A Paradigm Shift in

Diagnosing and Treating

ASD patients:

Autism is a treatable medical and metabolic disease with behavioral components

Prepared Statement: Congressional Autism Hearing Original: November 29, 2012, Last Updated: Jan 31, 2013

Cassandra L. Smith, PhD¹ ² Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University¹ Director of Research, Athena Biomedical Institute²

Kazuko Grace² Consumer Advocate, Athena Biomedical Institute²

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A Paradigm Shift in Diagnosing and Treating ASD patients:

Autism is a Treatable Medical and Metabolic Disease with Behavioral Components

A Paradigm Shift in Diagnosing and Treating ASD patients:

Autism is a Treatable Medical and Metabolic Disease with Behavioral Components

Prepared Statement: Congressional Autism Hearing November 29, 2012 Last updated in January 31, 2013

Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute

Kazuko Grace Abstract: Research on autism and autism spectrum disorders Athena Biomedical Institute (hereafter referred to as ASD), and other serious neurobehavioral www.athenabiomedicalinstitute.org disorders, has minimally influenced patient outcome or disease

prevention. Although research links many genetic and environmental factors to these diseases, no single or small number of factors is responsible for disease in the majority of patients. An increasing number of seemingly disparate factors (genetic, environmental, and epigenetic) linked to ASD are converging on specific medical anomalies. Additionally, research tells us that each patient is unique. The medical abnormalities found in ASD are serious and debilitating and, in some cases, have been known for a long time. However, diagnosis and treatment of ASD remains focused on behavioral abnormalities. The translation of research to the clinic means that a new diagnostic and treatment paradigm must be developed that acknowledges the unique spectrum of medical and behavioral symptoms present in each patient. Diagnosis and treatment of medical abnormalities will improve patient quality of life and behavior. Today, this new paradigm is only benefiting a small number of individuals that can either personally pay for treatment, or are patients in free non-insurance reimbursed clinics.

ABI©2012, 2013

Prepared Statement of Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute and Kazuko Grace, Consumer Advocate Athena Biomedical Institute

Contact Information: Email: [email protected] Tel: 617 571 3068

A Paradigm Shift in Diagnosing and Treating ASD patients:

Autism is a treatable medical and metabolic disease with behavioral components.

Abstract: Research on autism and autism spectrum disorders (hereafter referred to as ASD), and other serious neurobehavioral disorders, has minimally influenced patient outcome or disease prevention. Although research links many genetic and environmental factors to these diseases, no single or small number of factors is responsible for disease in the majority of patients. An increasing number of seemingly disparate factors (genetic, environmental, and epigenetic) linked to ASD are converging on specific medical anomalies. Additionally, research tells us that each patient is unique. The medical abnormalities found in ASD are serious and debilitating and, in some cases, have been known for a long time. However, diagnosis and treatment of ASD remains focused on behavioral abnormalities. The translation of research to the clinic means that a new diagnostic and treatment paradigm must be developed that acknowledges the unique spectrum of medical and behavioral symptoms present in each patient. Diagnosis and treatment of medical abnormalities will improve patient quality of life and behavior. Today, this new paradigm is only benefiting a small number of individuals that can either personally pay for treatment, or are patients in free non-insurance reimbursed clinics.

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Introduction: Research has linked many genetic, epigenetic and environmental factors to ASD and other serious neurobehavioral diseases like schizophrenia. Many factors should be linked to these diseases because the brain is the most complex and evolved organ, and will be sensitive to the greatest number of factors. Although research findings do not provide the simple sought after answer, useful new information on how to diagnose and treat serious symptoms in ASD should not be ignored.

Increasingly, seemingly disparate genetic, environmental, epigenetic and medical factors linked to ASD are converging on deficits in specific metabolic processes. The medical and metabolic abnormalities of ASD patients need to be diagnosed because available treatments can improve the quality of life and behavior of these patients, and in some cases prevent disease. Diagnostic regimes must be broad because research tells us that each patient is unique. "Biomedical" diagnosis and treatment is increasingly, albeit slowly, becoming recognized as important to these patients. Here, we will provide an overview of genetic, environmental and epigenetic factors linked to the medical and metabolic abnormalities present in ASD patients. A synopsis of the content of this paper is presented in Table 1.

The issues raised are important discussion points for not only ASD but also other serious neurobehavioral disorders like schizophrenia. Clearly, these suggestions entail a major change in the manner in which neurobehavioral diseases are viewed within the medical field, and by the public.

Appendix 1 has a glossary of scientific terms, abbreviations, and acronyms used in this document. The Addendum has a list of suggested testing for comprehensive assessment of each ASD patient. The Addendum is meant as a starting point for discussion.

Diagnosis and treatment is based on behavioral criteria: ASD is diagnosed by the presence of behavioral abnormalities that include impaired verbal and nonverbal communication and social interactions, and stereotypic behavior and interest. In most cases testing for genetic (e.g. genotyping, gene expression and epigenetic assessment) or medical (e. g. metabolite, screening for toxic chemical, infections, inflammation, digestions issues, and nutritional status) problems is not done, although these issues commonly occur in ASD. Likewise, treatment regimes focus on insurance covered behavioral interventions.

Genetic Liability Highly Variable: There are over 100 genetic loci linked to ASD. These genes code for proteins involved in a variety of processes including: brain development and function; metabolic pathways important in DNA and RNA replication; energy production; cellular responses to inflammation and infection, xenobiotic exposures, oxidative stress; and epigenomic programming.

Table 1. Synopsis: ASD is a group of complex medical and metabolic diseases with behavioral abnormalities.

1. Diagnosis and treatment of ASD patients are focused on behavioral abnormalities and not the cause of disease, or presenting medical and metabolic problems. 2. Many varied (genes, environmental, medical and metabolic) factors are linked to ASD. 2.1 The brain is the most highly evolved organ should have the greatest number of factors linked to its development and function. 2.2 Early life is uniquely and especially sensitive to disruption. 2.3 Heritability measurements in ASD are mis-interpreted as saying disease is mainly due to genetic causes. 3. Over 100 genes are linked to AS 3.1 Genes linked to ASD are involved in neurodevelopment and brain function, metabolic pathways, and known medical conditions. 3.2 Some genes linked to ASD are also linked to rare genetic diseases caused by known single gene mutations. 3.21 Some rare genetic diseases are preventable through early diagnosis and intervention. 3.3 ASD displays genetic anticipation with successive generations have more severe and early onsets of disease. 4. ASD is linked to elevated rates of mutation making each patient unique. 4.1 Genetic and environmental factors linked to ASD can increase mutation rates and contribute to genetic anticipation and the spectrum of symptoms. 5. Xenobiotic exposures that increase the presence of reactive oxygen species (ROS) are linked to ASD but are difficult to assess. 5.1 Both major and minor xenobiotic exposures can be important. 5.2 A subset of individuals may be sensitive to xenobiotics like the heavy metals mercury. 5.3 Sensitivity to exposure can have a genetic origin, or be confounded by other, even non- assessed environmental factors. 5.4 Sensitivity can be due to deficits in the detoxification process, or the production and cellular response to the accompanying oxidative stress. 6. Infection and inflammation, commonly seen in ASD, impede biological processes and hinder the bodies response to stress. 6.1 Infection and inflammation impede DNA and RNA synthesis. 6.2 Gastrointenstinal disorders can lead to malabsorption and malnutrition. 6.2 Malnutrition before 2 years of age leads to behavioral abnormalities that resists nutritional interventions. 7. Nutrition is under-appreciated in pharmaceutical industry and in medicine. 7.1 Typically, medical professionals are not well-versed in nutrition, and insurance does not pay for such consultations unless symptoms are overt and classical. 7.2 Early nutritional interventions can prevent some ASD. 7.3 Essential nutrients must be obtained from the diet or the billions of bacterial that inhabit our bodies. 8. Folate, methionine, transulfuration and dopamine metabolic pathways are closely linked to each other and to ASD in multiple ways. 8.1 These pathways are involved in DNA and RNA synthesis, epigenomic programming, energy production, the cellular response to oxidative stress, and dopamine metabolism. 8.2 Multiple essential nutrients are required by these pathways. 8.3 Usually, most ROS are produced in the mitochondria during ATP (energy) production 8.31 Mitochondrial defects are found in a subset of ASD patients. 5

8.32 ATP is required for all bodily responses, e.g. infection, inflammation, response to ROS etc. 8.4 Complex and system biology approaches are needed to understand the dynamics of metabolic pathways in ASD. 9. Epigenomic regulation of development and function is disrupted in ASD. 9.1 Epigenomics represents the interaction between the genome and the environment. 9.2 The greatest amount of epigenomic programming occurs early in live. 9.3 Many and varied epigenomic changes are linked to ASD that include DNA methylation, histone modification. 10. Treatment of medical and metabolic abnormalities will improve the quality of life and behavior of patients.

About 20% of ASD patients, have "secondary autism", because a rare genetic disorders with a single gene cause is present. Fragile X syndrome is the most common genetic disorder linked to (1-2%) ASD patients, and is the most common cause of hereditary mental retardation. Other single gene disorders linked to ADS are developmental diseases that impact brain function and development.

The new ASD diagnostic criteria proposed in the Diagnostic and Statistical Manual for Mental Illnesses (DSM-)-V, published by the American Psychiatric Association, increases the number of disorders placed under the ASD umbrella without regard to disease cause or treatment. For instance, Rett syndrome, a rare single gene disease linked to a global deficite in epigenetic programming is now classified as part of the ASD spectrum. Another group of genetic diseases linked to ASD involve “inborn-errors-of - metabolism” (Karnebeek and Stockler, 2011). In some of these diseases, early dietary interventions can prevent or reduce illness. An example of a severe brain disease prevented by early detection and treatment is phenylketonuria. In the United States, all newborns are tested for this disease because early and a simple dietary intervention (elimination of phenylalanine) can prevents brain damage and disease.

Diagnosis of ASD based on behavioral abnormalities discourages genetic testing for rare, treatable genetic diseases. Such a focus ignores research progress that defines subsets of patients where prevention is possible, and adds un-necessary complexity to basic and applied research and treatment.

Families segregating ASD with multiple family members affected by disease are identified (Piven et al., 1997). In some families, ASD does not appear to be multi- generational. Instead, less severe behavior abnormalities are seen in earlier generations reminiscent of "genetic anticipation". Genetic anticipation refers to diseases that become more severe, or have an earlier age of onset in successive generations. Genetic anticipation in ASD may not be surprising given the recent rapid increase in disease occurrence.

A contributor to genetic anticipation in ASD can be the elevated rate of mutations found in patients. Mutations that occur in egg and sperm will be passed to the next generation, and can contribute to increasing disease liability, severity, the myriad of symptoms, and genetic anticipation. Elevated mutation rates are found in other neurobehavioral diseases (e. g. Nguyen et al., 2003 and many more recent genetic studies)

Mutations in sperm accumulate with paternal age, and elder fathers have an increased number of children with ASD (For review see Smith et al., 2010, 2012). Further, many environmental factors (discussed below) linked to ASD have the potential to increase mutation rates and affect genetic anticipation. Thus, several lines of evidence link ASD to increased mutation rate. Importantly, each patient has a unique set of mutations and each patient must be tested for a broad range of mutational liabilities. Similar results are found for other serious neuropsychiatric diseases like schizophrenia.

In the past, some research funding agencies focused almost exclusively on genetic studies because ASD is said to have ~80% heritability. This was unfortunate because of the widespread misunderstanding of heritability measurements especially in neurobehavioral disorders. An ~80% heritability refers to the amount of disease phenotypic variation that is due to genetic variation and NOT the percent of disease due to genetic mutations. In addition, heritability does NOT measure the affect of environment on disease variability. For instance, ~80% of a disease phenotypic variability may be linked to both genetic and environmental factors. Today, more research funds are being spent on environmental factors linked to ASD, although the general perception remains that ASD is a genetic disease.

Environment Exposure History and Response Highly Variable: The industrial age included the introduction of more than 100,000 commercially important chemicals into the environment. Research on these chemicals is ongoing in a variety of arenas because of their potential to influence many biological and organ systems, and diseases. One outcome of environmental research has been the reduction or elimination of some dangerous compounds from the environment.

However, the ability to define the effect of an environmental factor on humans remains difficult because of the great genetic variability and environmental-history differences between individuals. Further, the affect of multiple, varied low dose exposures to different compounds may be important but is even more difficult to study. Other complexities include exposure length, dose, time of life, and the presence of confounding but un-assessed exposures and factors. For example, research has demonstrated that ambient temperature, a simple environmental factor, can affect outcome of an exposure.

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Although controversial, xenobiotic, heavy metal like mercury are linked to ASD. Some theorized that mercury in vaccines was responsible for the increase in ASD. Although, the Federal Vaccine Court ruled in 2010 that there was insufficient evidence to link vaccination to ASD, the controversy has not been put to rest. In 2011, Holland et al. reported an increased incidence of ASD in vaccinated children. The picture became more complicated because trans-generation mercury exposure was linked to ASD (Shandley and Austin, 2011). In this study, grandchildren of pink disease (infantile acrodynia) patients had an astounding 1 in 22 probability of becoming ill with ASD. Pink disease occurs in a subset of individuals with elevated mercury levels.

Many studies support the idea that a subset of individuals is sensitive to xenobiotics. Sensitivity may be due to defects in xenobiotic metabolism responsible for detoxification of these compounds. Other sensitivity is due to the production or removal of reactive oxygen species (ROS) and oxidative stress that is generated during the detoxification process.

Genetic testing has identified deficits in these metabolic processes in some ASD patients. Although direct and indirect affects may be difficult to sort out in individual patients, detection of faulty pathways, and interventions that improve these pathways are possible. For instance, the use of dietary anti-oxidants can improve the response to oxidative stress and treatment regimes that remove detectable xenobiotics and reduced ASD symptoms have been reported.

