Mitochondrial Diseases: Evaluation and Treatment · 1984: Syndromic Paper in Ann Neurology (MERRF,...
Transcript of Mitochondrial Diseases: Evaluation and Treatment · 1984: Syndromic Paper in Ann Neurology (MERRF,...
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Mitochondrial Diseases: Evaluation and Treatment Neurobiology of the Diseases of Children
Bruce H. Cohen, MD Professor of Pediatrics, Northeast Ohio Medical University
Director of Neurology, Akron Children’s Hospital
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Thanks to my Teachers and Mentors
Richard I. Kelley, MD, PhD
Gerald Berry, MD
Robert Cohn, MD
Darryl DeVivo, MD
Lewis P. Rowland, MD
Salvatore DiMauro, MD
John Shoffner, MD
Charles Hoppel, MD
Robert Naviaux, MD, PhD
Richard Haas, MD
Russ Saneto, DO, PhD
Sumit Parikh, MD
Marni Falk, MD
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Thanks to those that support my practice
All my patients
All my patient’s families
UMDF
Polly Irwin, RN
Fran Yanak, RN
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A Brief History DiMauro, JIMD, 2010
1962: Rolf Luft, et al describe a woman with hypermetabolism not caused by hyperthyroidism ◦ First description of a mitochondrial disease ◦ Concept of organelle medicine – organelle disease
1963: Engel develops Gomori Trichrome stain (RRF) 1963: Discovery of mtDNA (Nass and Nass) 1965: Shy and Gonatas describe ultrastructural findings in myopathies 1970s: Enzymology
PDHC, 1970 (Blass) CPT, 1973 (DiMauro)
Complex III, 1970 (Spiro) Complex IV, 1977 (Willems)
Nomenclature 1977: “mitochondrial encephalomyopathies” coined by Yehuda Shapira 1985: DiMauro’s general classification;
Substrate transport Substrate utilization Citric Acid cycle defects Defects in the ETC Defects in OXPHOS coupling
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A Brief History DiMauro, JIMD, 2010
1980s-1990s: “Lumpers” and “Splitters” Is MELAS one disease or are there variations on a theme, and if so, why?
1984: Syndromic Paper in Ann Neurology (MERRF, MELAS, KSS), Pavlakis et al 1988: Large scale deletion of mtDNA (Holt and Harding) 1988: LHON caused my mtDNA point mutations (Wallace) 1988-89: PEO, PEO+ and KSS linked to mtDNA deletions (Moraes, Zeviani) 1990: MERRF due to a mutation in tRNALys (Shoffner) 1990: MELAS due to a mutation in tRNALeu(UUR) 2001: 115 point mutations in mtDNA causing disease cataloged, 2006: >200 1996: Human pol-γ ‘discovered’ and POLG cloned and characterized (Copeland),
and in 2001 1st diseases described (Suomalainen) and in 2004 Alpers (Naviaux) Late 1990s-current: explosion of nuclear genes Late 2000s: Genotype-Phenotype match is not universal Late 2000s: “Unarguable” secondary mitochondrial disorders: FA, ETHE1 2010”s: Ecovariant disorders
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Despite good intentions experts may disagree, personal opinions regarding certainty of diagnosis may change over time and new testing may trump interpretation of prior test results
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Definite
Probable
Possible
Unlikely
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Stratifying Certainty
• Possible Mitochondrial Disorders
• Ecogenetic Mitochondrial Disorder
• Secondary Mitochondrial Diseases
• Primary Mitochondrial Diseases
History & Exam Biochemistry
Histology Molecular Genetics
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Illnesses with a classic mitochondrial phenotype Caused by pathogenic mutations in ◦ The mtDNA (point mutations or del/dup) ◦ The nDNA genes that provide the mitochondrial Framework Enzymes ◦ Matrix ◦ IMM ◦ Redox couples ◦ Cofactors
Trafficking and Channels Assembly Replication
And the mutations need to be ◦ Verified pathogenic PolyPhen, SIFT Unrelated families with the same phenotype
◦ Recessive mutations present in trans
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Illnesses with a classic or less-than classic mitochondrial phenotype
Does not meet the criteria of a primary mitochondrial disease
Un-Refuted pathology or biochemistry Mitochondria may be injured by toxins or external
factors, but not changing the mtDNA ◦ Exogenous: MPTP, Rotenone, Iron, 3-Nitroprusside ◦ Endogenous: Sulfur Oxide, Iron
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Illnesses that may have a classic or less-than classic mitochondrial phenotype
Do not meet the criteria of a primary mitochondrial disease
Pathology or biochemistry may or may not look mitochondrial
