Mitochondrial Disorders and Cerebral Folate Deficiency in

Autism Spectrum Disorder

Richard E. Frye, M.D., Ph.D.Director of Autism Research

Associate Professor of PediatricsArkansas Children’s Hospital

The Rise in AutismCDC estimates the prevalence of Autism Spectrum Disorder is as high as 7 per 1,000 or 1 in 150!

Debate over whether this is due to a shift in diagnosis or a true rise in the number of cases. Does it matter?? Either way there are a lot of children that need care.

Autism Defined

The Etiology of Autism: More than Genetic Disorders

Estimated Prevalence of Genetic AbnormalitiesCytogenetic Abnormalities 5%Fragile X 5%Rett Syndrome (Females only) 5% (~1% overall)Chromosomal Microarray 10%Total 21%

This leaves about 79%+ children with ASD without an identified genetic diagnosis.

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(Schaefer and Mendelson, Genetics in Medicine, 2008)

New Understanding of Autism

• Autism is defined as a collection of symptoms

• Symptoms of Autism are associated with underlying medical disorders in may cases

• In many cases, Autism is a multisystemic disorder with primary neurological manifestations.

• The rise in Autism cases is probably due to complex interactions between genetics, environment and the dynamics of physiological development.

Mitochondrial Dysfunction and Cerebral Folate Deficiency/Insufficiency is becoming

recognized as highly prevalent in autism spectrum disorder

This talk will review– Evidence for Mitochondrial Dysfunction in Autism

Spectrum Disorder– Biomarkers for mitochondrial dysfunction– Importance of cerebral folate deficiency/insufficiency

and the folate receptor autoantibody– How cerebral folate deficiency/insufficiency is

diagnosed and treated

Evidence for MitochondrialDisease and Dysfunction in Autism Spectrum Disorder

Biomarkers of Abnormal Energy Metabolism

in Children with Neurodevelopmental Disorders

A review of metabolic studies from 133 consecutive patients evaluated in a

medically-based autism clinic

Examined a wide range of metabolic markers in children with autism including markers of

fatty-acid oxidation disorders

6 Biomarkers Reviewed

4 Groups with high prevalence Identified

Lactate, Alanine-to-Lysine & Acyl-Carnitine 34.3%with AST 53.0%

Acyl-Carnitine Elevation Group

3-hydroxy-3-methylglutaryl is a metabolite of Acetyl-CoA, the starting point of the citric acid cycle. Suggests that the citric acid cycle is working inefficiently. It can also be seen in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency but at much higher levels.

Muscle Biopsy of child with acyl-carnitine elevations

Altered brain phospholipid and acylcarnitine profiles in propionic acid infused rodents: further development of a potential model of autism spectrum disorders. J Neurochem. 2010 Apr;113(2):515-29.

Derrick MacFabe et al.

Defining Mitochondrial DiseaseSeveral systematic criteria are used to diagnose mitochondrial disease.

Here we consider the Morava et al. (2006) criteria which uses the following findings:I. ClinicalII. Metabolic and neuroimaging III. Mitochondrial morphological

Patients are classified into 4 categories:• Not likely (<=1)• Possible (2-4 points)• Probable (5-7 points)• Definite (>=8 points).

•Score of 3+ suggests a muscle or skin biopsy should be done

I. Clinical signs and symptoms, 1 point/ symptom (max. 4 points)

Probably associated with ASD (% in ASD)

Might be associated with ASD

Probably not associated with ASD

A. Muscular(max. 2 points)

Muscle weakness (myopathies)

Abnormal EMG Exercise intoleranceRhabdomyolysis

Ophthalmoplegia† Facies myopathica

B. CNS(max. 2 points)

Develop delay (100%)Loss of skills (33%)Seizures (25%)

Extrapyramidal signsMyoclonus Pyramidal signs

Stroke-like episodeMigraineCortical Blindness Brainstem abnormal

C. Multisystem(max. 3 points)

GI tract (7-91.4%)Endocrine/growthFamilial (10.9%) Neuropathy

Heart Kidney

VisionHearing Hematological

† Scores 2 points. ‡ Scores 4 points.EMG = electromyography

(Frye and Rossignol, Ped Research, 2011)

II. Metabolic/imaging studies (max. 4 points)Probably associated with ASD (% in ASD)

Might be associated with ASD

Probably not associated with ASD

Elevated lactate† (17.1- 76.6%)Elevated L/P ratio (27.6%)Elevated alanine† (36.0%) Elevated lactate/MRS (11.1%)

Elevated CSF lactate†Elevated CSF proteinElevated CSF alanineUrinary TA excretion†Stroke-like picture/MRI

Ethylmalonic aciduriaLeigh syndrome/MRI† (0%)

† Scores 2 points. ‡ Scores 4 points.L/P = lactate/pyruvate; TA = tricarbon acid.; CSF = Cerebrospinal fluid

(Frye and Rossignol, Ped Research, 2011)

III. Mitochondria Morphology (max. 4 points)Probably associated with ASD (% in ASD)

Might be associated with ASD

Probably not associated with ASD

Abnormal mitochondria/EM† Reduced COX staining‡Ragged red/blue fibers‡

COX-negative fibers‡ Reduced SDH stainingSDH positive blood vessels†

† Scores 2 points. ‡ Scores 4 points.COX = cytochrome c oxidase; SDH = succinate dehydrogenase; EM = electron microscopy

(Frye and Rossignol, Ped Research, 2011)

Weissman et al 2008

Review of 25 children diagnosed with autism eventually diagnosed with a mitochondrial disorder

