Fall DAN!^TM 2003 Conference
*** Portland, Oregon *** October 3-5, 2003

Autism An Overview of DAN!^TM Findings on Causative Factors

Jon Pangborn, PhD

Prior to about 1980, the occurrence of autism was about 3 to 5 individuals per 10,000 births with variations that depended upon diagnostic criteria, who did the research, and geographical location.^1,2 At least two-thirds of those autistics had discernable problems from birth.³ Less than one-third showed regression of social skills, speech and behavior between ages one and two years. During the period 1980-85, the occurrence of autism doubled. By 1985, the occurrence of regressive autism about equaled that of the from-birth conditions,³ suggesting that an acquired condition was overtaking inborn errors or purely genetic conditions. By 1997, both types had increased, but the late-onset or regressive type was now at least 75% of the total occurrence.³ And the total occurrence had increased by a factor of ten, to 30 to 35 per 10,000 births, with some locales reporting twice that number.^4,5 A tentative conclusion is that regressive autism is due to exposure to stressors that worsen metabolic predispositions, and that is now the predominant type.

During the last three decades, at least ten genetic/metabolic disorders that may feature autism have been described. More information on most of these is included in the October 2002 DAN! Consensus Report available from ARI, San Diego.⁶ These ten conditions are outlined here so that the reader will grasp the concept that these ten very different metabolic conditions all are related to disorders in purine or pyrimidine nucleoside/nucleotide metabolism.

1. Phosphoribosylpyrophosphate (PRPP) synthetase superactivity (very rare), an Xq chromosomal fault exacerbated by impaired proline metabolism. PRPP is the sugar template on which purine and pyrimidine nucleotides are assembled, and superactivity of the synthase imbalances nucleosides/nucleotides.
2. Adenylosuccinate lyase deficiency is a 22q 13.1 chromosomal fault which impairs purine formation before inosine monophosphate formation and after adenylosuccinate formation. Elevated “succinylpurines” occur, adenosine, AMP, ADP, ATP imbalances occur and immune dysregulation is likely.
3. Histidinemia is a chromosomal fault at 12q22-23 which causes deficiency of formiminoglutamate (“FIGlu”) and consequently, limited amounts of special folate forms needed for purine synthesis. Occurrence is about one in 10,000 and victims may have poor auditory memory and poor response to verbal input.
4. Lesch-Nyhan disease, a fault at Xq26-27 with mutant activity of hypoxanthine-guanine-phosphoribosyltransferase. Occurrence is about one in 10,000 male births; purines are wasted as urate, and formation of cytidine triphosphate, CTP, is deficient in certain cells. Victims are typically self-destructive.
5. Fragile X syndrome has faults at Xq27-28. The problem was originally considered to be deficiency of 5,10-methylene THF which is required in pyrimidine metabolism to form thymidine from uridine. Fragile X is a leading genetic cause for mental retardation in males; some become autistic.
6. Rett Syndrome, a fault at Xq28 seen in females (males do not survive), with 21 different possible genetic mutations. Incidence is about one in 12,000 females. Gene binding of methylated cytosine and guanine is disordered, and progressive regression occurs starting at 6 to 18 months.
7. Dihydropyrimidine dehydrogenase (DPD) deficiency, a chromosomal fault at 1p22, results in uracil not forming enough dihydrouracil and thymine not forming enough dihydrothymine. Elevated uracil/thymine occurs in urine. Mild heterozygous DPD is common; severe cases with autism are uncommon.
8. Tuberous sclerosis, a fault at 16p13.3, results in formation of a protein called “tuberin”. Tuberin has homology for GTPase activating protein, and it can deplete guanosine as GTP, GDP, GMP by dephosphorylation. Incidence is one in 10,000 births with 25 to 60% featuring autistic traits.
9. Superactivity of pyrimidine 5’-nucleotidase (P5N) with several chromosomal links (4q26 cytosolic, 17p11.2 mitochondrial). This causes accelerated depletion of uridine monophosphate (UMP) and cytidine monophosphate (CMP); urate is typically subnormal. Incidence is unknown.
10. Phenylketonuria with possible faults at 12q22-24 and at 4q15.1-16.1 (biopterin), which is now a rare autism condition due to diagnosis and treatment in early infancy. Without treatment, as many as 40% of PKUs could become autistic. In PKU there is futile use (wasting) of GTP to form biopterin.

