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

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

Thanks to those that support my practice

All my patients

All my patient’s families

UMDF

Polly Irwin, RN

Fran Yanak, RN

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

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

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

Definite

Probable

Possible

Unlikely

Stratifying Certainty

• Possible Mitochondrial Disorders

• Ecogenetic Mitochondrial Disorder

• Secondary Mitochondrial Diseases

• Primary Mitochondrial Diseases

History & Exam Biochemistry

Histology Molecular Genetics

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

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

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

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

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

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”

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

What’s Changed in Diagnostics since 1982?

1982

Lactate Amino Acids

Organic Acids

CSF - Basic

Muscle Histology Karyotype

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

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

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

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

Diana DOB June 20, 1982

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

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)

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

Diana’s 2013 Evaluation – 2 Weeks

History CBC CMP

Lactic acid Amino acids Organic acids

Carnitine

Organ System

Evaluation

mtDNA Long-Range

PCR

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

What is the Diagnostic Approach?

History

Family History

Organ Functional

Testing

Genetic Biochemical & Microscopy

Analyte

Physical Examination

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

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

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

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

When to Consider NextGen Whole ExomeTesting?

Overlaps with NextGen Mitochondrial Panel When the mitochondrial phenotype is less

certain

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

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?

CIDEM, Dr. Charles Hoppel’s Lab

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

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

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

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

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

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