Committee to Review Adverse Effects of Vaccines An Institute of Medicine Workshop on the Safety of Vaccines. Mitochondrial Disorders

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1 Committee to Review Adverse Effects of Vaccines An Institute of Medicine Workshop on the Safety of Vaccines Mitochondrial Disorders Bruce H. Cohen, MD Cleveland Clinic August 26, 2009 special thanks to Robert Naviaux, MD, PhD, UCSD

2 What Are Mitochondria? subcellular organelles 1 micron in length cigar shaped (in vitro) complex structure in vivo comprised of outer membrane and a heavily infolded inner membrane Made from ~1500 structural and enzymatic proteins Most of these proteins are encoded by ndna fatty acid oxidation, urea acid cycle, citric acid cycle and other matrix enzymes inner and outer membrane structures ETC subunits must be imported and assembled using nuclear encoded proteins 13 proteins of the ETC are encoded by mtdna, resides in the mitochondria ~16.5kB, circular, all exon 37 genes 2rRNA 22 trna 13 structural proteins of the ETC heteroplasmy haplogroups

3 What do mitochondria do? (1)generate ATP (2)critical component of apoptosis (3)generate free radicals (4)Serves as a genetic barometer that allows immediate intracellular changes (seconds, minutes, days) and quick evolutionary changes (~1000s of years) (5)Roles in most neurodegenerative diseases and some cancers

4 TABLE 1 Red-Flag Findings in Mitochondrial Disease Neurologic Cerebral stroke-like lesions in a nonvascular pattern Basal ganglia disease Encephalopathy: recurrent or with low/moderate dosing of valproate Neurodegeneration Epilepsia partialis continua Myoclonus Ataxia MRI findings consistent with Leigh disease Characteristic MRS peaks Lactate peak at 1.3 ppm TE (time to echo) at 35 and 135 Succinate peak at 2.4 ppm Cardiovascular Hypertrophic cardiomyopathy with rhythm disturbance Unexplained heart block in a child Cardiomyopathy with lactic acidosis (5 mm) Dilated cardiomyopathy with muscle weakness Wolff-Parkinson-White arrhythmia Ophthalmologic Retinal degeneration with signs of night blindness, color-vision deficits, decreased visual acuity, or pigmentary retinopathy Ophthalmoplegia/paresis Fluctuating, dysconjugate eye movements Ptosis Sudden- or insidious-onset optic neuropathy/atrophy Gastroenterologic Unexplained or valproate-induced liver failure Severe dysmotility Pseudo-obstructive episodes Other A newborn, infant, or young child with unexplained hypotonia, weakness, failure to thrive, and a metabolic acidosis (particularly lactic acidosis) Exercise intolerance that is not in proportion to weakness Hypersensitivity to general anesthesia Episodes of acute rhabdomyolysis TABLE 2 Nonspecific Findings in Mitochondrial Disease Constitutional Failure to thrive Short stature Intrauterine growth retardation Microcephaly Neurologic Hypotonia Infantile spasms Intractable epilepsy Unexplained movement disorder Hearing loss (sensorineural) Axonal neuropathy Status epilepticus with an additional red-flag or nonspecific feature Coma Ototoxicity to certain medications Cardiovascular Tachycardia (postural or paroxysmal) Ophthalmologic Optic nerve hypoplasia, pigmentary retinopathy Gastroenterologic Chronic or cyclic vomiting Chronic unexplained constipation or diarrhea Dermatologic Symmetric lipomatosis Endocrine Hypothyroidism Hypoparathyroidism Idiopathic growth hormone deficiency Renal Renal tubular dysfunction (includes renal tubular acidosis and/or aminoaciduria) Nephrotic syndrome Imaging Unexplained basal ganglia lesions Unexplained central nervous system atrophy (cerebral or cerebellar) Unexplained leukodystrophy Family history Sudden infant death syndrome Multigenerational maternal inheritance pattern Richard H. Haas, Sumit Parikh, Marni J. Falk, Russell P. Saneto, Nicole I. Wolf, Niklas Darin, Bruce H. Cohen PEDIATRICS Volume 120, Number 6, December 2007