High rate of non-neurologic symptoms

High rate of fatigability – 68%

Unusual regression -- 60%

SuspectFatty AcidDisorder

CMP

Lactic AcidAmino AcidsAmmoniaAcyl-CarnitineCreatine KinaseUrine Organic Acids

CO2LFTsGlucose

Morning Fasting

If Lab AbnormalRepeat to Confirm

Pyruvic AcidCMAMitoMetmtDNA point mutationsStart Supplements L-Carnitine (Carnitor) Ubiquinol (Douglas Labs) B-Complex (Supra-Nu Thera)

SuspectMitochondrial

Disorder

No Diagnosis

Muscle Biopsy Histology Electron Microscopy Electron Transport Chain Studies mtDNA Content Studies

Testing of Specific GenesmtDNA sequencing

More Specific Diagnosis

Specific Therapy

RBC Zinc & Copper, BiotinTriglyceride & Cholesterol PanelUrine Acyl-Glycine & Ketones

Skin Biopsy with Fatty-acid oxidation and electron transport chain studies, MitoMet

No Diagnosis

A New Type of Mitochondrial Disorder:

Complex IV Hyperfunction.

Electron Transport Chain Studies

From 14 Children with ASD

and Mitochondrial Disease

defined by Morava et al. criteria

Corrected ETC Function

Maximum Corrected ETC Function by Subject

Why Is Mitochondrial Disease

Associated with Autism?

Brain growth peaks just after the first year of life. Brain growth requires energy. A deficit in energy during this essential time may result in a failure in brain development

Mitochondrial Dysfunction

Increased Free Radicals

Environmental Toxins Genetic Defects

Cell Damage and Death

Treatment of Mitochondrial Disorders

Treatment of Mitochondrial Disease

Treatment of Mitochondrial Disease

• Prevention of Regression– Avoid Dehydration– Avoid Fever– Avoid Viral Illness– Avoid Specific Drugs

Treatment of Mitochondrial Disease• Prevention of Regression

28 patients with ASD and mitochondrial disease. 17 individuals had a history of regression 71% (12 of 17) regressed with fever 24% (4 of 17) fever followed vaccination

Drug Interactions• Antibiotics to avoid:

– Linezolid and other oxazolidinone antibiotics

– Rifampin– Tetracycline– Chloramphenicol– Imipenem– Cephalogycin– Beta-lactam (penicillin and

cephalosporin)

• Other substances to avoid : – Alcohol– Cigarette smoke– monosodium glutamate– Acetaminophen– Antipsychotics– Fasting– Dehydration– Sleep Deprivation

• Antibiotics that are probably okay: – Fluoroquinolones

(Ciprofloxacin, floxin, levaquin),

– Macrolides (azithromycin, clarithromycin, erthromycin)

– Cephaloglycin, – Bactrim

Preventive Strategies: Drug Interactions

Treatment of Mitochondrial Disease

• Diets– Ketogenic Diet– Modified Atkins Diet– Complex Carbohydrate Supplementation

Treatment of Mitochondrial Disease• Secondary Effects of Mitochondrial Disorders

– Gastrointestinal Dysfunction- Dysmotility– Thyroid Dysfunction– Adrenal Dysfunction– Immune System Dysfunction– Cardiovascular Dysfunction– Brain Dysfunction

• Cerebral Folate Deficiency• Seizures• Neurotransmitter Deficiencies

– Acetylcholine – Excitatory-Inhibitory Balance

Cerebral Folate Deficiency

Antibodies to the FR1 (Folate Receptor 1) block folate from crossing the blood-brain barrier. Since this is an energy dependent process disorder of energy metabolism will also reduce folate transport into the central nervous system

(Ramaekers and Quadros, in press)

(Ramaekers et al., NEJM, 2005)

Symptoms of Classic Cerebral Folate Deficiency

Antibody Mediated Cerebral folate deficiency• Infantile-onset cerebral folate deficiency

• Low-IQ autism with neurological deficits

Energy Mediated Cerebral Folate Deficiency syndromes•Mitochondrial encephalopathies (deficits in mitochondrial function)

Unknown Mechanisms (both mitochondrial and antibody?)

•Rett Syndrome

Conditions Associated with Autism and Cerebral Folate Deficiency

Ramaekers et al., 2007 Neuropediatrics 38(6):276-81

Wide Range of Children with Autism Spectrum Disorder

Energy Mediated Cerebral Folate Deficiency syndromes

• Mitochondrial Complex IV Hyperfunction (Frye and Naveux, Journal of Pediatric Neurology, 2012)

The Expanding Association betweenAutism and Cerebral Folate Deficiency

More than half of children with Autism Spectrum Disorder referred to two autism specialty clinics test positive for

antibodies to the folate transporter (n=93)

Frye et al, Molecular Psychiatry, 2012

75% of children with Autism Spectrum Disorder referred to two autism specialty clinics test positive for one of the two

antibodies to the folate transporter

Frye et al, Molecular Psychiatry, 2012

44 children with Autism and Positive autoantibodies were treated with 2mg/kg of folinic acid in an open-label fashion for a mean of 4 months and compared to a wait list control group of children with autism and positive autoantibodies.

Reduced central nervous system folate results in decreased de novo purine synthesis which leads to decrease tetrahydrobiopterin (BH4) production

(Ramaekers et al., Neurology, 2003)

Cerebral folate deficiency

Treatment: Folinic Acid 1-2mg/kg/day

Ramaekers et al., 2008 Dev Med Child Neurol 50(5):346-52

Ramaekers et al., 2008 Dev Med Child Neurol 50(5):346-52

Questions?