In addition to the 10 genetic disorders outlined above, a recent and cogent finding is that when activation-induced cytidine deaminase is disabled (cytidine doesn’t convert to uridine on demand), lab animals soon exhibit problems with immune response. A huge expansion (100-fold) of anaerobic gut flora occurs, and ileal lymphoid hyperplasia develops.⁷   This seems to reproduce observations of the autistic GI tract made by Dr. Andrew Wakefield and others.^8,9,10 And in 1982, Dr. Gene Stubbs found apparent weakness in erythrocyte adenosine deaminase in autistics.¹¹ We now believe the adenosine deaminase problem to be either the alleles described by Persico,¹² or faults with its binding protein, CD26, also known as dipeptidylpeptidase IV (DPP4).¹³

All this leads to the conclusion that purine and pyrimidine nucleotide/nucleoside imbalances are the central and unifying problem in autism. Their roles in cellular perception and response are discussed in the October 2002 DAN! Consensus Report.¹⁴

Today, fewer than 20% of analytically tested autistics are typed as one of the 10 conditions listed above. Our problem in describing the causes of our present autism epidemic is one of finding predispositions and stressors that result in disordered purine and/or pyrimidine metabolism.

Many current clinical findings point directly at the area of human metabolism where transmethylation, transsulfuration and purine synthesis intersect. That area is methionine metabolism and associated folate, cobalamin and “one-carbon” chemistry. The evidence at hand suggests that, on average, 40 to 50% of today’s autistic individuals have problems in this area of metabolism. Some itemized evidence for this follows.

1. Therapeutic doses of supplemental vitamin B₆ and magnesium help over 45% of autistics (parent survey results, n=5284).¹⁵ B₆ as pyridoxal 5-phosphate is required three times on the route from methionine to sulfate and also three times enroute to taurine.
2. Melatonin helps 58% of autistics (parent survey results, n=375).¹⁵ Melatonin is formed by methylation of serotonin (via S-adenosylmethionine, SAM),   and methylation by SAM would be down-regulated if adenosine or adenosylhomo-cysteine were elevated at the cellular level.
3. Supplementing dimethylglycine (DMG) helps over 40% and trimethylglycine (TMG) helps 72% of those that DMG helps (DMG parent survey results, n=4725).¹⁶ DMG provides one-carbon pieces that are required for purine nucleotide synthesis, and DMG comes from TMG when TMG methylates homocysteine.
4. Cysteine and glutathione are low (so is metallothionein) per hundreds of reviewed lab tests, and sulfation is impaired.^17,18,19,20 So, the methionine ® cysteine conversion pathway is faulty or at least limited in many autistics.
5. Utilization of glutathione is subnormal, suggesting detoxification deficits and oxy-redox imbalances.^17,21
6. One enzyme in methionine metabolism, methionine synthase, (makes methionine from homocysteine via methylcobalamin) is shut down by miniscule amounts of mercury (e.g. Thimerosal).²²
7. Injection of high-dose methylcobalamin alleviates autistic traits in 40 to 70% (depending upon traits monitored and clinicians’ observations).^23,24 Methylcobalamin needs glutathione (made with cysteine) to be formed, and it promotes formation of methionine directly from homocysteine.²⁵
8. Dietary exorphins such as beta-casomorphin are elevated in many autistics.²⁶ The enzyme dipeptidylpeptidase IV (or DPP4) that digests these peptides is the same protein as lymphocyte activation factor CD26²⁷ which is intrinsically underexpressed on lymphocytes during milk allergy.²⁸  And, CD26 is the binding protein for adenosine deaminase.¹³ At the cellular level, adenosine virtually controls the methionine ® homocysteine sequence.
9. Use of digestive enzymes with DPP4 activity and removal of exorphin food sources (casein, gluten) help a significant subset of autistics (GF-CF diet helps 63%, n=933).^29,30,15
10. Immune dysregulation with autoantibody responses to multiple tissues and lymphocyte receptors (including DPP4/CD26) are commonly found in autistics.³¹

What phenotypical variants could be responsible for weakness or predisposition to problems in forming cysteine from methionine and consequently insufficiency in one-carbon parts for purine assembly, poor transmethylation and poor sulfation capabilities?