5 Symptoms of Mitochondrial Failure Brain seizures Autistic Spectrum Disorder poor attention and concentration dementia headaches strokes movement disorder behavorial, mood disorders altered tone and weakness Muscle strength pain fatigue cardiomyopathy Nerve autonomic dysfunction pain abnormal nerve conduction GI -GU gastric and intestinal dysmotility exocrine pancreatic dysfunction bladder dysmotility Eyes retinitis pigmentosa and optic atrophy Ears hearing loss Systemic and Endocrine growth - prevention of failure to thrive diabetes thyroid and parathyroid dysfunction Liver synthetic dysfunction

6 A Few Classic Mitochondrial Diseases Leigh Syndrome (mutations in mtdna and ndna) Kearn-Sayre Syndrome (deletion/duplication of mtdna) MELAS (primarily mtdna mutations) MERRF (primarily mtdna mutations) LHON (mtdna mutations) Alpers Spectrum- POLG mutations

7 Leigh Syndrome Healthy child until age 3 Non-specific viral infection Lost the ability to ambulate due to ataxia, hemiparesis then bilateral hemiparesis lactic acidosis eye movement and bulbar dysfunction dystonia neuropathy some recovery over 6 months followed by deterioration T8993G ATP6 (NARP) a number of mtdna mutations PDH deficiency Biochemical complex I defects Biochemical complex IV defects Others muscle biopsy most often normal or non-diagnostic!!! Leigh, D. : Subacute necrotizing encephalomyelopathy in an infant. J. Neurol. Neurosurg. Psychiat. 14: , 1951

8 Kearn Sayre Syndrome x Triad high frequency hearing loss cardiac conduction defect PEO Other features myopathy diabetes retinopathy dementia and seizures cardiomyopathy dysphagia and weight loss 5 kb deletion in all (>97%) of the mtdna from middle of ND5 to ATP8; (4977 base pairs from 8488 to 13460; 13 base pair repeat at mutation break point) Kearns T, Sayre G (1958). "Retinitis pigmentosa, external ophthalmophegia, and complete heart block: unusual syndrome with histologic study in one of two cases". A.M.A. Archives of Ophthalmology 60 (2): x

9 MELAS Variable Age of Onset (4 months through adult) FTT and DD in infancy, ADD common weakness and fatigability Short Stature Strokes and stroke-like episodes Progressive Dementia Hearing loss and DM Anorexia from autonomic gut neuropathy Migraine Myoclonic or tonic-clonic seizures WPW, hypertrophic > dilated cardiomyopathy Ophthalmoplegia RTA A3242G trnaleu (UUR) gene; 1: 6135 (Finnish) (80%) C3271G trnaleu (UUR) gene Pavlakis SG, Phillips PC, DiMauro S, et al: Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes: a distinctive clinical syndrome. Ann Neurol 1984 Oct; 16(4): 481-8

10 MERRF Mitochondrial Encephalopathy with Ragged Red Fibers myoclonic epilepsy myopathy ataxia fatty deposits on neck

11 LHON Leber Hereditary Optic Neuropathy Painless profound visual loss presenting in the late teens or early 20s Caused by mtdna Complex I genes, usually homoplasmic

12 Pathogenic Mitochondrial DNA Mutations Are Common in the General Population H. R. Elliott, D. C. Samuels, J. A. Eden, C. L. Relton, and P. F. Chinnery The American Journal of Human Genetics 83, , August 8, 2008 m.1555a/g (aminoglycoside deafness) m.3243a/g (MELAS) m.3460g/a (LHON) m.11778g/a (LHON) m.14484t/c (LHON) m.7445a/g (Deafness) m.8344a/g (MERRF) m.8993t/g (NARP - Leigh) m.13513g/a (MELAS-Leigh) m.14459g/a (Leigh - Leber) denovo mutation rates for the 10 most common mtdna mutations is > 1:1000 > 1:200 people carry 1 of these 10 pathogenic mtdna mutation

13 Alpers Syndrome Infantile fatal cerebral disorder that was first described by Bernard Alpers in 1931; poliodystrophy -- refractory seizures, developmental regression, cortical blindness, age of onset 3-7 years, some with identified developmental disabilities and others normal study showing mitochondrial ultrastructural changes in Alpers poliodystrophy 1976 Huttenlocher described the hepatic features (micronodular necrosis) and familial nature of the disorder; Huttenlocher variant or Alpers-Huttenlocher syndrome Based on pattern of illness - autosomal recessive many children present with epilepsia partialis continua or status epilepticus progressive neuropathy and spasticity Death 1-20 years into the course, often from liver failure B. J. Alpers. Diffuse progressive degeneration of the grey matter of the cerebrum. Archives of Neurology and Psychiatry, Chicago, 1931, 25:

