Practical Approaches To A Mito Diagnosis: Richard H. Haas M.B., B.Chir., M.R.C.P

Practical Approaches to
a Mito Diagnosis
Richard H. Haas M.B., B.Chir., M.R.C.P.
Director UCSD Mitochondrial Disease Laboratory
Co-Director UCSD Mitochondrial and
Metabolic Disease Center
Overview of Mitochondrial Diagnosis

Basic Mito Facts and Background, including what mitochondria do
Types of problems that can be caused by Mito dysfunction
Genetics, in brief
Mito Diseases - what are they and how are they classified
(OXPHOS, Leigh's, MELAS, etc.)
Inherent problems in diagnosis/diagnostic approaches of both
OXPHOS and mtDNA disease - Heteroplasmy
Testing, in brief, including advantages and limitations of new
nDNA gene sequencing
Clues to the diagnosis of mitochondrial disease for clinicians (and
families)
How does one arrive at a diagnosis of Mito disease? Combination of
clinical testing, biochemical testing, personal and family history, and
symptoms/clinical presentation
Why are more invasive tests (i.e. muscle biopsy) sometimes
necessary?
How is the field of mitochondrial medicine changing? Are there new
types of mitochondrial disease? What may the future look like for
this field and for patients/families?
Basic Mito Facts

Prokaryote (Bacterial) origin of
mitochondria & mtDNA symbiotic
relationship
1500 nuclear mitochondrial genes
2-10 mtDNA molecules per mitochondrion
100 10,000 mitochondria per nucleated
cell
mtDNA is maternally inherited
Kirkman MA, Yu-Wai P, Chinnery PF

Clin Med. 2008 Dec;8(6):601-6.
Major Mitochondrial Functions

Make ATP for cellular energy
oxidative phosphorylation
Metabolize
fats
carbohydrates
amino acids
Interconvert carbohydrates, fats and amino acids

Synthesize some proteins
Reproduce themselves (replicate), fusion/fission
Participate in apoptosis
Make free radicals
Innate Immunity
Human mtDNA 16569 bp
Patient photos
removed for
privacy purposes.
Oxphos Disease
A disease of energy metabolism resulting in
impairment of oxidative phosphorylation
Nuclear Gene Defects (80% of Child disease)

mtDNA Defects (60% of Adult disease)
Leigh Syndrome
Cytochrome Oxidase Deficiency
Experimental Treatment with TAU and DCA
patient photo removed for

privacy purposes.
Age 5
Age 8
Age 16
Graduating from HS
In June 2011
Leigh Syndrome: Subacute Necrotizing

Encephalomyelopathy
Leigh
Syndrome
15%
30%
25%
MELAS
Mitochondrial Encephalomyopathy with
Lactic Acidosis and Stroke-like Episodes
Severe Pediatric MELAS

90% Heteroplasmy
Age 19: prepubertal,
short stature, ataxia,
dementia, seizures
Multiple occipital
infarcts cortical
blindness
Deafness, myopathy,
cardiomyopathy
Plasma lactate 3.5 mM,
CSF lactate 5.5 mM
Calcified Basal Ganglia
Skoglund RR.
Neurology. 1979 May;29(5):717-20
Heteroplasmy
Wild Type
Mutant
Heteroplasmy in Fibroblasts
Cox I 488
Control
MELAS
3243
Porin 594
merge
How Does Genetic Mitochondrial

Disease Present ?
Acute/ Subacute
Severe Metabolic Crisis

Encephalopathy
Arrhythmia, Heart block
Opthalmoplegia, Blindness
Stroke
Chronic
Growth Retardation
Developmental Delay
Strabismus
Diabetes
Irritable Bowel Syndrome
Cardiomyopathy
Neuropathy, Ataxia
Hypotonia and Weakness
Exercise Intolerance
Dementia
Severity of Disease Affects Onset

