.0000192127.63013.8d

Mitochondrial pathology in inclusion body myositis Lindgren, et al. Neuromuscular DisordersDOI: 10.1016/j.nmd.2014.12.010

Other myopathies - myofibrillar

Impact of age/biopsy site

July 2015 BMC Neurology 15(1):114

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle. Pesce et al.

Free Radical Biology & Medicine, Vol. 30, No. 11, pp. 1223–1233, 2001

Paraspinal muscle

Takk for oppmerksomhet