Often simply ‘look-alike’ disorders ◦ Some may end up as primary mitochondrial after all Familial spastic paraparesis Rett, Angleman, Prader-Willi Familial Parkinson CNVs
◦ Channelopathies, myopathies, other biochemical disorders
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1999: 2 year old boy with mild developmental delays and dystonia
Moderate lactic acidemia (3-4 mM) and high alanine
MRI – moderate atrophy
Muscle biopsy – normal microscopy, very low ETC complex I activity and low state iii rates with substrates utilizing complex I
100% comfortable with complex I defect
….office visit in the summer of 2003
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Illnesses with a classic or less-than classic mitochondrial phenotype
Mutation in the mtDNA or nDNA not sufficient to cause illness
Mutation + Environmental Factor cause illness ◦ A1555G mtDNA ◦ POLG
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Legitimate classification ◦ Do not forget an alternative diagnosis may exist ◦ Often reasonable for treatment sake ◦ Not to be included in the literature unless labeled as such
Illnesses with a classic or less-than classic mitochondrial phenotype Does not meet the criteria of a primary, secondary or look-alike
mitochondrial disease Pathology or biochemistry may or may not look mitochondrial Some labeled because laboratory variants will on occasion
statistically fall outside the control range Some labeled because of systematic collection error or laboratory
error Some labeled because finances cannot support the evaluation Some difficult to “un-diagnose”
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1992: Complex I Deficiency and MELAS-Like
Katelyn D. – 1992 ◦ 4 year old girl, developmental delays, ataxia, intellectual disability, ASD ◦ Repeated events of hemiparesis with apparent headache and encephalopathy ◦ Persistent lactic acidosis, high alanine ◦ MRI – corpocephaly ◦ Muscle biopsy – ETC Enzymology c/w complex I deficiency ◦ Placed on UCSD Dichloroacetate study ◦ Event in 1993; fell asleep under a tree at a church picnic on a hot day and unarousable.
MRI showed bilateral cerebellar > corical patchy T2 changes (before FLAIR), which resolved. Return to baseline in a month – her last regression
◦ Genetic testing: normal karyotype, mtDNA, aCGH, glycosylation, further CSF and genetic testing declined
◦ Young adult with intellectual disability, limited language, very ataxic with myoclonus
2013: Possible Mitochondrial Disease
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What’s Changed in Diagnostics since 1982?
1982
Lactate Amino Acids
Organic Acids
CSF - Basic
Muscle Histology Karyotype
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What’s Changed in Diagnostics since 1982?
1982 2012
Lactate Amino Acids
Organic Acids
CSF - Basic
Muscle Histology Karyotype
Analytes CSF-Expanded
Muscle Histology
Muscle & Other Tissue Biochemistry
Neuroimaging with MRS Genetics
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What’s Changed in Diagnostics since 1982?
1982 2013
Lactate Amino Acids
Organic Acids
CSF - Basic
Muscle Histology Karyotype
Analytes CSF-Expanded
Muscle Histology
Muscle & Other Tissue Biochemistry
Neuroimaging with MRS Genetics
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What’s Changed in Genetics since 1982?
1982 2013
Karyotype
aCGH • FISH • Subtelomeric FISH • BAC Array • Oligo Array • SNP Array
mtDNA genome
sequencing, del/dup
Sequencing of Single Genes
Next Gen • Panels • Whole Exome • Whole Genome
Karyotype
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1982 2013
Nothing Commercial
• Muscle, Isolated Mitochondria, Fibroblasts, other tissues
ETC Enzymology
• OXPHOS: iii, iv • Membrane function • Isolated Mitochondria • Whole tissue preps
Polarography
• CPT, FAO • CAC • Gluconeogenesis Pathway • Glycogen Pathways • ANT
Enzyme Analysis
Supercomplex
Western Blot
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Diana DOB June 20, 1982
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Diana’s Story
• Food intolerance • Chronic diarrhea
Failure to thrive in first few months of life
• Unable to sit • Just beginning to attempt to roll Floppy baby
• Hb 8.1 • Febrile in the clinic
Referred to hematology for anemia
• Awake • RR ~ 80 Exam
• Lactic acid 8.1 mmol/l, CAC intermediates
Lab’s Obtained after Admission
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Diana: What Tools did we have in 1982?