Nutrition and exercise is an under considered but un-refuted environmental factor important for good health. Generally, the medical profession is not well versed in nutrition and many clinicians fail to appreciate or treat nutrients as pharmaceuticals. Similarly, medical insurance reimbursement for nutritional assessments and treatments are limited to those patients with classical and severe symptoms of malnutrition. Nutritional interventions in ASD have not been evaluated yet in many conventional evidence based studies such as those used for pharmaceutical drug testing. Meanwhile, parents of ASD patients continue to use nutritional interventions and, in some cases, report good outcomes. The generally lack of interest in nutra-pharmaceuticals research in both the pharmaceutical industry and the medical community is not surprising. Likewise, exercise is an under utilized but beneficial ASD treatment (Sowa and Meulenbroek, 2012) as it is for many common illnesses.

Under-nutrition and malnutrition before two years of age will lead to behavioral abnormalities that resist nutritional interventions later in life (Galler et al., 1996). Gastrointestinal problems and malnutrition are often present in ASD patients. Gastrointestinal problems, including infection and inflammation, can lead to malabsorption and malnutrition. Recently, the American Pediatric Association advocated for the inclusion of gastrointestinal disorder assessment and treatment in ASD (Buie et al., 2012). Although a start, it is not clear that this testing will occur early enough to prevent disease occurrence or to reduce severity (see phenylketonuria above).

Nutritional, genetic, environmental, medical and metabolic factors linked to ASD converge on the abnormalities in the Folate-Methionine-Transulfuration-Dopamine (FMTD) hub. Details are provided in Figure 1. In brief, the FMTD metabolic hub includes pathways involved in oxidative stress response, DNA and RNA synthesis, energy production, dopamine metabolism, and epigenomic programming.

Epigenetics Programming and ASD: Genetic and environmental factors linked to ASD interfere with epigenomic programming directly and indirectly. Epigenetics programming is a fuzzy term that refers to gene-environment interactions, and developmental programs that enable a single inherited genome to code for multiple cell type such as neurons, muscle and blood (for reviews see Abdolmaleky et al., 2008, Smith et al., 2010, Smith and Huang, 2012). The greatest amount of epigenomic programming occurs early in life. However, epigenomic programming is dynamic, and changes occur throughout life.

Interference in epigenomic programming in early in life has the greatest consequences because the largest number of cells and the earliest developmental programs are affected. Some affects may not be apparent until much later in life or even in another generation. The brain has the greatest amount of epigenetic programming, and the greatest sensitivity to factors that interfere with this process.

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Figure 1. The folate-methionine-transulfuration-dopamine (FMTD) metabolic hub. Nutrition, DNA and RNA synthesis, intracellular oxidative stress, epigenomic programming, and dopamine metabolism are closely linked in the FMTD metabolic hub. Abnormalities in these processes are linked to ASD. Details of the FMTD hub are provided here. Essential nutrients (dotted lines) that must be provided by the diet or from our microbiome are vitamins B9 (folate), B6, and B12; the amino acid methionine (met); and betaine/choline. Folate is converted to dihydrofolate (DHF), then tetrahydrofolate (THF) derivatives. The folate cycle (blue) directly participates in the synthesis of dTMP from dUMP and IMP (red *). dTMP is converted to dTTP and used for DNA synthesis. IMP is a precursor for the synthesis of all purine. Purines are required for DNA and RNA synthesis, and the energy currency of the cell, ATP and GTP. The major product of the met cycle (red) is S-adenosyl methionine (SAM). In epigenetic programming, methyl transferases (MT) enzymes use SAM as a methyl group donor. In the met cycle, SAM is made from met and ATP. Met is obtained exogenously, and can regenerated from homocysteine (HCY) by the addition of a methyl groups from THF or betaine (obtained from choline in the diet) by the enzymes methionine synthetase (MS), or betaine homocysteine methyl transferase (BHMT), respectively. The transulfuration pathway (green) synthesizes the major intracellular antioxidant, glutathione (GSH), and the amino acid cysteine (cys) from HCY. The enzyme MS has a second substrate, the demethylated dopamine D4 receptor (HCY-DRD4), that is converted to methylated DRD4 (met-DRD4) by the additional of a methyl group from THF (purple). The methylated DRD4 receptor (met-DRD4) covalently transfers the methyl group to lipopolysaccharides, changing cellular membrane characteristics.

Today a large research effort is focused on cataloguing the many epigenomic changes that occur in ASD. Ultimately, the number of epigenetic changes will be at least an order of magnitude greater than the observed genomic changes linked to ASD. Many epigenetic changes will affect the same pathways that have been identified in earlier genetic and environmental studies and are discussed above. Here, the discussion will focus on global epigenetic changes that are linked to ASD and likely contribute to the systemic and complex nature of disease.

The FMTD metabolic hub depicted in Figure 1 is the key global metabolic regulator of epigenetic programming. In the FMTD hub, S-adenosyl methionine (SAM), a sulfur containing metabolite, is the major methyl donor required for epigenomic programming. DNA methylation is the best-studied epigenetic process, and generally DNA methylation of gene promoter regions is associated with a loss of expression. Methyl groups from SAM are donated to a wide variety of large (e.g. DNA, RNA, proteins, lipids and polysaccharides) and small molecules (e. g. dopamine). Some xenobiotic detoxification reactions utilize SAM. The FMTD hub is sensitive to nutrition, infection, inflammation, oxidative stress, and toxic exposures.

Dopamine metabolism, a major process in the brain, is linked directly to the FMTD metabolic hub and ASD (for review Smith et al., 2010, 2012). Direct links include epigenetic programming of genes involved in dopamine metabolism, degradation of dopamine by a methyl transferase (COMT), and methylation of the dopamine receptor D4 (DRD4 receptor). Methyl transferase (MT) enzymes that use SAM or folate as a methyl donor occur in all these reactions.

The FMTD hub is connected to the greatest number of intracellular reactions because two metabolites, ATP and SAM, are the most used cofactors in the cell. During evolution, the extensive metabolic wiring optimized the FMTD and imparted unique characteristics: robustness to familiar conditions, and sensitivity to unfamiliar conditions.

The industrial age introduced new types and higher levels of xenobiotics. Some xenobiotics that affect the FMTD hub were not encountered previously during evolution. Xenobiotic exposures will increase the level of reactive oxygen species (ROS) and oxidative stress. These exposures affect the FMTD hub because glutathione (GSH), a sulfur-containing metabolite product of the FMTD hub, is the primary intracellular antioxidant. Heavy metal exposures increase the level of reactive oxygen species (ROS), and bind sulfur containing endogenous anti-oxidants.

Most ROS within a cell are produced as a side product of energy production (ATP) in the mitochondria. Increases in processes that require ATP, like xenobiotic metabolism and an oxidative stress response, will increase the level of ROS in the cells. Infection and inflammation will increase the level of intracellular ROS and oxidative stress directly, and indirectly because the biological response to these events includes an increase in DNA and RNA synthesis, and energy production. Deficits in mitochondrial function are detected in a subset of ASD patients (Anitha et al., 2012, Villafuerte, 2011)

Multiple essential nutrients (e.g. B vitamins, amino acids, and betaine/choline) required within the FMTD hub must be acquired exogenously. Nutritional deficiencies including both folate and betaine/choline are linked to behavioral and brain abnormalities, ASD and schizophrenia (Smith et al., 2010, 2012, Blusztajn and Mellott, 2012)

Exogenous sources for essential nutrients include the diet and the microbiome. The microbiome refers to the billions of microorganism that share our bodies. Treatments with antibiotics to fight infection can change the microbiome detrimentally. Although, this is a new area of research, abnormal microbiomes are detected in ASD (e. g. Parracho et al., 2005), and other neuropsychiatric patients (Severance et al., 2012).

Severe gastrointestinal disorders in both adults and children, including ASD, are linked to changes in the microbiome (e. g. Parracho et al., 2005; Benache et a., 2012), for instance, infection by the pathogenic microorganism Clostridium difficile. Recently, fecal transplants were used to successfully treat adult patients with C. difficile infections (Stollman and Surawicz, 2012; Gought et al., 2011). Fecal transplants in ASD have yet to

11 be done, and are likely to be able to treat less severe microbiome imbalances. At this time, the contribution of diet and the microbiome to human nutrition (or other internal processes) is largely unknown, although dietary deficiencies alone are known to cause disease.

Abnormal homocysteine (HCY) levels in blood are detected in many ASD patients. HCY is a key FMTD metabolite with changes found many common diseases (e. g. neuropsychiatric, cancer, and cardiovascular). In most cases, normalization of HCY levels through simple (single) nutritional interventions has not been accompanied by a reduction in disease symptoms. Hence, HCY levels are no longer monitored clinically.

The affect of nutritional intervention in neurobehavioral disease is not clear. Folate and methionine supplementation alter psychotic symptoms (up and down, respectively) in adult schizophrenia patients. Further, some pharmaceuticals useful in treated schizophrenia affect the FMTD hub directly. An example is the increased incidence of ASD like symptoms in children from mothers treated with valporate (to control seizures) during pregnancy (Bromely et a., 2009). As a consequence of this observation, valporate treatment of pregnant rodents is used to create rodent model of ASD (Bambini-Junior et al., 2011). Valporate affects the production of SAM and epigenetic programming.

Some clinical tests, including the measurement of single metabolites, are used routinely to monitor health overall, while others are only used when warranted by presenting symptoms. Research argues for monitoring ASD patient health using both old (e.g. HCY), and newly identified important metabolites.

Global monitoring approaches used in systems biology can help understanding disease in individual ASD patients. SNP testing and DNA sequencing which provide direct information about genetic make-up is now in the clinical arena. Gene expression, a state- of-the art research method, monitors cell function and has been useful for diagnosing and treating cancer patients. Epigenomic changes and treatments based on this knowledge are revolutionizing the treatment of cancer, especially blood displasias. In contrast to other diseases, in cancer, the affected tumor tissue is available for analysis, and more recent work has detected the DNA footprint of solid tumor cancer cells in blood.

As brain tissue is not readily available, metabolic monitoring of ASD patients will need to be done with blood, or another readily available tissue. Changes in blood are monitored for many diseases, and are linked already to ASD. Clinical tests of blood for folate, HCY and other metabolites are done on blood, already. Whether changes are a direct or indirect consequence of disease, blood has, and can serve as a sentinel of mental health.

The new paradigm: ASD is a group of complex medical and metabolic diseases with behavioral abnormalities. Increasingly, the medical profession is recognizing that the standard-of-care for ASD should include detection and treatment of non-behavioral abnormalities. ASD patients have multiple and varying medical and metabolic problems that need to be detected and treated. Today, ASD clinics that try to treat medical and metabolic aspects of disease fall outside current insurance reimbursement schemes, and in some cases mainstream medicine.

ASD is an illness that costs the government about $60 billion per year in the US and is likely to increase. Schizophrenia, a similar common neurobehavioral disorder costs about $160 billion per year. Both ASD and schizophrenia are common, severe and lifelong. Research progress has increased our understanding of what processes are abnormal for these diseases. However, the funds spent on research do not reflect the relative cost of these diseases, and research results, for the most part, have yet to be translated to the clinic. An estimated 20% of schizophrenia and ASD patients have underlying but undetected medical disorders. It is a disgrace that patients are not tested and treated for underlying medical and metabolic problems.

The U. S. National Institute of Health is moving towards a paradigm of prevention rather than treatment of disease. Genetic make-up, infections, inflammation, toxic exposures, malnutrition, diet and exercise influence the health of all individuals. Research increasingly and repeatedly links environmental factors to many common disorders. Appropriate diet and exercise will improve health for all individuals, and reduce disease. For instance, a ~40% decrease in breast cancer can be realized through diet and exercise. The translation of recent research observations to the clinic, whether for breast cancer, ASD or schizophrenia can reduce disease.