• Weakness in adenosine-processing enzymes. Adenosine and ATP/adenosine ratio control the methionine-SAM-adenosylhomocysteine-homocysteine-methionine cycle.
• Weakness of the methionine-sequence enzymes: methionine synthase, methionine adenosyltransferase, betaine-homocysteine methyltransferase, etc.
• Limitations in folate-cobalamin-one carbon transformations: glutathionylcobalamin formation, 5,10-MeTHF reduction, glutamate-formiminotransferase (uses FIGlu from histidine), formiminotransferase cyclodeaminase, etc.

Also, weak methylation enzymes like catechol-O-methyltransferase, “COMT”, would be further inhibited by adenosine or adenosylhomocysteine disorders.

Several triggers or circumstances that initiate or worsen the autistic condition are:

• Milk allergy – decreases lymphocyte expression of CD26 (DPP4), which can decrease lymphocyte adenosine deaminase activity, raise adenosine and decrease local lymphocyte methionine metabolism.
• Strep infection with release of streptokinase (SK) (rapid release with antibiotic use); SK binds to lymphocyte CD26. Antibiotics can also affect glycosylation on intestinal DPP4, allowing uptake of exorphin peptides.
• Mercury (Thimerosal) exposure shuts down methionine synthase because GSH is deficient and Hg detox is impaired. Deficient cysteine means deficient thioneins and deficient GSH; Hg and Cu can inactivate DPP4 in the intestinal tract, allowing uptake of exorphin peptides.
• Dietary casein and gluten, with weakened DPP4, can provide exorphin peptides that attenuate or confuse cellular perception.

THERAPEUTIC PRIORITIES

1. Reduce/eliminate false neurotransmitters and deceptive messengers that attenuate or confuse cellular perception.
• Clean up and adjust the diet
• Trial of casein avoidance (casomorphins)
• Trial of gluten avoidance (gliadorphin, gluteomorphin)
• Use digestive enzymes (with DPP4 activity)
• Use basic nutritional supplements: zinc, calcium, magnesium, vitamin B₆, “Super Nu-Thera”, “D-Plex”, melatonin (if sleep is an issue), vitamin C, vitamin A
2. Clear the mouth, throat and gastrointestinal tract of dysbiotic flora that have taken up residence because of immune dysregulation and maldigestion – improves cellular response.
• Treat bacterial dysbiosis
• Treat fungal/yeast dysbiosis
• Use immune support (colostrum, transfer factor) and antivirals (turmeric for measles) if appropriate
• Secretin trial
• Continue digestive enzymes and basic nutritional supplements
3. Further improve cellular response (neurons, lymphocytes, secretory GI cells) – more advanced nutritional support.
• Trials of DMG, TMG, with/without B₁₂, folate
• Methylcobalamin
• Amino acid supplements
• External enhancement of sulfur (Epsom salt baths)
4. Get rid of toxic inhibitors – detoxification trials, therapy if indicated
• Lab tests: hair, blood, urine, stool – use these to assess toxic burden and to monitor detoxification progress
• Use provocative challenges to mobilize toxics: DMSA, TTFD, (intravenous) GSH, others at physician’s discretion.
• Beware of yeast/fungal reinfection of GI tract when oral sulfur agents are used (esp. lipoic acid, DMSA)
• Try oral sulfur supplements: GSH, cystine, NAC, “MT-Promoter” (with cystine)
• Should be able to normalize blood Cu/Zn ratio at this point if not already normalized
5. Investigate residual metabolic needs/disorders – rule out purine/pyrimidine metabolism errors: see previous text for conditions 1-10 and refer to DAN! October 2002 Consensus Report. Genomics testing for phenotypical variants and heterozygous conditions may reveal pertinent subacute metabolic weaknesses.
6. Sensory training, special education and other social-behavioral-educational programs should now be more successful in most cases.