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15 Hudson and Chinnery Hum Mol Genet 2006;15:R October 2006

16 So How Common Are These Diseases? The gene frequency of the most common mtdna mutation causing MELAS, A3243G, is 1:1000 The disease frequency of MELAS due to the A3243G mutation is 1:5000 The gene frequency (recessive alleles only) in northern western European populations in the POLG gene is about 2%, resulting in a disease frequency of about 1:10,000 Total estimated disease frequency of only the two most common genes: mtdna MELAS + POLG = 1:4400 but the majority of those diagnosed with mitochondrial disorders do not have Leigh, KSS, MELAS, MERRF, LHON or Alpers 2. in most centers, the ability to make a firm genetic diagnosisis on the order of 10-15% 3. in the clinic we must live with the diagnostic uncertainty

17 Clinical History Physical Examination Family History Analytes (Blood, Urine, CSF studies) Diagnosis End Organ (MRI/MRS, Echocardiogram) Tissue Histology (Microscopy, Immunohistochemsitry, EM, Western Blot, Blue Native Gel Electrophoresis) Biochemistry (OXPHOS, ETC in Mitochondria) Molecular Genetics

18 My Typical Initial Lab Evaluation 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 Skin biopsy for EM and Fibroblast Culture: acylcarnitne probe Dysmorphic + Cognitive Problems? acgh OR Fragile X Rett Prader-Willi / Angelman CSF Neurotranmitter Disorder Disorders of Glycosolation Specific mtdna Disorder? Specific mtdna point mutation or Southern Blot LR-PCR Maternal Pattern? mtdna-spectrum? Whole Mito Genome + Haplogroup Typing Specific ndna Disorder? Specific ndna gene sequence

19 When Should We Think of Something Else? Mitochondrial Cytopathies

20 Unproven Suspicions (Difficult Truths to Accept) The common final pathway to all cellular demise may be a mitochondrial-triggered event Common disorders may have at the core of the pathophysiology mitochondrial dysfunction The innate immune response may be a key mitochondrial function

21 False Positive Clinical Diagnosis Failure to recognize well-characterized (common, rare and extraordinarily rare) non-mitochondrial genetic disorders (includes failure to perform acgh) Improper sample handling Poor laboratory technique Incorrect interpretation of laboratory results analyte blood results genetic results; mutations of uncertain signficance - many are reported pathology or protein study results Failure to perform proper laboratory studies and rely on less specific or nonspecific data to make the diagnosis Disuse of muscle from any cause will lead to decreased mitochondrial enzymatic function Reliance on the use of muscle or skin mitochondrial enzyme analysis without taking all factors, including clinical, into consideration Although psychiatric disturbance is very common in mitochondrial disorders, overcalling chronic fatigue syndrome, malingering and conversion disorders as mitochondrial disorders

22 Questions What is the natural history of these disorders? What percentage of children with these disorders present with encephalopathy? Is the long-term prognosis for children with these disorders who have an acute encephalopathy different (worse?) than children without these disorders who have an acute encephalopathy? Are there developmental time periods during which metabolic stress is more likely to have adverse consequences, particularly chronic sequelae, in children with mitochondrial disorders? What is the current thinking about possible adverse effects of vaccination in these children is the fever the triggering event or is the immune reactions per se a significant metabolic stressor?

23 The animal cell Mitochondrial Proteome is Tissue Specific proteins are imported into the mitochondria the number of imported proteins is tissue specific the specific proteins and isoforms are tissue specific mtdna copy number is regulated by developmental state and cellular needs

24 mtdna Biomarkers of Disease aging inflammation and viral attacks diabetes and the metabolic syndrome heart and endothelial disease Parkinson disease bipolar depression cancer drug toxicity HAART, prenatal prophylaxis post-chemotherapy syndromes Autoimmune diseases

25 Knock-out of a Single RC Subunit (cox4) in Yeast Produced 1000 Protein Changes Spots resolved 970 Spots (40%) changed more than 150% 529 (22%) were decreased ( fold) 441 (19%) were increased ( fold) Courtesy of Robert Naviaux, MD, PhD