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References:

Abdolmaleky HM, Smith CL, Faraone SV, Shafa R, Stone W, Glatt SJ and Tsuang MT (2004) Methylomics in psychiatry: Modulation of gene-environment interactions may be through DNA methylation. American journal of medical genetics Part B, Neuropsychiatric genetics 127B:51-59. Abdolmaleky HM, Zhou JR, Thiagalingam S and Smith CL (2008) Epigenetic and pharmacoepigenomic studies of major psychoses and potentials for therapeutics. Pharmacogenomics 9:1809-1823. Anitha A, Nakamura K, Thanseem I, Yamada K, Iwayama Y, Toyota T, Matsuzaki H, Miyachi T, Yamada S, Tsujii M, Tsuchiya KJ, Matsumoto K, Iwata Y, Suzuki K, Ichikawa H, Sugiyama T, Yoshikawa T and Mori N (2012) Brain region-specific altered expression and association of mitochondria-related genes in autism. Molecular autism 3:12. Bambini-Junior V, Rodrigues L, Behr GA, Moreira JC, Riesgo R and Gottfried C (2011) Animal model of autism induced by prenatal exposure to valproate: behavioral changes and liver parameters. Brain research 1408:8-16. Benach JL, Li E and McGovern MM (2012) A microbial association with autism. mBio 3. Blusztajn JK and Mellott TJ (2012) Choline nutrition programs brain development via DNA and histone methylation. Central nervous system agents in medicinal chemistry 12:82-94. Buie TR, Fuchs III GJ, Furuta GT, Kooros K, Levy J, Lewis JD, Weshil BK and Winter H (2010) Recommendation for evaluation and treatment of common gastrointestinal problems in children with ASDs. Pediatrics 125: 219-S29. Galler JR, Shumsky JR and Morgane PJ (1996) Malnutrition and brain development, in Physiology and Pathophysiology pp 196-212, B. C. Decker Inc. Gough E, Shaikh H and Manges AR (2011) Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 53:994-1002. Holland M, Conte L, Krakow R and Colin L (2011) Unanswered questions from the vaccine injury compensation program: A review of the compensated cases of vaccine-induced brain injury. Pace Environmental Law Review 28:480-466. Nguyen GH, Bouchard J, Boselli MG, Tolstoi LG, Keith L, Baldwin C, Nguyen NC, Schultz M, Herrera VL and Smith CL (2003) DNA stability and schizophrenia in twins. American journal of medical genetics Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 120B:1-10. Parracho HM, Bingham MO, Gibson GR and McCartney AL (2005) Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. Journal of medical microbiology 54:987-991. Piven J, Palmer P, Jacobi D, Childress D and Arndt S (1997) Broader autism phenotype: evidence from a family history study of multiple-incidence autism families. The American journal of psychiatry 154:185-190. Severance EG, Alaedini A, Yang S, Halling M, Gressitt KL, Stallings CR, Origoni AE, Vaughan C, Khushalani S, Leweke FM, Dickerson FB and Yolken RH (2012) Gastrointestinal inflammation and associated immune activation in schizophrenia. Schizophrenia research 138:48-53. Shandley K and Austin DW (2011) Ancestry of pink disease (infantile acrodynia) identified as a risk factor for autism spectrum disorders. Journal of toxicology and environmental health Part A 74:1185-1194. Sowa ME and Meulenbroek R (2012) Effect of physical exercise on Autism Spectrum Disorder: A meta-analysis. Research in Autism 6:46-57. Smith CL, Bolton A and Nguyen G (2010) Genomic and epigenomic instability, fragile sites, schizophrenia and autism. Current genomics 11:447-469. Smith C and Huang K (2012) Epigenomics of neurobehavioral diseases, in Epigenetics of Human Disease (Tollesfsbol T ed) pp 127-152, Elsevier, Amsterdam, The Netherlands. Stollman N and Surawicz C (2012) Fecal transplant for Clostridium difficile. Archives of internal medicine 172:825; author reply 825-826. van Karnebeek CD and Stockler S (2012) Treatable inborn errors of metabolism causing intellectual disability: a systematic literature review. Molecular genetics and metabolism 105:368-381. Villafuerte S (2011)Suggestive evidence on the genetic link between mitochondria dysfunction and autism. Acta psychiatrica Scandinavica 123:95

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Appendix 1. Glossary of Scientific Terms and Acronyms

Anti-oxidant - molecules that protect cells from chemical damage caused by free radicals ASD - autism spectrum disorder Betaine - a small neutral metabolite that donates methyl groups to HCY BHMT - betaine homocysteine methyl transferase enzyme

CH3-lipopolysaccharide - methylated lipopolysaccharide found in the cell membrane Cys - cysteine, a sulfur containing amino acid DSM - Diagnostic and Statistical Manuel for Mental Diseases published by the American Psychiatric Association DHF - dihydrofolate Dopamine - neurotransmitter DRD4 - dopamine receptor D4 dTMP - deoxythymidine mononucleoside, a precursor in dTTP synthesis dTTP - deoxythymidine triphosphate, a precursor used in DNA synthesis dUMP - deoxyuridine monophosphate, a precursor for dTMP synthesis Epigenetics - a fuzzy scientific term that involves gene-environment interactions, and processes that allow a single genome to develop into multiple cell types Fragile X disease - most common cause of hereditary mental retardation Federal Vaccination Court - refers to the Office of Special Masters of the U. S. Federal Claims Court that administers a no-fault system for litigations involving vaccine injury FMTD metabolic hub - folate-methionine-transulfuration-dopamine Folate - vitamin B9 Gene expression - measurement of mRNA or protein levels Genetic anticipation - phenomenon observed in some genetic diseases where severity increases, and/or age of onset decreases in successive generations. GSH - glutathione, a tripeptide composed of glycine-cysteine-glutamine with a gamma peptide bond between the gamma carboxyl group of glutamine and the amino group of cysteine HCY - homocysteine HCY-DRD4 - demethylated DRD4 receptor MET-DRD4 - methylated DRD4 receptor IMP - inosine monophosphate, a precursor to the synthesis of all purines Inborn-errors-of-metabolism - a large number of single gene diseases linked to metabolism Met - methionine; a sulfur containing amino acids MS - methionine synthase enzyme MT - methyl transferase enzyme Oxidative stress - refers to an imbalance in the level of reactive oxygen specie (ROS)s and the biological detoxification pathways ROS - reactive oxygen species Phenylalanine - amino acid Phenylketonuria - inborn error of metabolism disease Pink disease - a disease found in a subset of individuals with elevated levels of mercury characterized by pink hands or feet Rett Syndrome - single gene disorder with a mutation in MeCP (methylated cytosine binding protein) gene SAM - S-adenosyl methionine, the major methyl donor in the cell THF - tetrahydrofolate Transulfuration pathway - metabolic pathway that converts homocysteine (HCY) to glutathione (GSH) and cysteine an amino acid Vitamin - small molecular compound essential to cells that must be obtained exogenously Xenobiotic - small molecular weight molecule not ordinarily present in a biological system Xenobiotic metabolism - pathway that detoxifies a xenobiotic

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Addendum I

Suggested Medical and Metabolic Assessments Useful for ASD

A Paradigm Shift in Diagnosing and Treating ASD patients:

Autism is a Treatable Medical and Metabolic Disease with Behavioral Components

Addendum I

Suggested Medical and Metabolic Assessments Useful for ASD

Prepared Statement: Congressional Autism Hearing Original: November 29, 2012 Last updated: January 31, 2013

Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute

Kazuko Grace Athena Biomedical Institute www.athenabiomedicalinstitute.org

Prepared Statement of Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute and Kazuko Grace, Consumer Advocate Athena Biomedical Institute Contact Information: Email: [email protected] Tel: 617 571 3068

A Paradigm Shift in Diagnosing and Treating ASD patients:

Autism is a treatable medical and metabolic disease with behavioral components.

Addendum I

Suggested Medical and Metabolic Assessments Useful for ASD

Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

Suggested Medical and Metabolic Assessments Useful for ASD

Cassandra L. Smith, and Kazuko Grace Boston University and Athena Biomedical Institute

______Introduction: Most governmental funds are directed towards studies on the causes of ASD while translation of research results to patients is minimal. Research has revealed some important ways to improve patient treatment today. However, the answer is not simple.1 Each patient is unique and can have a unique variety of medical, metabolic, epigenomic and behavioral symptoms.2 Hence, complex diagnosis and treatment regimes need to be applied to patient care. Each patient must be viewed from a holistic rather than the traditional reductionist perspective, requiring input from multidisciplinary fields characteristics of “systems biology”. Systems biology is a field of biology that focuses on understanding the many varied complex interactions that occur in the body. Today’s clinician is not well versed in all aspects of disease and research outcomes, and needs direct input from researchers to insure maximal benefit to the patient.3

There is a revolution in research on disease prompted by the human genome project. Arguably, patients likely to benefit the most from the new finding and technologies are those with neurobehavioral illnesses like ASD. It is especially egregious not to identify patients with inborn-errors-of-metabolism in time for treatment interventions that may prevent a severe, lifelong, and untreatable illness.4

In most cases, insurance does not cover diagnostic procedures and treatments for the medical, metabolic and epigenomic abnormalities linked to ASD. Patients who can self-fund benefit from the accumulated knowledge of research. However, testing alone is prohibitively expensive.5

The American Academy of Pediatrics believes that children's medical care should be accessible, family-centered, continuous, comprehensive, coordinated, compassionate, and culturally effective.6 7 Comprehensive health care insurance coverage of ASD will reduce the financial burdens on families with ill children.8 Admittedly, this will be expensive. However, early diagnosis and treatment can limit, and some cases, prevent disease, reduce the estimated ~60 billion dollar per year expense for ASD. 9 10 11 12

This Addendum includes testing for a plethora of abnormalities found in ASD patients with variable presentations. Because each patient is unique, a wide range of testing is necessary to optimized individual treatments; however, we recognize the financial burden imposed by all this testing. We present this list as a starting point for discussion to develop an optimized testing hierarchy and treatment regime that must include patient and advocate participation.

Behavior is influenced by medical and metabolic abnormalities: Today, autism behavioral analysis includes hearing, speech and language, motor social skills. Other testing that may uncover underlying medical or metabolic abnormalities that affect behavioral is not done.13

A symptom of ASD is communication deficits, and some patients are non- verbal. Putting aside the general issue that children have difficulty describing symptoms, ASD imposes an additional handicap. Children with ASD may not reveal health problems ranging from pain from the stomach, bladder, ear, infections etc. Instead, these problems may be expressed as behavioral issues such as aggressiveness, screaming etc. Many parents experience emergency room trip for their children with ASD but disturbing behavioral problems may prevent medical personnel from searching for and treating medical issues that can disrupt behavior. Most physicians do not know that up to 20% of ASD patients have underlying undiagnosed and untreated medical illness. 14

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

Research Funds: ASD is at epidemic proportions. Schizophrenia, an orphan neurobehavioral disease similar to ASD also occurs in epidemic proportions. Comprehensive medical testing of schizophrenia patients are predicted to reveal underlying disease in 20% of patients, including inborn errors of metabolism that in some cases are treatable. Today, comprehensive medical testing is not done on schizophrenia patients. Research funds spent by the US government are minimal, and not in line with the estimated ~160 million dollars per expense of schizophrenia. Further, the scarce research resources tend to be concentrated in specific centers.

Unfortunately, the field of mental health has been rift with soothsayers that are subsequently proven wrong, but who controlled the direction of research and treatment. Concentrating research funds in the hands of a few investigators prevents innovation. The historic low funding rates for research and treatment of neurobehavioral disease limits innovation and improvements in clinical treatments. Pharmaceutical companies have moved away from developing new drugs for neurobehavioral diseases because of unusually high expense. If comprehensive testing is not done on ASD, the situation continues to evolve towards the same unsatisfying and frustrated outcome seen in schizophrenia where patients are abandoned and viewed generally as being hopelessly ill.15

A new standard of care for ASD: Today, the question is what should the standard of care be for ASD? Certainly, the underlying genetic liabilities associated with disease need to be determined, and testing is easy and relatively inexpensive. Medical abnormalities in ASD should be assessed by conventional testing, and by targeted analysis of metabolites linked to diseases. For instance, immune and inflammatory responses can be monitored with well-established laboratory tests. Some metabolite levels are measured clinically already, but others metabolite abnormalities linked to ASD need to be added to the testing repertoire.

Gene expression, epigenomic and metabolomics testing is important because these measurements represent functional test that reflect the combined affects of genetic, epigenetic and environmental factors. Gene expression and epigenomic testing is well established and quite helpful in distinguishing between different types of cancer. In ASD gene expression testing might be done using a sentinel, readily available tissue like blood, or saliva. Recent findings show that solid tumors leave detectable DNA residues in the blood.

The molecular functional studies can reveal past and present toxic exposures. Some toxic chemical can be detected directly and removed. Individuals with sensitivities can be identified and monitored. Comprehensive diagnostic testing using the new genomic approaches can reveal a wide range of metabolic conditions that warrant treatment.

Prospective: Below we provide a suggested list of assessment that should be considered in the treatment of ASD patients. This list is long and comprehensive, and is meant to serve as a starting point of discussion for a new treatment paradigm.16

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

Addendum I

Suggested Medical and Metabolic Assessments Useful for ASD

Table of Contents Introduction ...... 19 1. EVALUATION PROCESS ...... 25 a. Physical Exam and History ...... 25 i. When full metabolic evaluation should be performed ...... 25 2. DIAGNOSTIC TESTING ...... 26 a. Metabolic Evaluation ...... 26 i. Genetic Genotype/Phenotype Testing ...... 27 ii. Genetic Disease Testing (secondary autism) ...... 27 iii. IEM (Inborn Errors of Metabolism) ...... 28 b. Metabolic Biomedical Phenotype Testing ...... 29 i. Amino Acids ...... 29 ii. Cholesterol & bile acids ...... 30 iii. Creatine ...... 32 iv. Fatty Acid Metabolism Disorders ...... 31 v. Glucose transport & regulation ...... 31 vi. Hyperhomocysteinemia ...... 31 vii. Lysosomes ...... 32 viii.. Metals ...... 32 ix. Mitochondrial Dysfunction ...... 34 x. Neurotransmission ...... 35 xi. Organic acids ...... 36 xii. Pyrimidines ...... 37 xiii. Hormone Metabolism ...... 37 xiv. Epsilon-trimethyllysine hydroxylase deficiency ...... 37 xv. Urea cycle ...... 38 xvi. Vitamins/co-factors ...... 38 c. Drug Metabolism ...... 40 i. Risperidone ...... 40

ii. Risperdal ...... 40 iii. Ibuprofen ...... 40 iv. Acetaminophen ...... 40 v. Aspirin ...... 40 d. Functional Testing ...... 40 i. Chemistries ...... 40 ii. Mitochondrial Function ...... 41 iii. Organic Acids ...... 41 iv. Amino Acids ...... 44 v. Lipid Metabolism ...... 47 vi. Elemental Analysis ...... 47 vii. Vitamins and Metabolic Function ...... 48 viii. Metabolic and Essential Fatty Acids ...... 48 ix. Hormone Metabolism ...... 50 x. Gastrointestinal Function ...... 50 xi. Detoxification Function ...... 51 xii. Immune function ...... 51 e. Mast Cell ...... 56 f. Peptides...... 56 g. Neurotoxins ...... 57 i. Heavy Metals ...... 57 ii. Biotoxins ...... 57 iii. Xenobiotics (man-made environmental toxins) and Food Preservatives .... 57 3. FAMILY TESTING ...... 58 a. Mother ...... 58 b. Father ...... 58 c. Siblings ...... 58

4. LIST OF ABBREVIATIONS ...... 60

5. REFERENCES ...... 63

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

Suggested Medical and Metabolic Assessments Useful for ASD

1. Evaluation Process a. Physical Exam and History i. When full metabolic evaluation should be performed: 1. Sleep: sleep disturbance, abnormal snoring, pauses in breathing or instances of abnormally low breathing 17 18 19 2. Body weights: obesity (overweight) or underweight 3. Height, weight and head circumference 20 4. Eating/Feeding disorders: food aversion, narrow food preferences, poor appetite, vomiting, appetite loss 21 5. Repetitive infections 22 6. MIA (maternal immune activation) risk factor to ASD like behavior 23 7. Repetitive asthma event 24 8. Repetitive behaviors 25 26 9. "Allergic-like" symptoms 27 10. Gastrointestinal: Diarrhea and/or constipation, abdominal discomfort, abnormal odor of stool 28 11. Hearing: Healing loss, sound sensitivity 12. Eyes: Light sensitivity, Blurred vision, yellowing of the eyes 13. Joint and Muscle: poor muscle tone, poor eye-hand coordination, chewing/swallowing problems, pain 14. physical and motor problems 29 30 15. Abnormal body Sensitivities: Pain, tempters, touch 16. Autonomic disturbances: excessive sweating, poor circulation 17. Seizures 31 32 18. Skin: rashes, itches, bumps, color 33 19. Neonatal jaundice 34 20. Head Circumference: too large or too small 35 21. Hand and feet: cold hand and feet, soft and/or waved nail

22. Vitals: elevated heart rate, low/high blood presser, continuous low-grade fever, low or high oxygen level 23. Increase incidence of allergies: Foods, inhalant, chemical, mold 24. Urine: Abnormal odors, dark color 25. Hair: Steely, spots of gray hair 26. Rapid breathing or shortness of breath 27. Family History: Metabolic Disease in maternal, paternal, and siblings 28. Others as pediatricians feel necessary With any developmental delay is occurring.