MITOCHONDRIAL DISEASE
SEVERE
MODERATE
MILD
INFANCY
CHILDHOOD
TEENAGE
ADULT
Severe Lactic Acidosis
Leigh's Syndrome
MELAS
Parkinson's Disease
Diagnosis of Mitochondrial Disease

Clinical Symptoms
Physical Exam
Family History
Metabolic Tests
Blood, Urine, CSF
Tissue Biopsy
(Skin, Muscle,
Liver, Heart)
Diagnosis
Organ Evaluation
(MRI/MRS,
EKG/Echo)
Molecular Genetics
Biochemistry
Oxphos Studies
ETC analysis
Metabolic & Other Tests

Blood, Urine & CSF
CPK
Lactate and Pyruvate
Ammonia
Plasma Amino Acids
Plasma Acyl-carnitine profile
Plasma Carnitine
Urine Organic Acids
DNA Studies
Available Mito Tests

Test
Tissue
Histology/EM
Muscle, Liver, Heart
Carnitine, CoQ
Nuclear DNA
mtDNA
Blood, All Tissues

All Tissues, Muscle Best
Electron Transport
Assays
Polarographic Assay
Muscle Fresh/Frozen,
Fibroblasts, Liver, Heart
Fresh muscle, Liver, Heart
or Mitochondria
Enzyme Assay
All Tissues, Mitochondria,

Fibroblasts
Protein Immunoassay Mitochondria (Blue Native)
Tissue (Clear Native)
Immunocytochemistry
Fibroblasts/ Muscle Tissue
Immunohistochemistry
Tissue Diagnosis
Available Tissues
Blood
Saliva (Buccal Epithelial Cells)
Urine Sediment
Muscle
Skin Fibroblasts
Other Tissues
Liver
Heart
Heteroplasmy
Wild Type
Mutant
The Basics of Tissue Testing for

Mitochondrial Disease
Tissues for mtDNA Testing

The Heteroplasmy Problem
Blood
Saliva
Urine
Muscle
% of Mutation
Saliva Collection (Oragene)
Muscle Biopsy Problems

Histochemistry
Often normal in Pediatric cases
Electron Microscopy (EM)

May help but often difficult to get
ETC Assays
Lab to lab variation
Very susceptible to sample handling
Leigh
Syndrome
15%
30%
25%
Figure 3. Residual activity of complex I CS ratios in the 66 skeletal muscle biopsies

analyzed in this patient series
Bernier, F.P. et al. Neurology 2002;59:1406-1411
Ragged Red Fiber Myopathy
Neurometabolic Evaluation
SS Age 23 months
Referred age 16 months with

Global delay & hypotonia
Plasma lactate 4.1 mM
CPK 155 U/L
Urine organic acids mild
increase in 3-OH isovalerate
and glutamate.
Plasma acylcarnitines
C5OH, C3 and C2.
Biotinidase normal
Leukocyte carboxylases
normal
Muscle PCR Msp-I Digest

SS
PC
WT
NARP 8993 T>C or G

60-70%
338
169
Summary
Tissue sampling for mitochondrial disease is
dictated by the tests required. Nuclear DNA
testing requires only blood
Blood, saliva and urine for mtDNA testing
are all feasible but heteroplasmy presents a
problem
Muscle biopsy remains the Gold Standard
for electron transport chain assay and for
mtDNA testing
Fresh muscle offers the opportunity to
perform functional polarography and to
isolate mitochondria for electron transport
and protein study
Probability of Mitochondrial
Disease
Clinical + Biochemical Criteria
Definite
Probable
Possible
Unlikely
Wolf N., Smeitink J.A.