Genetic • Karyotype
Analyte • Lactic acid & Pyruvic acid • Amino acids • Organic acids • Ammonia • Betahydroxybuterate and acetoacetate
Pathology • Light microscopy • Immunohistochemistry
Biochemical • Enzymology • Polarography
Normal kayrotype
Elevated lactate
Elevated lactate:pyruvate ratio
Elevated alanine
Elevated urine lactate, fumerate, citrate
Low state iii respiration on fibroblast polarography (R. Kelley)
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Diana’s Course
Sideroblastic anemia
Moderate Intellectual Deficiency
Myopathy
Ptosis and full PEO
Neuropathy
Progressive Hearing Loss
Diabetes Mellitus
Cardiac conduction block
Cardiomyopathy
Definitive Diagnosis in 1993 (common 4977 bp deletion in mtDNA)
Died on 9/4/1994
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Diana’s 2013 Evaluation – 2 Weeks
History CBC CMP
Lactic acid Amino acids Organic acids
Carnitine
Organ System
Evaluation
mtDNA Long-Range
PCR
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Pearson Syndrome
Pearson HA, et al. (1979). "A new syndrome of refractory sideroblastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction". J. Pediatr. 95 (6): 976–84. Rotig A, et al. (1989). "Mitochondrial DNA deletion in Pearson's marrow/pancreas syndrome". Lancet 1 (8643): 902–3. Commercial testing: Southern Blot ~1992
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What is the Diagnostic Approach?
History
Family History
Organ Functional
Testing
Genetic Biochemical & Microscopy
Analyte
Physical Examination
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A Typical Lab Evaluation: 1989-
Blood Lactic Acid Amino Acids Total and Free carnitine with acylcarnitine profile B12 level Methylmalonic acid Ammonia CK CMP + ? HBA1C CBC CoQ10 (WBC) Free T4 + TSH
Urine Routine Urine Analysis Organic Acids Amino Acids Carnitines + Acylcarnitine Profile Acylglycine Guanidoacetate + Creatine Purine and Pyrmidines
Dysmorphic +
Cognitive Problems? aCGH
Maternal Pattern? mtDNA-spectrum?
Whole Mito Genome
Specific mtDNA Disorder?
Specific mtDNA point mutation or LR-PCR
OR Fragile X SCN1A Rett Prader-Willi / Angelman CSF Neurotranmitter Disorder Disorders of Glycosolation
Specific nDNA gene sequence
Specific nDNA Disorder?
Skin biopsy for EM & Fibroblast Culture: acylcarnitne probe
Muscle Biopsy
NextGen Expanded Nuclear
Gene Testing
CSF
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When to order mtDNA sequencing?
Developmental Regression with lactic acidosis⎃ or elevated CSF lactate KSS phenotype (muscle better tissue than blood) LHON, NARP, MELAS, MERRF or Leigh phenotype
◦ where screening for the common mutations is not informative ◦ as an initial test
Epilepsia Partialis Continua -Status Epilepticus-Refractory Seizures when POLG is normal
Caudal-Cephalic MRI progression maternal inheritance pattern or similarly affected siblings Developmental Regression in the setting of prior normal development and non-
dysmorphism > 3 objective signs in > 2 organ systems
⎃ elevated alanine, alanine:lysine > 4, abnormal organic acids (CAC, 3-MG, lactate, pyruvate); note that lactate is often elevated in mutations involving tRNA but not the mtDNA mutations that encode for respiratory chain proteins
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When to Consider POLG? Child Adult Developmental Regression Epilepsia Partialis Continua -Status
Epilepticus-Refractory Seizures Cephalic-Caudal MRI progression Valproate-induced liver toxicity Cortical Blindness Myoclonus Ataxia Neuropathy Liver Failure
• PEO • myopathy • psychiatric illness • Parkinsonism or EP movement • ataxia • dysarthria • seizures • DM • ataxia - dysarthria • neuropathy • myoclonus • dementia
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When to Consider NextGen Panel Mitochondrial Testing?