REFERENCES

1. Coleman M and C Gillberg The Biology of the Autistic Syndromes, Praeger Pub (1985) p 53-56
2. “Changes in the Population of Persons with Autism and Pervasive Developmental Disorders… Dept. Develop. Services, California, Health and Human Services Agency (March 1, 1999), Sacramento CA
3. Autism Research Review vol. 14, no 1 (2000), ARI, p. 3,6
4. “Autistic Spectrum Disorders”, Dept. Develop Services, California Health and Human Services Agency (Apri8l 30043), Sacramento CA, p 9.
5. Blaxill M “The Rising Incidence of Autism” from The Global Crisis in Autism Science, June 2001. See also DAN! May 2001 Boston Conference Proceedings
6. Pangborn JB and SM Baker, Biomedical Assessment Options for Children with Autism and Related Problems DAN! Consensus Report, ARI (Oct 2002) p 21-44.
7. Fagarasan S et al. Science, 298 15 Nov 2002 p 1424-1427)
8. Wakefield AJ, A Anthony et al. “Enterocolitis in Children with Developmental Disorders” Am.J.Gastroenterology 95 no.9 (2000) p 2285-2295
9. Torrente F, P Ashwood et al. “Small intestinal enteropathy with epithelial, IgG and complement deposition in children with regressive autism” Molec.Psych 7 (2002) p 375-382
10. Bingham M “Autism and the Human Gut Microflora” Food Microbial Sciences Unit, Univ of Reading, Berkshire UK May 2002
11. Stubbs G, M Litt et al. “Adenosine Deaminase Activity Decreased in Autism” J.Am Acad Child Psych 21 (1982) p 71-74
12. Persico AM, R Militerni et al. “Adenosine Deaminase Alleles and Autistic Disorder…” Am J Med Genetics 96 (2000) p 784-790
13. Pangborn JB and SM Baker, op.cit, p 51-6
14. Pangborn JB and SM Baker, op.cit. p 9-17
15. ARI “Parent Ratings of Behavioral Effects of Biomedical Interventions” ARI Publ 34 April 2003
16. Edelson S “TMG/DMG Response Data” report to DAN! Philadelphia Think Tank, May 2003
17. Pangborn J “Detection of Metabolic Disorders in People with Autism” NSAC 1984 Annual Conference Proceedings, San Antonio TX (July 1984) p 32-51.
18. Pangborn J, observations of amino acid analyses on autistics, American BioScience Lab, Doctors’ Data Lab, Great Smokies Diagnostic Lab, SmithKline Beecham, Quest Labs, 1980-present.
19. Owens SC “Understanding the Sulfur System” Spring DAN! 2003 Conference Proceedings, Philadelphia PA, May 16, 2003 p 65-76
20. Waring RH andLV Klovrza “Sulfur Metabolism in Autism” J .Nutr & Environ Med 10 2000 p 25-32.
21. Michelson AM in Pathology of Autism AP Autor Ed, Academic Press (1982) p 277-279
22. Waly M, H Olteanu et al. “P13-Kinase Regulates Methionine Synthase: Activation by IGF-1 or Dopamine and Inhibition by Ethanol, Heavy Metals and Thimerosal” Dept. Pharmaceutical Sciences, Northeastern Univ, Boston MA 20902 Communication provided by Dr. Richard Deth.
23. Neubrander J “Biochemical Context and Clinical Use of Vitamin B₁₂” DAN! 2003 Philadelphia Conference Proceedings, May 2003 p 103-117
24. Informal Clinician reports to author
25. Rosenblatt DS and WA Fenton, Chapter 155 in Scriver et al., eds, The Metabolic and Molecular Bases of Inherited Disease, 8^th ed, McGraw-Hill (2001) p 3910, 3913
26. Reichelt KL, AM Knivsberg et al. “Nature and Consequences of Hyperpeptiduria and Bovine Casomorphins Found in Autistic Syndromes” Dev. Brain Dysfunct 7 (1994) p 71085
27. Handbook of Proteolytic Enzymes, AJ Barrett, ND Rawlings and JF Woessner eds, Academic Press (1998) p 378-382
28. Schade RP et al. “Cell surface expression of CD25, CD26 and CD30 by allergen-specific T cells is intrinsically different in cow’s milk allergy” J.Allergy Clin. Immunol. 109 Feb 2002 p 357-362
29. Pangborn JB, DAN 2000 San Diego Conference Proceedings September 2000 report on Klaire Labs DPPIV-activity enzyme p 76; DAN! 2001 San Diego Practitioner Conference Proceedings (October 2001), report on Kirkman Labs DPPIV-activity enzyme, p 81
30. Knivsberg AM, KL Reichelt et al. “A Randomized, Controlled Study of Dietary Intervention in Autistic Syndromes” Nutritional Neuroscience 5 (2002) p 251-261
31. Vojdani A, JB Pangborn et al. “Infections, Toxic Chemicals and Dietary Peptides Binding to Lymphocyte Receptors and Tissue Enzymes are Major Instigators of Autoimmunity in Autism” (2003), in press.

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