Neurology. 2002 Nov 12;59(9):1402-5
Epidemiology
>1:200 Children are Born with Potentially

Pathogenic mtDNA Mutations
The American Journal of Human Genetics 83, 254260, August 8, 2008

Screened for just 10 (5%) of >200 known pathogenic mtDNA mutations.
mtDNA Disease
(<50% of Total)
9.2 per 100,000 Retired Adults
16.5 per 100,000
Working Adults and Children
Total Prevalence = 25.7 per 100,000
= 1 in 4,000 (3,891)
mtDNA + nDNA Disease

Birth Incidence
1 in 2,000 will Develop Disease
1 in 4,000 Before Age 10
1 in 4,000 After Age 10
Epidemiology of Mitochondrial DNA Disease
Expanding the Phenotype

A never-ending process
The Dynamic Nature of Mitochondrial Networks

Control Fibroblast
Severe Complex I Deficiency
From Nhu-an Pham et al. Microsc.Microanal. 10, 247-260, 2004
David Chan
Caltech
Mitochondrial fusion and fission

Mitochondrial fusion GTPases
Mitofusin 2
(MF2) Charcot-Marie-Tooth disease CMT2A
HMSN VI
Optic atrophy 1 (OPA1)

Autosomal Dominant Optic Atrophy
Fission proteins
Dynamin Related Protein 1 (DRP1)
Infantile mitochondrial cytopathy with lactic
acidemia VLCFA, optic atrophy and hypotonia
Autism:
tRNA Lys G8363A mtDNA Point Mutation
Four year-old boy with history of normal pre-,
peri- and postnatal courses

Normal development until 18 months of age
Progressive loss of expressive language and
language comprehension
Gradual increase in disruptive behavior,
hyperkinesis, and self injurious behavior
Mild motor clumsiness but no ataxia
Normal plasma lactate
Sister with Leigh Disease
Graf W.D. et al. J Child Neurol. 2000 Jun;15(6):357-61
Autism
Spectrum
Autism and Mitochondria
1:110
Classical
Autism
Definite
Mito Disease
>1:5000
5 - 8%
Possible Mito
Disease
(Mito Dysfunction)
Probable
Mito Disease

Practical Approaches To A Mito Diagnosis: Richard H. Haas M.B., B.Chir., M.R.C.P

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Practical Approaches to

Overview of Mitochondrial Diagnosis

Basic Mito Facts

Kirkman MA, Yu-Wai P, Chinnery PF

Major Mitochondrial Functions

Interconvert carbohydrates, fats and amino acids

Human mtDNA 16569 bp

Nuclear Gene Defects (80% of Child disease)

patient photo removed for

Leigh Syndrome: Subacute Necrotizing

Severe Pediatric MELAS

How Does Genetic Mitochondrial

Severe Metabolic Crisis

Severity of Disease Affects Onset

Severe Lactic Acidosis

Diagnosis of Mitochondrial Disease

Metabolic & Other Tests

Available Mito Tests

Muscle, Liver, Heart

Blood, All Tissues

All Tissues, Mitochondria,

The Basics of Tissue Testing for

Tissues for mtDNA Testing

Saliva Collection (Oragene)

Muscle Biopsy Problems

Electron Microscopy (EM)

Figure 3. Residual activity of complex I CS ratios in the 66 skeletal muscle biopsies

Bernier, F.P. et al. Neurology 2002;59:1406-1411

Ragged Red Fiber Myopathy

Referred age 16 months with

Muscle PCR Msp-I Digest

NARP 8993 T>C or G

Wolf N., Smeitink J.A.

>1:200 Children are Born with Potentially

The American Journal of Human Genetics 83, 254260, August 8, 2008

mtDNA + nDNA Disease

Epidemiology of Mitochondrial DNA Disease

Expanding the Phenotype

The Dynamic Nature of Mitochondrial Networks

Severe Complex I Deficiency

From Nhu-an Pham et al. Microsc.Microanal. 10, 247-260, 2004

Mitochondrial fusion and fission

Optic atrophy 1 (OPA1)

tRNA Lys G8363A mtDNA Point Mutation

Four year-old boy with history of normal pre-,

peri- and postnatal courses

Graf W.D. et al. J Child Neurol. 2000 Jun;15(6):357-61

Autism and Mitochondria

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