1. Mitochondrial Disorders and
Cerebral Folate Deficiency in
Autism Spectrum Disorder
Richard E. Frye, M.D., Ph.D.
Director of Autism Research
Associate Professor of Pediatrics
Arkansas Children’s Hospital

2. The Rise in Autism
CDC 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.

4. Autism Defined

6. The Etiology of Autism: More than Genetic Disorders
Estimated Prevalence of Genetic Abnormalities
Cytogenetic 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.
.
(Schaefer and Mendelson, Genetics in Medicine, 2008)

7. 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.

8. 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

9. Evidence for Mitochondrial
Disease and Dysfunction in
Autism Spectrum Disorder

12. 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

13. 6 Biomarkers Reviewed
4 Groups with high prevalence Identified
Lactate, Alanine-to-Lysine & Acyl-Carnitine 34.3%
with AST 53.0%

15. 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.

16. Muscle Biopsy of child with acyl-carnitine elevations

17. 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.

19. Defining Mitochondrial Disease
Several systematic criteria are used to diagnose mitochondrial
disease.
Here we consider the Morava et al. (2006) criteria which uses
the following findings:
I. Clinical
II. 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

20. I. Clinical signs and symptoms, 1 point/ symptom (max. 4 points)
Probably associated Might be associated Probably not
with ASD (% in ASD) with ASD associated with ASD
A. Muscular Muscle weakness Abnormal EMG Ophthalmoplegia†
(max. 2 points) (myopathies) Exercise intolerance Facies myopathica
Rhabdomyolysis
B. CNS Develop delay (100%) Extrapyramidal signs Stroke-like episode
(max. 2 points) Loss of skills (33%) Myoclonus Migraine
Seizures (25%) Pyramidal signs Cortical Blindness
Brainstem abnormal
C. Multisystem GI tract (7-91.4%) Heart Vision
(max. 3 points) Endocrine/growth Kidney Hearing
Familial (10.9%) Hematological
Neuropathy
† Scores 2 points. ‡ Scores 4 points.
EMG = electromyography
(Frye and Rossignol,
Ped Research, 2011)

21. II. Metabolic/imaging studies (max. 4 points)
Probably associated with ASD Might be associated Probably not associated
(% in ASD) with ASD with ASD
Elevated lactate† (17.1- 76.6%) Elevated CSF lactate† Ethylmalonic aciduria
Elevated L/P ratio (27.6%) Elevated CSF protein Leigh syndrome/MRI†
Elevated alanine† (36.0%) Elevated CSF alanine (0%)
Elevated lactate/MRS (11.1%) Urinary TA excretion†
Stroke-like picture/MRI
† Scores 2 points. ‡ Scores 4 points.
L/P = lactate/pyruvate;
TA = tricarbon acid.;
CSF = Cerebrospinal fluid
(Frye and Rossignol,
Ped Research, 2011)

22. III. Mitochondria Morphology (max. 4 points)
Probably associated with Might be associated with Probably not associated
ASD (% in ASD) ASD with ASD
Abnormal mitochondria/EM† COX-negative fibers‡
Reduced COX staining‡ Reduced SDH staining
Ragged red/blue fibers‡ SDH 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)

25. 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%

26. Morning Fasting Suspect Pyruvic Acid
CO2 Mitochondrial CMA
CMP LFTs Disorder MitoMet
mtDNA point mutations
Glucose Start Supplements
Lactic Acid L-Carnitine (Carnitor)
Amino Acids Ubiquinol (Douglas Labs)
Ammonia B-Complex (Supra-Nu Thera)
Acyl-Carnitine No Diagnosis
Creatine Kinase If Lab
Urine Organic Acids Muscle Biopsy
Abnormal
Histology
Suspect Repeat to
Electron Microscopy
Fatty Acid Confirm
Electron Transport Chain Studies
Disorder mtDNA Content Studies
RBC Zinc & Copper, Biotin
More Specific Diagnosis
Triglyceride & Cholesterol Panel
Urine Acyl-Glycine & Ketones Testing of Specific Genes
No Diagnosis mtDNA sequencing
Skin Biopsy with Fatty-acid
oxidation and electron transport Specific Therapy
chain studies, MitoMet

27. A New Type of Mitochondrial Disorder:
Complex IV Hyperfunction.

28. Electron Transport Chain Studies
From 14 Children with ASD
and Mitochondrial Disease
defined by Morava et al. criteria

29. Corrected ETC Function

30. Maximum Corrected ETC Function by Subject

31. Why Is
Mitochondrial Disease
Associated with Autism?

32. 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

33. Mitochondrial Dysfunction
Environmental Toxins Genetic Defects
Increased Free Radicals
Cell Damage and Death

34. Treatment of
Mitochondrial Disorders

35. Treatment of
Mitochondrial Disease

37. Treatment of
Mitochondrial Disease
• Prevention of Regression
– Avoid Dehydration
– Avoid Fever
– Avoid Viral Illness
– Avoid Specific Drugs

38. 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

39. Drug Interactions
• Antibiotics to avoid: • Antibiotics that are probably
– Linezolid and other oxazolidinone okay:
antibiotics – Fluoroquinolones
– Rifampin (Ciprofloxacin, floxin, levaquin),
– Tetracycline – Macrolides
– Chloramphenicol (azithromycin, clarithromycin, ert
hromycin)
– Imipenem
– Cephaloglycin,
– Cephalogycin
– Bactrim
– Beta-lactam (penicillin and
cephalosporin)
• Other substances to avoid :
– Alcohol
– Cigarette smoke
– monosodium glutamate
– Acetaminophen
– Antipsychotics
– Fasting
– Dehydration
– Sleep Deprivation

40. Preventive Strategies: Drug Interactions

41. Treatment of
Mitochondrial Disease
• Diets
– Ketogenic Diet
– Modified Atkins Diet
– Complex Carbohydrate Supplementation

42. 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

43. Cerebral Folate Deficiency

44. 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)

45. Symptoms of Classic Cerebral Folate Deficiency
(Ramaekers et al., NEJM, 2005)

46. Conditions Associated with Autism and
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

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

48. The Expanding Association between
Autism and Cerebral Folate Deficiency
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)

49. 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

50. 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

51. 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.

52. Reduced central nervous system folate results in
decreased de novo purine synthesis which leads to
decrease tetrahydrobiopterin (BH4) production
(Ramaekers et al., Neurology, 2003)

53. Cerebral folate deficiency
Treatment: Folinic Acid 1-2mg/kg/day

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

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

56. Questions?