2. Diagnostic Testing a. Metabolic Evaluation i. Genetic Genotype/Phenotype Testing 36 37: AANAT, ABCD1, ACAT1, ACE, ACP*1A, ACP1, ACSL4, ACTA, ADA, ADCY5, ADNP, ADRB2, AGT, AGT1, AGTR1, AGTR2, AHCY, ALA, ALT, ANoA, AP3B2, APO E2, APO E3, APO E4, APOC3, ARID1B, ASMT, ASMT1, ASMTL, ATD, ATXN1, BCKDK, BCOR, BDNF 38, BHMT, BHMT2, BMPR2, BRSK2, BRWD1, C2Oortf7, C4B 39, CACNA1D, CACNA1E, CADPS2, CALCR, CBS, CCP1, CD19, CD36, CD44, CD8, CDC42BPB, CDH10, CDH5, CDKL5, CETP, CHD3, CHD7, CHD8, CHDH, CNOT4, CNTFR, CNTNAP1, CNTNAP2, COL1A1, COMT 40, CPT2, CTA1, CTH, CTNNB1, CTT1, CUL3, CUL5DAT1, CYBA*8, CYBA, CYBB, CYP 1B1, CYP1A1*2A, CYP1A1*2C, CYP1A1, CYP1A2, CYP1B1, CYP21A2, CYP27B1, CYP2A6, CYP2C19*2, CYP2C19*3, CYP2C19, CYP2C9*2, CYP2C9*3, CYP2D6 41 , CYP2D6*3, CYP2E1*5A, CYP3A4*17, CYP3A4*1B, CYP3A4*3, CYP3A4, DHPR, DISC1, DLX1, DLX2, DNMT3B, DR13, DRD2, DRD342 43, DSCR1, DVR, DYRK1A, Dyrk1a, EAAT3, EHD2, eNOS, Factor I, Factor II, Factor V, FBXO10, FOXL2, FOXP1, FOXP2, FTSJ1, G6PD, G6PDH, GABAᴀ 44, GABRA1 45, GABRA2, GABRA4, GABRA5, GABRA6, GABRA6, GABRB1 46, GABRB1, GABRB3, GABRB3, GABRD, GABRD, GABRG, GABRG, GABRG1, GABRG1,GABRG2,

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

GABRP, GABRR1, GABRR2, GAD 1, GAD 2, GAD, GATA3, GCH1, GCLC, GCLM, GCPII, GDI1, GNB3, GNMT, GP3a, GP3APL(A), GPX1, GPX2, GRIP1 47, GRIP2, GRM5 48, GSS, GSTM149, GSTO1, GSTP1, GPS1, GRINL1A, HADH2, HCCS, HCE2 (CES2), HLA 50, HLA-A2, HLA-DR4, HLA-DQ8, HMX1, HOXA1, HTR1D, HDGFRP2, HDLBP, HYR1, IFN-γ, IFN-γ/α/β, IL-1 β 51, IL-2, IL-4, IL-5, IL-6 52, IL-8, IL-10, IL-12, IL-13, IL-12p40, IL-12p70, IL-13, IL-17, IL-23, ITGA4, ITGB3, KATNAL2, LPL, MAOA, MARK1, MAT, MCP-1, MDMA, MECP2, MERRF, MET 53, Mic B, MIF, mMT-I, MnSOD, MS(MTR), MS-MTRR, MSR(MS_MTRR), MT, MT1 (L/E), MT-2, MT3, MTF1, MTHFR 54 55, MTHFS, MT-ND1, MT- ND2, MT-ND3, MT-ND4, MT-ND5, MT-ND6, MTR, MTRR, MT-T52, MBD5, MDM2, MLL3, NLGN1, NOTCH3, NR4A2, NTNG1, NTNG1, NADH2, NAPQI, NAT 1, NAT 2, NDUFA1, NDUFA11, NDUFAF2, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NNMT, NOS, NPY1, NPY5, NRCAM, NTRK2, OTC, OXTR, OPRL1, PCDHB4, PDCD1, PSEN1, PTEN, PTPRK PAI-1, PCV2a, PCV2b,56 PCBD1, PEMT, PITX1, PON1, PON2, PPAR-g2, PTEN, PTS, QDPR, RELN, RFC1, RGMA, RORA 57, RPS6KA3, RUVBL1, SESN2, SETBP1, SAHH (AHCY), SAM, SCN1A, SCN1B, SELE, SELS (SEPS1), SHMT1, SLC19A3, SLC25A12, SLC40A1, SLC6A4, SNRPN, SOD2, SOD2, SOD3, STK39, SUOX, SVF1, TCN2, TCOF1, TDF, TGFBR1, TGFBR2, TGF-β 58, TGF-β1, TGIF, TH, TH-1, TH-2, TIMM8A, TM4SF2 (TSPAN7), TMEM1, TMLHE, TNF – alpha, TPH1, TPH2 59 60, UMPS, UPP1, VCX3A, VDR, ZnT1, α7nAChR 61, others.

ii. Genetic Disease Testing (secondary autism): 1. Genetic Disease with ASD like features including: a. Celiac Disease 62 b. G6PD (Glucose-6-Phosphate Dehydrogenase) Deficiencies c. Mitochondria Disease d. Pink Disease 63 e. Rett syndrome 64 f. Sickle Cell Anemia

g. Tourette syndrome 65 h. Wilson’s Disease i. others 2. Genetic Disease with secondly ASD a. Down’s Syndrome b. Fragile X Syndrome c. others iii. IEM (Inborn Errors of Metabolism): 66 1. Abnormal intestinal permeability 67 2. BH4 deficiency 68 3. Biotin responsive basal ganglia disease 4. C4B deficiency 69 5. Carnitine palmitoyltransferase II deficiency (CPT-II) 6. Cerebral folate receptor deficiency 7. Co enzyme Q10 deficiency 8. Congenital intrinsic factor deficiency 9. Dysfunction of mitochondrial β-oxidation 70 10. Fatty aldehydes Sjögren–Larsson syndrome 11. Glucose transport & regulation (GLUT1) deficiency syndrome 12. Glucose-6-Phosphate Dehydrogenase deficiency 13. Hyperinsulinism hyperammonemia syndrome 14. Hyperphenylalaninemia 71 15. Imerslund Gräsbeck syndrome 16. Immune / Autoimmune Dysfunction 72 73 74 17. Lysosomal Gaucher disease 18. Menkes disease-occipital horn syndrome 19. Mitochondria dysfunction 75 76 77 20. Mitochondrial Oxidative Phosphorylation (OXPHOS) Dysfunction 21. Neurotransmitters DHPR (Dihydropteridine reductase) deficiency

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

22. peripheral blood (PB) monocytes and specific polysaccharide antibody deficiency (SPAD) 78 23. Phenylketonuria 79 24. PHGDH deficiency: D-3-phosphoglycerate dehydrogenase (serine deficiency) 25. PSAT deficiency: phosphoserine aminotransferase deficiency (serine deficiency) 26. PSPH (phosphoserine phosphatase) deficiency (serine deficiency) 27. PTPS (6 Pyruvoyl Tetrahydropterin Synthase) deficiency (biopterin deficiency) 28. Pyridoxine dependent epilepsy 29. Smith–Lemli–Opitz Syndrome 30. SPR (Sepiapterin Reductase) deficiency 31. Thiamine-responsive encephalopathy 32. Tyrosine hydroxylase deficiency 33. Vitamins/co-factors Biotinidase deficiency

b. Metabolic Biomedical Phenotype Testing: 80 (a) OMIM#81, (b) Biochemical deficiency, (c) Gene(s). Remark: l.o.: late-onset form, AD: autosomal dominant, AR: autosomal recessive, Mt: mitochondrial i. Amino Acids: 1. Branched-chain ketoacid dehydrogenase kinase deficiency a. 614901 b. Branched-chain ketoacid dehydrogenase kinase deficiency 82 c. BCKDK (16p11.2) 2. HHH syndrome (hyperornithinemia, hyperammonemia, homocitrullinemia) a. 238970 b. Ornithine translocase c. SLC25A15 (AR) 3. l.o. Non-ketotic hyperglycinemia

a. 605899 b. Aminomethyltransferase/glycine decarboxylase/glycine cleavage system H protein c. AMT/GLDC/GCSH (AR) 4. Phenylketonuria a. 261600 b. Phenylalanine hydroxylase c. PAH (AR) 5. PHGDH deficiency (Serine deficiency) a. 601815 b. Phosphoglycerate dehydrogenase c. PHGDH (AR) 6. PSAT deficiency (Serine deficiency) a. 610992 b. Phosphoserine aminotransferase c. PSAT1 (AR) 7. PSPH deficiency (Serine deficiency) a. 614023 b. Phosphoserine phosphatase c. PSPH (AR) 8. Tyrosinemia type II a. 276600 b. Cytosolic tyrosine aminotransferase c. TAT (AR) ii. Cholesterol & bile acids 1. Cerebrotendinous xanthomatosis a. 213700 b. Sterol-27-hydroxylase c. CYP27A1 (AR) 2. Smith–Lemli–Opitz Syndrome a. 270400 b. 7-Dehydroxycholesterol reductase c. DHCR7 (AR)

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

iii. Creatine 1. AGAT deficiency a. 612718 b. Arginine: glycine amidinotransferase c. GATM (AR) 2. Creatine transporter Defect a. 300352 b. Creatine transporter c. SLC6A8 (X-linked) 3. GAMT deficiency a. 612736 b. Guanidino-acetate-N-methyltransferase c. GAMT (AR) iv. Fatty Acid Metabolism Disorders 1. Sjögren–Larsson syndrome a. 270200 b. Fatty aldehyde dehydrogenase c. ALDH3A2 (AR) v. Glucose transport & regulation 1. GLUT1 deficiency syndrome a. 606777 b. Glucose transporter blood–brain barrier c. SLC2A1 (AR) 2. Hyperinsulinism hyperammonemia syndrome a. 606762 b. Glutamate dehydrogenase superactivity c. GLUD1 (AR) vi. Hyperhomocysteinemia 1. Cobalamin C deficiency a. 277400 b. Methylmalonyl-CoA mutase and homocysteine :methyltetrahydrofolate methyltransferase

c. MMACHC (AR) 2. Cobalamin D deficiency a. 277410 b. C2ORF25 protein c. MMADHC (AR) 3. Cobalamin E deficiency a. 236270 b. Methionine synthase reductase c. MTRR (AR) 4. Cobalamin F deficiency a. 277380 b. Lysosomal cobalamin exporter c. LMBRD1 (AR) 5. Cobalamin G deficiency a. 250940 b. 5-Methyltetrahydrofolate-homocysteine, S- methyltransferase c. MTR (AR) 6. Homocystinuria a. 236200 b. Cystathatione β-synthase c. CBS (AR) 7. l.o. MTHFR deficiency a. 236250 b. Methylenetetrahydrofolate reductase deficiency c. MTHFR (AR) vii. Lysosomes 1. α-Mannosidosis a. 248500 b. α-Mannosidase c. MAN2B1 (AR) 2. Aspartylglucosaminuria a. 208400