Strong story for mitochondrial disease with negative prior testing
Strong story for neurogenetic disorder with a large differential
Evidence on a muscle ETC enzyme test of an abnormality, but with normal mtDNA Complex I nuclear genes and assembly genes Complex IV nuclear genes and assembly genes
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When to Consider NextGen Whole ExomeTesting?
Overlaps with NextGen Mitochondrial Panel When the mitochondrial phenotype is less
certain
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Parents Identified
poor hearing and low tone
from birth
• Evaluated at UCSD – Bruce Barshop
• Identified Need for Muscle Biopsy
Failure to thrive
diagnosed by 2 months
• Kinky Hair • Seen by Dr. Menkes • Amino aciduria • Slow developmental
progress • no speech or language Google Search:
Taryn LA Ink
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Bjornstad syndrome: pili
torti and congenital hearing
loss
GRACILE Syndrome: growth retardation, aminoaciduria,
cholestasis, iron overload, lactic acidosis and early death
Does Genotype Drive Phenotype?
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CIDEM, Dr. Charles Hoppel’s Lab
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Case Solved
Bjornstad syndrome: pili torti and congenital hearing loss
GRACILE Syndrome: growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis and early death
Both diseases are due to a single gene (BCS1L), codes for an assembly protein of complex III of the ETC, responsible for putting Fe into the Fe/S core
Muscle biopsy showed a severe complex III deficiency
Compound hetrozygote for G183A and G235A
Lesson Learned: Nothing special about this story – same as POLG and probably all the other genes
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Lessons Learned
1 Gene – 1 Mutant Allele – 1 Disease no longer applies
This was big news 5 years
ago
This story may unfold in the other 1000+ nuclear genes
that are part of the MitoCarta
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Reasons to Do a Muscle Biopsy
Availably of an Excellent Diagnostic Lab
Myopathy or Neuropathy
Concern for Inflammatory or Vasculopathy
Genetic Testing was Not Diagnostic
Genetic Testing was Near Diagnostic
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Thoughts on Muscle Biopsy
If you are going to do it, do it right
Choose your lab correctly for frozen muscle ETC testing
ETC on homogenize muscle has some value but should
never be interpreted without the context
of other data
ECT and OXPHOS on isolated
mitochondrial valuable but limited
availability
Isolated mitochondrial
studies will become more valuable as we head deeper into the
genomic era
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What is the Future of Diagnostic Testing? Legacy testing is not going away
Genetic Testing will move in front of muscle biopsy but create new issues with diagnosis
NextGen Panels and Whole Exome will keep us busy for the next 5 years •Sorting out disease causing mutations •Multigenic disorders •Understanding “incomplete penetrance” and “variable expressivity” •nDNA – mtDNA interactions; rRNA as an example •Role for testing pre-theraputics •Cancer Chemotherapy •Antibiotic Therapy • Immune Modifying Therapy
Whole Genome is in the future (once we understand gene expression, siRNA, etc.)
Protein studies on fresh muscle will evolve
The old techniques of muscle testing (polarography and enzymeolgy) will find new value
Real-Time Analysis •Oxygen extraction •Near-infrared approach •Other techniques as outlined in Marni Falk’s talk
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Vitamins - What Makes Sense (to me)
CoEnzyme Q10 5 - 20 mg/kg/day ÷ tid
L-Carnitine 30-100 mg/kg/day ÷ tid; 990 mg tid max
Riboflavin 100-600 mg/day qHS
Lipoic Acid 10 mg/kg/day ÷ bid
Creatine Monohydrate 100 mg/kg/day; 5 gms max
MELAS L-Arginine 100-300 mg per kg per day L-Citrulline 100-300 mg per kg per day
Kearn-Sayre Syndrome + Folinic Acid 5-50 mg a day
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