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
Recognisable PhenotypeGenotype
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