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

b. Aspartylglucosaminidase c. AGA (AR) 3. Aspartylglucosaminuria a. 208400 b. Aspartylglucosaminidase c. AGA (AR) 4. Gaucher disease type III a. 231000 b. ß-Glucosidase c. GBA (AR) 5. Hunter syndrome (MPS II) a. 309900 b. Iduronate-2-sulfatase c. IDS (X-linked) 6. Sanfilippo syndrome B (MPS IIIb) a. 252920 b. N-acetyl-glucosaminidase c. NAGLU (AR) 7. Sanfilippo syndrome C (MPS IIIc) a. 252930 b. Acetyl-CoA glucosamine-N-acetyl transferase c. HGSNAT (AR) 8. Sanfilippo syndrome D (MPS IIId) a. 252940 b. N-acetyl-glucosamine-6-Sulfatase c. GNS (AR) 9. Sly syndrome (MPS VII) a. 253220 b. β-glucuronidase c. GUSB (AR) 10. Niemann–Pick disease type C a. 257220 b. Intracellular transport cholesterol & sphingosines

c. NPC1 NPC2 (AR) viii. Metals 1. Aceruloplasminemia a. 604290 b. Ceruloplasmin (iron homeostasis) c. CP (AR) 2. Menkes disease/Occipital horn syndrome a. 304150 b. Copper transport protein (efflux from cell) c. ATP7A (AR) 3. Wilson disease a. 277900 b. Copper transport protein (liver to bile) c. ATP7B (AR) ix. Mitochondrial Dysfunction 83 1. Co enzyme Q10 deficiency a. 607426 b. Coenzyme Q2 or mitochondrial parahydroxybenzoatepolyprenyltransferase; aprataxin; prenyl diphosphate, synthase subunit 1; prenyl diphosphate synthase subunit 2; coenzyme Q8; coenzyme Q9 c. COQ2, APTX, PDSS1,PDSS2, CABC1, COQ9 (most AR) 2. MELAS a. 540000 b. Mitochondrial energy deficiency c. MTTL1, MTTQ, MTTH, MTTK, MTTC, MTTS1, MTND1, MTND5,MTND6, MTTS2 (Mt) 3. PDH complex deficiency a. OMIM# according to each enzyme subunit deficiency:312170; 245348; 245349 b. Pyruvate dehydrogenase complex (E1α, E2, E3) c. PDHA1 (X-linked),DLAT (AR), PDHX (AR)

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

4. Mitochondrial Elongation Factor G1 a. 609060 b. Combined oxidative phosphorylation deficiency 1 c. GFM1 (3q25.32) 5. Carnitine palmitoyltransferase II a. 600649 b. palmitoyltransferase II Dificiency Infantile c. CPT2 (1p32.3) x. Neurotransmission 1. DHPR deficiency (biopterin deficiency) a. 261630 b. Dihydropteridine reductase c. QDPR (AR) 2. GTPCH1 deficiency (biopterin deficiency) a. 233910 b. GTP cyclohydrolase c. GCH1 (AR) 3. PCD deficiency (biopterin deficiency) a. 264070 b. Pterin-4α-carbinolamine dehydratase c. PCBD1 (AR) 4. PTPS deficiency (biopterin deficiency) a. 261640 b. 6-Pyruvoyltetrahydropterin synthase c. PTS (AR) 5. SPR deficiency (biopterin deficiency) a. 612716 b. Sepiapterin reductase c. SPR (AR) 6. SSADH deficiency a. 271980 b. Succinic semialdehyde dehydrogenase c. ALDH5A1 (AR)

7. Tyrosine Hydroxylase Deficiency a. 605407 b. Tyrosine Hydroxylase c. TH (AR) xi. Organic acids: 1. 3-Methylcrotonyl glycinuria a. GENE OMIM # 210200; 210210 b. 3-Methylcrotonyl CoA carboxylase (3-MCC) c. MCC1/MCC2 (AR) 2. 3-Methylglutaconic aciduria type I a. 250950 b. 3-Methylglutaconyl-CoA hydratase c. AUH (AR) 3. β-Ketothiolase deficiency a. 203750 b. Mitochondrial acetoacetyl-CoA thiolase c. ACAT1 (AR) 4. Cobalamin A deficiency a. 251100 b. MMAA protein c. MMAA (AR) 5. Cobalamin B deficiency a. 251110 b. Cob(I)alamin adenosyltransferase c. MMAB (AR) 6. Ethylmalonic encephalopathy a. 602473 b. Mitochondrial sulfur dioxygenase c. ETHE1 (AR) 7. l.o. Glutaric acidemia I a. 231670 b. Glutaryl-CoA dehydrogenase c. GCDH (AR)

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

8. Glutaric acidemia II a. 231680 b. Multiple acyl-CoA dehydrogenase c. ETFA, ETFB, ETFDH (AR) 9. HMG-CoA lyase deficiency a. 246450 b. 3-Hydroxy-3-methylglutaryl-CoA lyase c. HMGCL (AR) 10. mHMG-CoA synthase deficiency a. 605911 b. Mitochondrial 3-hydroxy-3-Methylglutaryl-CoA synthase c. HMGCS2 (AR) 11. l.o. Propionic acidemia a. 606054 b. Propionyl-CoA carboxylase c. PCCA/PCCB (AR) 12. SCOT deficiency a. 245050 b. Succinyl-CoA 3-oxoacid CoA transferase c. OXCT1 (AR) xii. Pyrimidines 1. Pyrimidine 5-nucleotidase superactivity a. 606224 b. Pyrimidine-5-nucleotidase Superactivity c. NT5C3 (AR) xiii. Hormone Metabolism 1. Smith-Lemli-Opitz syndrome (SLOS) 84 a. 270400 b. Smith-Lemli-Opitz syndrome c. DHCR7 xiv. Epsilon-trimethyllysine hydroxylase deficiency 85 a. 300872, 209850 (Autism susceptibility 1)

b. Epsilon-trimethyllysine hydroxylase deficiency c. TMLHE (Xq28) xv. Urea cycle 86 1. l.o. Argininemia a. 207800 b. Arginase 87 c. ARG1 (AR) 2. l.o. Argininosuccinic aciduria a. 207900 b. Argininosuccinate lyase c. ASL (AR) 3. l.o. CPS deficiency a. 237300 b. Carbamoyl phosphate synthetase c. CPS1 (AR) 4. Citrullinemia type II a. 605814 b. Citrin (aspartate–glutamate carrier) c. SLC25A13 5. l.o. NAGS deficiency a. 237310 b. N-acetylglutamate synthetase c. NAGS (AR) 6. l.o. OTC Deficiency a. 311250 b. Ornithine transcarbamoylase c. OTC (X-linked) 7. L.o. Citrullinemia a. 215700 b. Argininosuccinate Synthetase c. ASS1 (AR) xvi. Vitamins/co-factors 1. Biotin responsive basal ganglia disease

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

a. 607483 b. Biotin transport c. SLC19A3(AR) 2. Cerebral folate receptor-α deficiency a. 613068 b. a.o. Cerebral folate transporter c. FOLR1 (AR) 3. Congenital intrinsic factor deficiency a. 261000 b. Intrinsic factor deficiency c. GIF (AR) 4. Holocarboxylase synthetase deficiency a. 253270 b. Holocarboxylase synthetase c. HLCS (AR) 5. Imerslund Gräsbeck syndrome a. 261100 b. IF-Cbl receptor defects (cubulin/amnionless) c. CUBN & AMN (AR) 6. Molybdenum co-factor deficiency type A a. 252150 b. Sulfite oxidase & xanthine dehydrogenase & aldehyde oxidase c. MOCS1, MOCS2, (AR) 7. Pyridoxine dependent epilepsy a. 266100 b. Pyridoxine phosphate oxidase c. ALDH7A1 (AR) 8. Thiamine responsive encephalopathy a. 606152 b. Thiamine transport c. SLC19A3 (AR) 9. Biotinidase deficiency

a. 253260 b. Biotinidase c. BTD (AR) c. Drug Metabolism:88 i. Risperidone 1. rs1176713 is a SNP, also known as g.14396A>G, in the 5- hydroxytryptamine (serotonin) receptor 3A HTR3A gene ii. Risperdal 1. rs8179183 is a SNP in the leptin receptor LEPR gene. iii. Ibuprofen 1. SNP rs1057910(A), located in the cytochrome p450 CYP2C9, CYP2C9*1. rs1057910(C), CYP2C9*3, Ile359Leu or A1075C, iv. Acetaminophen 1. rs1467558(A;G) v. Aspirin 1. rs5918(C), rs3798220 d. Functional Testing: 89 90

i. Chemistries: 1. Complete Blood Count (CBC) is a standard, broad screening test used to check for disorders such as anemia, abnormal clotting, and infection. CBC is performed on the blood: WBC, RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, RDW, Platelet Count, MPV and Differential (Absolute and Percent - Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils), Ferritin 2. Basic Comprehensive Metabolic Panel (CMP) is a standard panel of tests that provides important information about the current status of the kidneys, liver, blood sugar, blood proteins, and electrolyte and acid/base balance. CMP is performed on

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

the blood. Basic Metabolic Panel: Albumin, Albumin/Globulin Ratio (calculated), Alkaline Phosphatase, ALT, AST, BUN/Creatinine Ratio (calculated), Calcium, Carbon Dioxide, Chloride, Creatinine with GFR Estimated, Globulin (calculated), Glucose, Potassium, Sodium, Total Bilirubin, Total Protein, Urea Nitrogen

ii. Mitochondrial Function 1. Acylcarnitines, blood plasma and whole blood 91 a. Carnetine b. Acryl free carnitine ratio 2. Amino Acids, plasma a. Alanine b. Alanine/Lysine ratio c. Glycine d. Proline e. Tyrosine f. Sorcosine 3. OAT, urine a. TCA intermediates b. Ethylmalonate c. 3- dimethyl glutaconate d. Dicarboxylic acid

iii. Organic Acids: 92 93 1. General Indicators of Gastrointestinal Dysbiosis: a. Citramalic Acid b. 5-Hydroxy-methyl-furoic Acid c. 3-Oxoglutaric Acid d. Furan-2,5-dicarboxylic Acid e. Furancarbonylglycine f. Tartaric Acid g. Arabinose

h. Carboxycitric Acid i. Tricarballylic Acid j. 2-Hydroxyphenylacetic Acid k. 4-Hydroxyphenylacetic Acid l. 4-Hydroxybenzoic Acid m. Hippuric Acid n. 4-Hydroxyhippuric Acid o. 3-Indoleacetic Acid p. HPHPA 94 (3-(3-hydroxyphenyl)-3-hydroxypropionic acid) q. 4-Cresol r. DHPPA(dihydroxyphenylpropionic acid)

2. Oxalate Metabolism: a. Glyceric Acid b. Glycolic Acid c. Oxalic Acid

3. Glycolytic Cycle Metabolites: a. Lactic Acid b. Pyruvic Acid c. 2-Hydroxybutyric Acid

4. Krebs Cycle Metabolites: a. Succinic Acid b. Fumaric Acid c. Malic Acid d. 2-Oxoglutaric Acid e. Aconitic Acid f. Citric Acid

5. Neurotransmitter Metabolism: a. HVA and VMA

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

b. 5-Hydroxyindoleacetic Acid c. Quinolinic Acid d. Kynurenic Acid (KYNA) e. Quinolinic Acid / Kynurenic Acid Ratio f. Quinolinic acid / 5-HIAA Ratio

6. Pyrimidine Metabolism: a. Uracil b. Thymine

7. Ketone and Fatty Acid Oxidation: a. 3-Hydroxybutyric Acid b. Acetoacetic Acid c. 4-Hydroxybutyric Acid d. Adipic Acid e. Suberic Acid f. Sebacic Acid g. Ethylmalonic Acid h. Methylsuccinic Acid i. Nutritional Markers: j. Methylmalonic Acid k. Pyridoxic Acid l. Pantothenic Acid m. Glutaric Acid n. valproic acid (Depakene), or celiac disease o. Ascorbic Acid p. 3-Hydroxy-3-methylglutaric Acid q. N-Acetylcysteine Acid r. Methylcitric Acid

8. Indicators of Detoxification: a. Pyroglutamic Acid b. Orotic Acid

c. 2-Hydroxyhippuric Acid

9. Amino Acid Metabolites: a. 2-Hydroxyisovaleric Acid b. 2-Oxoisovaleric Acid c. 3-Methyl-2-oxovaleric Acid d. 2-Hydroxyisocaproic Acid e. 2-Oxoisocaproic Acid f. Mandelic Acid g. Phenyllactic Acid h. Phenylpyruvic Acid i. Homogentisic Acid j. 4-Hydroxyphenyllactic Acid k. N-Acetylaspartic Acid l. Malonic Acid m. Methylglutaric Acid n. 3-Methylglutaconic o. 3-Hydroxyglutaric

10. Bone Metabolism: a. Phosphoric Acid iv. Amino Acids: 1. Plasma Amino Acids:95 a. 1-Methylhistidine b. 3-Methylhistidine c. Alanine d. Alpha-amino-N-butyric acid e. Alpha-aminoadipic acid f. Ammonia g. Anserine (dipeptide) h. Arginine i. Argininosuccinic acid

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

j. Asparagine k. Aspartic acid l. Beta-alanine m. Beta-aminoisobutyric acid n. Carnosine (dipeptide) o. Citrulline p. Cyst(e)ine q. Cystathionine r. Ethanolamine s. Gamma-aminobutyric acid t. Glutamic acid u. Glutamine v. Glycine w. Histidine x. Homocystine y. Hydroxyproline z. Isoleucine aa. Leucine bb. Lysine cc. Methionine dd. Ornithine ee. Phenylalanine ff. Phosphoethanolamine gg. Phosphoserine hh. Proline ii. Sarcosine jj. Serine kk. Taurine ll. Threonine mm. Tryptophan nn. Tyrosine oo. Urea pp. Valine

2. Urine Amino Acids: a. 1-Methylhistidine b. 3-Methylhistidine c. Alanine d. Alpha-amino-N-butyric acid e. Alpha-aminoadipic acid f. Ammonia g. Anserine (dipeptide) h. Arginine i. Argininosuccinic acid j. Asparagine k. Aspartic acid l. Beta-alanine m. Beta-aminoisobutyric acid n. Carnosine (dipeptide) o. Citrulline p. Cyst(e)ine q. Cystathionine r. Ethanolamine s. Gamma-aminobutyric acid t. Glutamic acid u. Glutamine v. Glycine w. Histidine x. Homocystine y. Hydroxyproline z. Isoleucine aa. Leucine bb. Lysine cc. Methionine dd. Ornithine ee. Phenylalanine