26 mtdna Haplogroups Were Environmentally Selected and Confer Risk and Resistance To Disease Haplogroups ensure that the OXPHOS core proteins are inherited en bloc, without recombination Balance between coupling and uncoupling substitutions evolve to suit the environment temperature, elevation, sunlight, water availability, food resources, infectious agents Root mtdna changes represent fundamental trade-off decisions in fitness Dozens of examples now exist; Ann Surg Sep;246(3):406-11; discussion 411-4, Canter, et al 745 consecutive patients admitted for trauma, analysis on 666 patients T4216 (ND1 Y304) increased mortality from trauma OR=2.63 increased male fertility, decreased DM2 C4216 (ND1 H304) Haplogroups J and T decreased trauma mortality OR=0.38 decreased male fertility, LHON increased insulin resistance and DM2 Lancet (9503): Mitochondrial DNA and survival after sepsis sequentially patients admitted for sepsis -542 age-matched controls -mitochondrial haplotyping performed (all but 2) -death vs survival at 6 months Results: -no difference in frequency of Haplogroup H -those belonging to Haplogroup H was a predictor of survival conferring a 2.12 fold (95% CI ) in increasing survival at 180 days Interestingly Haplogroup H is the most recent addition in Europe but pardoxically the most common Association of Mitochondrial Allele 4216C With Increased Risk for Complicated Sepsis and Death After Traumatic Injury. J Trauma: Injury, Infection, and Critical Care 2009, 66; OR

27 Leigh Encephalopath y At Presentation Cognitive Delay Prior to Onset MELAS LHON KSS Alpers Microdeletion

28 What is the natural history of these disorders? mtdna - progressive MELAS, MERRF, KSS LHON ndna - progressive 30+ defined other ndna disorders - progressive typically mtdna maintaince (Alpers and others) ETC assembly (BCS1L and others) fusion and fission (Mitofusion and others) Cofactor synthesis (CoQ2 and others) FAO, CAC, UC enzymes - quickly fatal with stress acgh mitochondrial disorders disorders -microdeletion are closely disorders mimic mitochondrial that either are may decrease mitochondrial function but not to the same disorders, nonprogressive at least when observed over many years degree -

29 4 year old girl presenting in 1992 with: -static cognitive delay -lactic acidosis -seizures -hemiplegic migraine MRI shows disorder of neuronal migration complex I defect -profound encephalopathy after heat stress with demyelination and prolonged stupor, recovery in a month -stable on dichloroacetate -no dementia -extensive genetic evaluation noninformative

30 What can we say about the rate of progression? Even for a well-characterized disease due to a specific mutation, the rate of progression is variable Example - extended family with MELAS Example - spectrum of survival in KSS Example - POLG patient

31 What percentage of children with these disorders present with encephalopathy? Leigh =100% MELAS ~ 100% KSS ~ rare as presenting sign Alpers ~ 100% LHON ~ 0% acgh >90% (often drawn as part of the evaluation for developmental delay and dysmorphism)

32 Brain the most common organ affected in children Embryopathy Global developmental delays Atypical cerebral palsy Autism and pervasive developmental disorders Seizures Acquired Seizures Atypical migraine Stroke and stroke-like episodes Neuropsychiatric symptoms Dementia

33 Is the long-term prognosis for children with these disorders who have an acute encephalopathy different (worse?) than children without these disorders who have an acute encephalopathy? 1. Children with embryopathic brains tend not to have dementia, unless it is epileptic-induced dementia 2. Children with normal brains that have acute encephalopathy and are found to have mtdna or ndna mitochondrial disorders tend to have a worse overall prognosis than if an extensive search does not demonstrate a mutation

34 Are there developmental time periods during which metabolic stress is more likely to have adverse consequences, particularly chronic sequelae, in children with mitochondrial disorders? 1. FAO, CAC, OA, UCDs: infancy is the typical presentation for the most serious disorders but most of these are screened for with Newborn Screening Programs 2. mtdna disorders: wide variable age of disease presentation, more severe disorders by default present earlier, so taking this variable out of the question, age does not seem to be a variable 3. ndna disorders: wide variable age of disease presentation

35 Stress 1. Infection 2. Temperature (fever, ambient temperature, humidity issues) 3. Dehydration 4. Starvation 5. Sleep 6. Medication (anesthetics)

36 Metabolic Medicine Our Knowledge is Only the Tip of the Iceberg