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

ff. Phosphoethanolamine gg. Phosphoserine hh. Proline ii. Sarcosine jj. Serine kk. Taurine ll. Threonine mm. Tryptophan nn. Tyrosine oo. Urea pp. Valine

v. Lipid Metabolism: 96 1. Total cholesterol: a. LDL b. HDL 2. Apolipoprotein A-I (Apo A-1) 3. Apolipoprotein B (Apo B) 4. Lipoprotein (a) (Lp (a)) 5. Homocysteine 6. Triglycerides

vi. Elemental Analysis: 97 1. RBC 98: Boron, Chromium, Calcium, Copper, Iron, Magnesium, Manganese, Molybdenum, Phosphorus, Potassium, Selenium, Vanadium, Zinc 2. WBC: Calcium (Ca), Magnesium (Mg), Copper (Cu), Zinc (Zn), Manganese (Mn), Lithium (Li), Selenium (Se), Strontium (Sr), Molybdenum (Mo) 3. Serum Elements: Calcium, Magnesium, Sodium, Potassium, Phosphorus, Iron 4. Urine: Barium, Boron, Calcium, Chromium, Cobalt, Copper, Iron, Lithium, Magnesium, Manganese, Molybdenum,

Phosphorus, Potassium, Selenium, Sodium, Strontium, Sulfur, Vanadium, Zinc, Zirconia 5. Hair 99: Calcium, Magnesium, Sodium, Potassium, Copper, Zinc, Manganese, Chromium, Vanadium, Molybdenum, Boron, Iodine, Lithium, Phosphorus, Selenium, Strontium, Sulfur, Barium, Cobalt, Iron, Germanium, Rubidium, Zirconium; Ratios: Calcium/Magnesium, Sodium/Potassium, Zinc/Copper, Zinc/Cadmium, Calcium/Phosphorus vii. Vitamins and Metabolic Function : 100 101 1. CoQ10 2. Folic Acids 102 3. Vitamin A 4. Vitamin B: a. Vitamin B1 (Thiamine) b. Vitamin B3 (Niacin) c. Vitamin B6 (Pyridoxine) d. Vitamin MeB12 (Methylcobalamin) 5. Vitamin C 6. Vitamin D: 103 a. Vitamin D, 25-OH, Total b. Vitamin D, 25-OH, D3 c. Vitamin D, 25-OH, D2 7. Vitamin E 8. Vitamin H (Biotin) 9. Vitamin K viii. Metabolic and Essential Fatty Acids: 104 105 1. Total Saturated 2. Total Monounsaturated 3. Total Polyunsaturated 4. Total Omega 3 5. Total Omega 6

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

6. Total Fatty Acids 7. Omega 3 Series: a. Alpha-Linolenic b. Eicosapentaenoic c. Docosapentaenoic d. Docosahexaenoic 8. Omega 6 Series: a. Linoleic b. Gamma-Linolenic c. Dihomo-Gamma-Linolenic d. Arachidonic e. Docosapentaenoic f. Docosatetraenoic 9. Omega 9 Series a. Eicosatrienoic 10. Monosaturated Series a. Lauroleic b. Myristoleic c. Palmitoleic d. Hexadecenoic e. Vaccenic f. Oleic g. Nervonic 11. Saturated: Caprylic, Lauric, Myristic, Palmitic, Stearic, Arachidic, Docosanoic, Tetracosanoic, Hexacosanoic 12. Branched-chain: Pristanic, Phytanic 13. Ratios: Triene-to-Tetraene 14. Behenic 15. Elaidic 16. Margaric 17. Nervonic 18. Pentadecanoic

ix. Hormone Metabolism: 1. Thyroid 106 107 108 a. Free T3 b. Free T4 c. Reverse T3 d. TSH 2. Plasma Leptin 109 3. Melatonin (N-acetyl-5-methoxytryptamine) 110 4. Growth Hormone 111 a. IGF-1 b. IGF-2 c. IGFBP-3 d. Growth hormone binding protein (GHBP) e. dehydroepiandrosterone (DHEA) f. DHEA sulphate (DHEAS)

x. Gastrointestinal Function: 112 1. Intestinal Permeability 113 2. Comprehensive Digestive Stool Analysis: 114 a. Bacteriology Culture, aerobic b. Bacteriology Culture, aerobic x 3 c. Bacteriology Culture, anaerobic d. Beneficial SCFAs e. Beta-Glucuronidase f. Bile Acids g. Calprotectin h. Cryptosporidium EIA i. Deoxycholic Acid j. Entamoeba histolytica k. Eosinophil Protein X (EPX) l. Giardia lamblia EIA m. LithoCholic Acid n. Pancreatic Elastase

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

o. Parasite Identification, Concentrate Prep p. Parasite Identification, Trichrome Stain q. Putrefactive SCFAs r. Yeast Culture s. n-Butyrate % t. pH u. Clostridium difficile Toxin A and B v. H. pylori Stool Antigen w. Shiga Toxin E. coli

xi. Detoxification Function: 115 1. Total Oxidant Level 2. Uric Acid: a. Uric Acid Urine b. Uric Acid Blood 3. Glutathione 116 117 118: a. Total Glutathione b. Reduced Glutathione c. Peroxidase Glutathione (GPX) 4. Superoxide Dismutase (SOD) 5. Cysteine/Sulfate Ratio 6. Cysteine/Cystine Ratio 7. Melatonin 8. Glucose-6-Phosphate Dehydrogenase (G6PD) 119 9. Metallothionein antibodies (anti-MT) a. antinuclear antibodies against nucleolar antigens (ANoA) b. antilaminin antibodies (ALA) c. antibodies to metallothionein protein (anti-MT)

xii. Immune function 120 1. Immune Deficiencies: 121 a. Immunoglobulins IgA, IgM, IgE, IgG

b. IgG Subclasses 1, 2, 3 and 4 c. Monoclonal antibodies 122 d. B Lymphocyte Antigen D8/17 123 2. AutoImmune 124 a. plasma progranulin 125 b. Serum Neurokinin A / Anti-ribosomal P protein antibodies 126 3. Allergies 127 a. Food Allergies 128 i. IgE Antibodies: Almond, Adzuki Bean, Almond, Apple, Apricot, Asparagus, Avocado, Banana, Barley, Beef, Beet, Blueberry, Broccoli, Buckwheat, Cabbage, Cane Sugar, Carrot, Casein, Cashews, Celery, Cheese, Chicken, Coconut, Cod Fish, Cocoa, Coffee, Corn, Crab, Cranberry, Eggplant, Egg White, Egg Yolk, Flax, Garbanzo Bean, Garlic, Gluten, Goat’s Milk Cheese, Grape, Grapefruit, Green Bean, Green Pepper, Halibut, Hazelnut, Honey, Kidney Bean, Lamb, Lemon, Lentil, Lettuce, Lima Bean, Lobster, Mango, Milk, Millet, Mushroom, Oat, Onion, Orange, Papaya, Pea, Peach, Peanut, Pear, Pecan, Pineapple, Pinto Bean, Pistachio, Plum, Pork, Potato, Pumpkin, Radish, Raisin, Rice, Rye, Salmon, Sardine, Sesame, Shrimp, Soybean, Spinach, Strawberry, Sunflower, Sweet Potato, Tomato, Turkey, Tuna, Walnut, Watermelon, Wheat, Whey, Yogurt, Yeast (Bakers), Yeast (Brewers), Zucchini ii. IgG Food Antibodies: Abalone, Almond, Apple, Apricot, Asparagus, Avocado, Baker’s Yeast (Saccharomyces cerevisiae), Bamboo Shoot, Banana, Barley, Beef, Black Pepper, Bonito,

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Buckwheat, Burdock (Gobo), Beet, Blueberry, Brewer’s Yeast (Saccharomyces cerevisiae), Broccoli, Buckwheat, Cherry, Chestnut, Chicken, Clam, Cocoa, Coconut, Coffee, Corn, Crab, Cucumber, Curry Powder, Cabbage, Candida albicans, Cane Sugar, Carrot, Cashews, Casein, Celery, Cheese, Chicken, Cod fish, Cranberry, Duck, Eggplant, Egg White, Egg Yolk, Flax, Garbanzo Beans, Garlic, Ginger, Gliadin, Goat’s Milk Cheese, Grape, Grapefruit, Green Bean, Green Pepper, Green Tea, Halibut, Hazelnut, Honey, Jack Mackerel, Kiwi, Kombu (Kelp), Kidney Bean, Lamb, Lemon, Lentil, Lettuce, Laver (Nori), Lotus Root, Lima bean, Lobster, Mackerel, Mango, Melon, Miso, Milk, Millet, Mozzarella Cheese, Mushroom, Mushroom- Enoki, Mushroom-Shiitake, Mustard, Oat, Olive, Onion, Oolong Tea, Orange, Oyster, Pacific Saury, Papaya, Pea, Peach, Peanut, Pear, Pecan, Pineapple, Pinto Bean, Pistachio, Plum (Prune), Pork, Potato, Pumpkin, Radish, Radish-Daikon, Red Pepper, Rice, Rye, Salmon, Sardine, Seaweed (Wakame), Sesame, Shrimp, Sorghum, Soybean, Spinach, Squid, Strawberry, Sunflower, Sweet Potato, Tomato, Tuna, Vanilla Bean, Turkey, Wheat Gluten, Walnut, Watermelon, Wheat, Whey, Yogurt

iii. Spices IgG: Allspice – IgG, Basil – IgG, Bay leaf – IgG, Black Pepper – IgG, Cayenne Pepper – IgG, Cinnamon – IgG, Cloves – IgG, Cumin – IgG, Curry – IgG, Dill – IgG, Fennel seed – IgG, Ginger – IgG, Horseradish – IgG, Marjoram –

IgG, Mustard – IgG, Nutmeg – IgG, Oregano – IgG, Paprika – IgG, Parsley – IgG, Peppermint – IgG, Rosemary – IgG, Sage – IgG, Thyme – IgG, Total IgG, Vanilla – IgG

iv. IgE Inhalant Allergies: Alder Tree- IgE, Australian Pine Tree- IgE, Bahia Grass- IgE, Bermuda Grass- IgE, Birch Tree- IgE, Brome Grass- IgE, Canary Grass- IgE, Cat dander- IgE, Cocklebur- IgE, Cockroach- IgE, Common Ragweed- IgE, Cottonwood Tree- IgE, Cultivated Oat Grass- IgE, Dandelion- IgE, Dog dander- IgE, Elm Tree- IgE, English Plantain- IgE, Eucalyptus Tree- IgE, Giant Ragweed- IgE, Johnson Grass- IgE, June Grass (Kentucky Blue)- IgE, Lamb's quarters- IgE, Maple Tree- IgE, Mesquite Tree- IgE, Mite Generic- IgE, Mold Generic- IgE, Mountain Cedar Tree- IgE, Nettle- IgE, Oak Tree- IgE, Olive Tree- IgE, Orchard Grass- IgE, Pecan Tree- IgE, Perennial Rye Grass- IgE, Red Top- IgE, Rough Marsh Elder- IgE, Rough Pigweed- IgE, Russian Thistle- IgE, Scale- IgE, Sweet Vernal Grass- IgE, Timothy Grass- IgE, Total IgE, Walnut Tree- IgE, Western Ragweed- IgE, White Mulberry Tree- IgE

4. Inflammation a. Acute-phase reaction (APR) markers i. C-Reactive Protein (CRP) ii. S100 protein

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Addendum I - Suggested Medical and Metabolic Assessments Useful for ASD

5. Opiate Receptors: Gluten / Casein Peptides 129 a. Gliadorphin (peptide from wheat) b. Casomorphin (peptide from dairy)

6. Infection (blood test): 130 a. Virus:131 i. Polyomaviruses 1. BK virus (BKV) 132 2. Cytomegalovirus (CMV) 3. Epstein Barr virus (EBV) 4. Human Herpes Virus-6 (HHV-6) 5. Human Herpes Virus-7 (HHV-7) 6. JC virus (JCV) 7. Simian virus 40 (SV40) 133 8. Varicella-Zoster virus (ZVZ) 9. PCV2b 10. Herpes simplex virus type 1 (HSV-1) 11. Herpes simplex virus type 2 (HSV-2) ii. Retroviruses iii. Rubeola/Measles Edmonston Strain Inactivated Cell Extract

b. Bacteria: i. Mycoplasma pneumonia ii. Chlamydia pneumonia 134 iii. Toxoplasma gondii iv. Group A β-hemolytic Streptococci (GABHS) 135 1. DNase antibodies in serum (ADB) 2. Antistreptolysin O titer (ASO) c. Vector-Born:136 i. Borrelia burgdorferi c6 peptide antibodies by ELISA ii. Lyme disease Western blot

d. Mycology: i. Aspergillus fumigatus

7. Plasma Chemokines 137 e. Mast Cell 138 i. Corticotropin-releasing hormone (CRH) ii. mitochondrial DNA, IgE/anti-IgE iii. 24 hours to measure vascular endothelial growth factor (VEGF) release by ELISA or for 6 hours or quantitative PCR f. Peptides i. Neuropeptides / Brain Inflammation (13 amino acid neuropeptide) 1. Neurotensin (NT) 139 ii. Beta-amyloid peptide iii. Beta-Amyloid Precursor Protein 140 1. Tau, 2. Tubulin 3. Synuclein 4. Amyloid Precursor Protein (APP) 141 5. Apo E 6. AD/PD related Proteins 7. Calmodulin 8. (sAPP-α ELISA)142 iv. Monoclonal Antibodies 1. Beta Amyloid 2. Tubulin 3. Tau and Related proteins

g. Neurotoxins 143

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i. Heavy Metals: 144 145 146 1. Porphyrins: 147 a. Coproporphyrin I and III (CP) b. Heptacarboxy (7-CP) c. Hexacarboxy (6-CP) d. Pentacarboxy (5-CP) e. Precoproporphyrin (PreCP) f. Uroporphyrins (UP) 2. Hair 148 149: Aluminum 150, Antimony, Arsenic, Beryllium, Bismuth, Cadmium, Lead, Mercury, Platinum, Thallium, Thorium, Uranium, Nickel, Silver, Tin, Titanium 3. RBC: Arsenic, Cadmium, Lead, Mercury, Thallium 4. WBC: Arsenic (As), Barium (Ba), Cadmium (Cd), Cobalt (Co), Lead (Pb), Mercury (Hg), Nickel (Ni), Platinum (Pt), Silver (Ag), Thallium (Tl), Uranium (U) 5. Urine 151: Aluminum, Antimony, Arsenic, Beryllium, Bismuth, Cadmium, Lead, Mercury, Nickel, Platinum, Thallium, Thorium, Tin, Tungsten, Uranium 6. Spot Urine: Urine protein to creatinine ratio (PrCP) 152 7. Fecal: Antimony, Arsenic, Beryllium, Bismuth, Cadmium, Copper, Lead, Mercury, Nickel, Platinum, Thallium, Tungsten, Uranium

ii. Biotoxins: Biotoxins are poisons that come from plants or animals. mold, black mold, tetanus toxin, botulinum toxin, ascaridin (from intestinal parasites), unspecified toxins from streptococci, staphylococci, lyme disease 153, clamydia, tuberculosis, fungal toxins and toxins produced by viruses

iii. Xenobiotics (man-made environmental toxins) and Food Preservatives: 154

Thimerosal155, Bisphenol A (BPA), Oxybenzone, Parabens, Phthalates, Butylated Hydroxyanisole (BHA), Perfluorooctanoic Acid (PFOA), Perchlorate, Decabromodiphenyl Ether (DECA), Asbestos, The Hazards Lurking at Home, Oxybenzone, Fluoride, Parabens, Phthalate, Butylated Hydroxyanisole (BHA), Perfluorooctanoic Acid (PFOA), Perchlorate, Decabromodiphenyl Ether (DECA), Asbestos, The Hazards Lurking at Home, dioxin, phthalates, formaldehyde, insecticides, wood preservatives, Polychlorinated biphenyl (PCB)156, Polybrominated diphenyl ethers (PBDEs) 157, Pesticide 158, aspartame, caramel colorings, fluoride, methyl-and propyl-paraben, etc.

3. Family Testing a. Mother 159 i. Metabolism Genes: MTHFR, COMT, MTRR, BHMT, FOLR2, CBS, TCN2, etc. 160 ii. Genotype and Phenotype Genetic Tests: 161 iii. Neuroinflammation 162 iv. Drug Metabolism (Pregnancy) 163 v. Comprehensive Metabolic Analysis 164 1. Organic Acids 165 vi. Heavy Metals: 1. Mercury 166 2. Lead 167 vii. Essential Minerals 168 viii. Vitamins 1. Folic Acids 169 2. Vitamin D 170 171 ix. Vitamins and Minerals (Prenatal Vitamins) 172 x. Xenobiotics173 174

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xi. Infection 175 176 xii. Antioxidants 1. Glutathione 177

b. Father i. Genotype and Phenotype genetic testing 178 179 ii. Infection 180

c. Sibling(s) 181 i. Genotype and Phenotype genetic testing 182

4. List of abbreviations 4-AA: 4-aminoantipyrine 5-CP:. Pentacarboxy 5-HT: serotonin 5'-IMP: 5'-inosine monophosphate 5'-NT: 5'-nucleotidase 6-CP: Hexacarboxy 7-CP: Heptacarboxy AD Alzheimer’s disease ADA: adenosine deaminase Ag: Silver APO A-1 Apolipoprotein A-I APO B Apolipoprotein B As: Arsenic ASD Autism spectrum disorders Ba: Barium BBB Blood brain barrier BHA: Butylated Hydroxyanisole Perfluorooctanoic Acid BKV: BK virus BPA: Thimerosal , Bisphenol A Ca: Calcium Cd: Cadmium CMV: Cytomegalovirus CNS Central nervous system Co: Cobalt CP: Coproporphyrin CPS-1: carbamoyl phosphate synthetase-1 Cr: creatine CRP: C-reactive protein Cu: Copper DECA: Decabromodiphenyl Ether EBV: Epstein Barr virus EHSPT: N-ethyl N-(2-hydroxy-3-sulfopropyl)-3-methylaniline

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FAP functional abdominal pain GABA: gamma-aminobutyric acid GDH: glutamate dehydrogenase GERD gastroesophageal reflux disease GFCF gluten-free, casein-free Gln: glutamine Glu: glutamate GRADE Grading of Recommendations Assessment, Development, and Evaluation GS: glutamate synthase GSH: Glutathione H2O2: hydrogen peroxide Hg: Mercury HHV-6: Human Herpes Virus-6 HHV-7: Human Herpes Virus-7 HPLC: high-performance liquid chromatography HSV-1: Herpes simplex virus type 1 HSV-2: Herpes simplex virus type 2 IBS irritable bowel syndrome IDO Indoleamine 2,3-dioxygenase Ig immunoglobulin IL Interleukin JCV: John Cunningham Virus Li: Lithium Lp (a) Lipoprotein (a) MeB12: Methylcobalamin Mg: Magnesium Mn: Manganese Mo: Molybdenum MPTP: mitochondrial permeability transition pore mtCK: mitochondrial creatine kinase Ni: Nickel NLH nodular lymphoid hyperplasia

NMDA N-Methyl-D-aspartic acid NO: nitric oxide NOS not otherwise specified OTC: Ornithine Transcarbamylase PANDAS Pediatric autoimmune diseases associated with strep Pb: Lead PBDEs: Polybrominated Diphenyl Ethers PCB: Polychlorinated Biphenyl PCr; phosphorylcreatine PCV2a: Porcine circovirus genotype 2a PCV2b: Porcine circovirus genotype 2b PDD pervasive developmental disorder PFOA: Perfluorooctanoic Acid PLS LYME patients with prior treatment and persistent symptoms PNP: Purine Nucleoside Phosphorylase POD: peroxidase PreCP: Precoproporphyrin Pt: Platinum Redox Reduction-Oxidation Se: Selenium SIV: Simian Immunodeficiency Virus Sr: Strontium SV40: Simian Virus 40 Tl: Thallium TNF Tumor necrosis factor U: Uranium UP: Uroporphyrins XOD: xanthine oxidase Zn: Zinc ZVZ: Varicella-Zoster virus α-KG: α-ketoglutaric acid

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5. References:

1 Olivié H (2012) The medical care of children with autism: Eur J Pediatr. 2012 May;171(5):741-9. doi: 10.1007/s00431-011-1669-1. Epub 2012 Jan 18

2 Herbert MR.(2010) Contributions of the environment and environmentally vulnerable physiology to autism spectrum disorders.: Curr Opin Neurol. 2010 Apr;23(2):103-10. doi: 10.1097/WCO.0b013e328336a01f.

3 Hu VW. (2012) Subphenotype-dependent disease markers for diagnosis and personalized treatment of autism spectrum disorders: Dis Markers. 2012 Aug 31 PMID: 22960334

4 Clara D.M.V. Karnebeek, Sylvia Stockler: (2011) Treatable inborn errors of metabolism causing intellectual disability: A systematic literature review Molecular Genetics and Metabolism: Molecular Genetics and Metabolism 105 (2012) 368–381

5 Michael L. Ganz, MS, PhD (2007) The Lifetime Distribution of the Incremental Societal Costs of Autism: Arch Pediatr Adolesc Med. 2007;161(4):343-349

6 Paul S. Carbone Diane D. Behl Virgina Azor Nancy A. Murphy (2010) The Medical Home for Children with Autism Spectrum Disorders: Parent and Pediatrician Perspectives: J Autism Dev Disord (2010) 40:317–324.

7 AMERICAN ACADEMY OF PEDIATRICS: POLICY STATEMENT: Organizational Principles to Guide and Define the Child Health Care System and/or Improve the Health of All Children: (2002) Medical Home Initiatives for Children With Special Needs Project Advisory Committee: Pediatrics 2002;110;184-186

8 Susan Parish, Kathleen Thomas, Roderick Rose, Mona Kilany, and Robert McConville (2012) State Insurance Parity Legislation for Autism Services and Family Financial Burden. Intellectual and Developmental Disabilities: June 2012, Vol. 50, No. 3, pp. 190-198.

9 Autism Society estimates based on UK study by Jarbrink K, Knapp M, (2001) London School of Economics: "The economic impact on autism in Britain," Autism, 5 (1): 7-22.

10 Press Releases (2006) Autism Has High Costs to U.S. Society: Harvard University School of Public Health, Department of Society, Human Development, and Health

11 Michael L. Ganz, MS, PhD (2007) The Lifetime Distribution of the Incremental Societal Costs of Autism: Arch Pediatr Adolesc Med. 2007;161:343-349

12 Michael L. Ganz, MS, PhD (2008) The Costs of Autism Technical Appendix: Harvard University School of Public Health, Department of Society, Human Development, and Health

13 C. Gillberg and, E. Billstedt (2000) Autism and Asperger syndrome: coexistence with other clinical disorders: Acta Psychiatrica Scandinavica, Volume 102, Issue 5, pages 321–330, November 2000

14 Clara D.M.V. Karnebeek, Sylvia Stockler: (2011) Treatable inborn errors of metabolism causing intellectual disability: A systematic literature review Molecular Genetics and Metabolism: Molecular Genetics and Metabolism 105 (2012) 368–381

15 Louise W. Kao, Gregory P. Moore (1999) The Violent Patient: Clinical Management, Use Of Physical And Chemical Restraints, And Medicolegal Concerns: Emergency Medicine Practice, November 1999 Volume 1, Number 6.

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16 Louisa Flintoft (2009) Complex disease: Autism clues from genome-wide studies: Nature Reviews Genetics 10, 346-347

17 Hollway JA, Aman MG.(2011) Sleep correlates of pervasive developmental disorders: a review of the literature: Res Dev Disabil. 2011 Sep-Oct;32(5):1399-421. Epub 2011 May 14.

18 Andersen IM, Kaczmarska J, McGrew SG, Malow BA. (2008) Melatonin for insomnia in children with autism spectrum disorders: J Child Neurol. 2008 May;23(5):482-5. Epub 2008 Jan 8.

19 Juthamas Wirojanan, M.D. Sebastien Jacquemont, M.D. Rafael Diaz, Ph.D.,1,4 Susan Bacalman, L.C.S.W.,Thomas F. Anders, M.D.,Randi J. Hagerman, M.D.,and Beth L. Goodlin-Jones, Ph.D.: (2009) The Efficacy of Melatonin for Sleep Problems in Children with Autism, Fragile X Syndrome, or Autism and Fragile X Syndrome: J Clin Sleep Med. 2009 April 15; 5(2): 145–150.

20 James L. Mills, Mary L. Hediger, Cynthia A. Molloy, George P. Chrousos, Patricia Manning-Courtney, Kai F. Yu, Mark Brasington and Lucinda J. England (2007) Elevated levels of growth-related hormones in autism and autism spectrum disorder: Clinical Endocrinology (2007) 67, 230–237

21 Tang B, Piazza CC, Dolezal D, Stein MT (2012) Severe feeding disorder and malnutrition in 2 children with autism: J Dev Behav Pediatr. 2011 Apr;32(3):264-7.

22 Angelidou A, Asadi S, Alysandratos KD, Karagkouni A, Kourembanas S, Theoharides TC. (2012) Perinatal stress, brain

inflammation and risk of autism-Review and proposal: BMC Pediatr. 2012 Jul 2;12:89.

23 Hsiao EY, McBride SW, Chow J, Mazmanian SK, Patterson PH (2012) Modeling an autism risk factor in mice leads to permanent immune dysregulation: Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12776-81

24 Volkmar FR, State M, Klin A.(2009) Autism and autism spectrum disorders: diagnostic issues for the coming decade: J Child Psychol Psychiatry. 2009 Jan;50(1-2):108-15

25 Eric Hollander, M.D.; Gina DelGiudice-Asch, M.D.; Lorraine Simon, M.A.; James Schmeidler, Ph.D.; Charles Cartwright, M.D.; Concetta M. DeCaria, Ph.D.; Jee Kwon, B.A.; Charlotte Cunningham-Rundles, M.D., Ph.D.; Floresta Chapman, R.N.; John B. Zabriskie, M.D. (1999) B Lymphocyte Antigen D8/17 and Repetitive Behaviors in Autism: Am J Psychiatry 1999;156:317-320.

26 TANYA MURPHY, M.D., AND WAYNE GOODMAN, M.D. (2002) Genetics of Childhood Disorders: XXXIV. Autoimmune Disorders, Part 7: D8/17 Reactivity as an Immunological Marker of Susceptibility to Neuropsychiatric Disorders: J. AM. ACAD. CHILD ADOLESC. PSYCHIATRY, 41:1, JANUARY 2002

27 Theoharides TC, Angelidou A, Alysandratos KD, Zhang B, Asadi S, Francis K, Toniato E, Kalogeromitros D. (2012) Mast cell activation and autism: Biochim Biophys Acta. 2012 Jan;1822(1):34-41.

28 Harris C, Card B.(2012) A pilot study to evaluate nutritional influences on gastrointestinal symptoms and behavior patterns in children with Autism Spectrum Disorder: Complement Ther Med. 2012 Dec;20(6):437-40

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29 Matson ML, Matson JL, Beighley JS.(2011) Comorbidity of physical and motor problems in children with autism: Res Dev Disabil. 2011 Nov-Dec;32(6):2304-8. Epub 2011 Sep 3.

30 Zingerevich C, Greiss-Hess L, Lemons-Chitwood K, Harris SW, Hessl D, Cook K, Hagerman RJ. (2009) Motor abilities of children diagnosed with fragile X syndrome with and without autism: J Intellect Disabil Res. 2009 Jan;53(1):11-8. Epub 2008 Sep 3.

31 Theoharis C Theoharides and Bodi Zhang (2011) Neuro- inflammation, blood-brain barrier, seizures and autism: J Neuroinflammation. 2011; 8: 168

32 Gabriele Tripi, Sylvie Roux, Tatiana Canziani, Frédérique Bonnet Brilhault, Catherine Barthélémy, Fabio Canziani (2007) Minor physical anomalies in children with autism spectrum disorder: Early Hum Dev, doi:10.1016/j.earlhumdev.2007.04.005

33 Jacobine E Buizer-Voskamp, Lude Franke, Wouter G Staal, Emma van Daalen,4Chantal Kemner, Roel A Ophoff, Jacob AS Vorstman, Herman van Engeland, and Cisca Wijmenga (2010) Systematic genotype– phenotype analysis of autism susceptibility loci implicates additional symptoms to co-occur with autism: Eur J Hum Genet. 2010 May; 18(5): 588–595

34 Cappellini MD, Fiorelli G. (2009) Glucose-6-phosphate dehydrogenase deficiency: Lancet. 2008 Jan 5;371(9606):64-74.

35 Chiu, Sufen MD, PhD; Wegelin, Jacob A. PhD; Blank, Jeremy BS; Jenkins, Megan BS; Day, Josh BA; Hessl, David PhD; Tassone, Flora PhD; Hagerman, Randi MD (2007) Early Acceleration of Head Circumference

in Children with Fragile X Syndrome and Autism: Journal of Developmental & Behavioral Pediatrics: February 2007 - Volume 28 - Issue 1 - pp 31-35

36 Dennis K. Kinney, Ph.D.,Daniel H. Barc, Bogdan Chayka, M.D., Siena Napoleon, and Kerim M. Munir, M.D., M.P.H., D. Sc.: (2010) Environmental Risk Factors for Autism: Do They Help Cause De Novo Genetic Mutations That Contribute to the Disorder?: Med Hypotheses. 2010 January; 74(1): 102–106

37 Brian J. O’Roak, Laura Vives, Santhosh Girirajan, Emre Karakoc, Nik Krumm, Bradley P. Coe, Roie Levy, Arthur Ko, Choli Lee, Joshua D. Smith, Emily H. Turner, Ian B. Stanaway, Benjamin Vernot, Maika Malig, Carl Baker,1 Beau Reilly, Joshua M. Akey, Elhanan Borenstein, Mark J. Rieder, Deborah A. Nickerson, Raphael Bernier, Jay Shendure, and Evan E. Eichler (2012) Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations: Nature. 2012 April 4; 485(7397): 246–250.

38 Balmiki Ray, Justin M. Long, Deborah K. Sokol, and Debomoy K. Lahiri (2011) Increased Secreted Amyloid Precursor Protein-α (sAPPα) in Severe Autism: Proposal of a Specific, Anabolic Pathway and Putative Biomarker: PLoS One. 2011; 6(6): e20405.

39 Thayne L Sweeten, Daniel W Odell, J Dennis Odell, and Anthony R Torres (2008) C4B null alleles are not associated with genetic polymorphisms in the adjacent gene CYP21A2 in autism: BMC Med Genet. 2008; 9: 1.

40 Gadow KD, Roohi J, DeVincent CJ, Kirsch S, Hatchwell E. (2009) Association of COMT (Val158Met) and BDNF (Val66Met) gene polymorphisms with anxiety, ADHD and tics in children with autism

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spectrum disorder.: J Autism Dev Disord. 2009 Nov;39(11):1542-51. Epub 2009 Jul 7.

41 C T Correia, J P Almeida, P E Santos, A F Sequeira, C E Marques, T S Miguel, R L Abreu, G G Oliveira and A M Vicente: (2010) Pharmacogenetics of risperidone therapy in autism: association analysis of eight candidate genes with drug efficacy and adverse drug reactions: The Pharmacogenomics Journal: 10, 418-430

42 Krom M, Staal WG, Ophoff RA, Hendriks J, Buitelaar J, Franke B, de Jonge MV, Bolton P, Collier D, Curran S, van Engeland H, van Ree JM: (2008) A common variant in DRD3 receptor is associated with autism spectrum disorder: Biol Psychiatry. 2009 Apr 1;65(7):625-30

43 Wouter G. Staal, Mariken de Krom, and Maretha V. de Jonge (2012) Brief Report: The Dopamine-3-Receptor Gene (DRD3) is Associated with Specific Repetitive Behavior in Autism Spectrum Disorder (ASD): J Autism Dev Disord: 42(5): 885–888. PMCID: PMC3324694

44 S. Hossein Fatemi, Teri J. Reutiman, Timothy D. Folsom and Paul D. Thuras: (2009) GABAA receptor downregulation in brains of subjects with autism: J Autism Dev Disord: 39(2): 223–230

45 D. Q. Ma,1 P. L. Whitehead, M. M. Menold, E. R. Martin, A. E. Ashley- Koch, H. Mei, M. D. Ritchie, G. R. DeLong, R. K. Abramson, H. H. Wright, M. L. Cuccaro, J. P. Hussman, J. R. Gilbert, and M. A. Pericak-Vance: (2005) Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism: Am J Hum Genet. 77(3): 377– 388

46 Fatemi SH, Folsom TD, Reutiman TJ, Thuras PD: (2009) Expression of GABA(B) receptors is altered in brains of subjects with autism: 8(1):64-9

47 Rebeca Mejias, Abby Adamczyk, Victor Anggono, Tejasvi Niranjan, Gareth M. Thomas, Kamal Sharma, Cindy Skinner, Charles E. Schwartz,e Roger E. Stevenson,eM. Daniele Fallin, Walter Kaufmann, Mikhail Pletnikov, David Valle, Richard L. Huganir, and Tao Wanga: (2011) Gain- of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism: Proc Natl Acad Sci U S A. 2011 March 22; 108(12): 4920–4925

48 Mejias R, Adamczyk A, Anggono V, Niranjan T, Thomas GM, Sharma K, Skinner C, Schwartz CE, Stevenson RE, Fallin MD, Kaufmann W, Pletnikov M, Valle D, Huganir RL, Wang T. (2011) Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism.: Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4920-5. Epub 2011 Mar 7.

49 Steven Buyske, Tanishia A Williams, Audrey E Mars, Edward S Stenroos, Sue X Ming, Rong Wang, Madhura Sreenath, Marivic F Factura, Chitra Reddy, George H Lambert and William G Johnson (2006) Analysis of case-parent trios at a locus with a deletion allele: association of GSTM1 with autism: BMC Genetics 2006, 7:8 doi:10.1186/1471-2156-7- 8

50 Torres AR, Westover JB, Rosenspire AJ. (2012) HLA Immune Function Genes in Autism: Autism Res Treat. 2012;2012:959073. Epub 2012 Feb 15.

51 Paul Ashwood,1,6,* Paula Krakowiak,2 Irva Hertz-Picciotto,2,6 Robin Hansen,3,6 Isaac Pessah,4,6 and Judy Van de Water5,6 (2010)

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Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome: Brain Behav Immun. 2011 January; 25(1): 40–45.

52 Theoharis C Theoharides and Bodi Zhang: (2011) Neuro- inflammation, blood-brain barrier, seizures and autism: J Neuroinflammation. 2011; 8: 168.

53 Daniel B. Campbell,James S. Sutcliffe,Philip J. Ebert, Roberto Militerni, Carmela Bravaccio, Simona Trillo,Maurizio Elia,Cindy Schneider,Raun Melmed, Roberto Sacco, Antonio M. Persico,and Pat Levit (2006) A genetic variant that disrupts MET transcription is associated with autism: Proc Natl Acad Sci U S A. 2006 November 7; 103(45): 16834–16839.

54 Sergiu P. Paşca, Eleonora Dronca, Tamás Kaucsár, Elena C. Crǎciun, Emõke Endreffy, Beatrix K. Ferencz, Felicia Iftene, Ileana Benga, Rodica Cornean, Ruma Banerjee, Maria Dronca: (2008) One carbon metabolism disturbances and the C677T MTHFR gene polymorphism in children with autism spectrum disorders: Article first published online: DOI: 10.1111/j.1582-4934.2008.00463

55 S. Jill James, Stepan Melnyk, Stefanie Jernigan, Mario A. Cleves, Charles H. Halsted, Donna H. Wong, Paul Cutler, Kenneth Bock, Marvin Boris, J. Jeffrey Bradstreet, Sidney M. Baker, and David W. Gaylor: (2006) Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism: Am J Med Genet B Neuropsychiatr Genet: 141B(8): 947–956. doi: 10.1002/ajmg.b.30366

56 Saha D, Huang L, Bussalleu E, Lefebvre DJ, Fort M, Van Doorsselaere J, Nauwynck HJ. (2012) Antigenic subtyping and epitopes'

competition analysis of porcine circovirus type 2 using monoclonal antibodies: Vet Microbiol. 2012 May 25;157(1-2):13-22.

57 AnhThu Nguyen, Tibor A. Rauch, Gerd P. Pfeifer,and Valerie W. Hu (2010) Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain: FASEB J. 2010 August; 24(8): 3036–3051.

58 Paula Goines1,2,3 and Judy Van de Water (2010) The Immune System’s Role in the Biology of Autism: Curr Opin Neurol. 2010 April; 23(2): 111–117.

59 Yang SY, Yoo HJ, Cho IH, Park M, Kim SA.(2012) Association with tryptophan hydroxylase 2 gene polymorphisms and autism spectrum disorders in Korean families:73(4):333-6. doi: 10.1016/j.neures.2012.05.012

60 Egawa J, Watanabe Y, Nunokawa A, Endo T, Kaneko N, Tamura R, Sugiyama T, Someya T. (2012) A detailed association analysis between the tryptophan hydroxylase 2 (TPH2) gene and autism spectrum disorders in a Japanese population: Psychiatry Res. 30;196(2-3):320-2

61 Palma E, Conti L, Roseti C, Limatola C. (2012) Novel approaches to study the involvement of α7-nAChR in human diseases. Curr Drug Targets: 13(5):579-86

61 Stephen J. Genuis, MD, FRCSC, DABOG, DABEM, FAAEM, and Thomas P. Bouchard, BSc: (2010) Celiac Disease Presenting as Autism: Journal of Child Neurology Volume 25 Number 1 January 2010 114-119

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63 Shandley K, Austin DW (2011) Ancestry of pink disease (infantile acrodynia) identified as a risk factor for autism spectrum disorders : J Toxicol Environ Health A. 2011;74(18):1185-94.

64 LaSalle JM, Yasui DH. (2009) Evolving role of MeCP2 in Rett syndrome and autism: Epigenomics. 2009 Oct;1(1):119-30.

65 Clarke RA, Lee S, Eapen V. (2012) Pathogenetic model for Tourette syndrome delineates overlap with related neurodevelopmental disorders including Autism: Transl Psychiatry. 2012 Sep 4;2:e158. doi: 10.1038/tp.2012.75.

66 Clara D.M.V. Karnebeek, Sylvia Stockler: (2011) Treatable inborn errors of metabolism causing intellectual disability: A systematic literature review Molecular Genetics and Metabolism: Molecular Genetics and Metabolism 105 (2012) 368–381

67 P D'Eufemia, M Celli, R Finocchiaro, L Pacifico, L Viozzi, M Zaccagnini, E Cardi, O Giardini: (2008) Abnormal intestinal permeability in children with autism: DOI: 10.1111/j.1651- 2227.1996.tb14220.x

68 P D'Eufemia, M Celli, R Finocchiaro, L Pacifico, L Viozzi, M Zaccagnini, E Cardi, O Giardini: (2008) Abnormal intestinal permeability in children with autism: DOI: 10.1111/j.1651- 2227.1996.tb14220.x

69 Anthony R. Torres, Jonna B. Westover, and Allen J. Rosenspire (2012) HLA Immune Function Genes in Autism: Autism Res Treat. 2012; 2012: 959073

70 Tonya Clark-Taylor, Benjamin E Clark-Taylor: (2004) Is autism a disorder of fatty acid metabolism? Possible dysfunction of mitochondrial β-oxidation by long chain acyl-CoA dehydrogenase: Medical Hypotheses Volume 62, Issue 6, June 2004, Pages 970–975

71 Barbara Manzi, MD, Anna Livia Loizzo, MD, Grazia Giana, MD, Paolo Curatolo, MD (2008) Autism and Metabolic Diseases: J Child Neurol March 2008 vol. 23 no. 3 307-314

72 Torres AR, Sweeten TL, Cutler A, Bedke BJ, Fillmore M, Stubbs EG, Odell D.(2006) The association and linkage of the HLA-A2 class I allele with autism: Hum Immunol. 2006 Apr-May;67(4-5):346-51. Epub 2006 Apr 3.

73 Careaga M, Van de Water J, Ashwood P (2010) Immune dysfunction in autism: a pathway to treatment: Neurotherapeutics. 2010 Jul;7(3):283-92

74 J-Y Jung,1 I S Kohane,1,2,3 and D P Wall (2011) Identification of autoimmune gene signatures in autism: Transl Psychiatry. 2011 December; 1(12): e63.

75 Guevara-Campos J, González-Guevara L, Briones P, López-Gallardo E, Bulán N, Ruiz-Pesini E, Ramnarine D, Montoya J. (2010) Autism associated to a deficiency of complexes III and IV of the mitochondrial respiratory chain: Invest Clin. 2010 Sep;51(3):423-31

76 Poling JS, Frye RE, Shoffner J, Zimmerman AW. (2006) Developmental regression and mitochondrial dysfunction in a child with autism: J Child Neurol. 2006 Feb;21(2):170-2.

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77 Jacqueline R. Weissman, Richard I. Kelley, Margaret L. Bauman, Bruce H. Cohen, Katherine F. Murray, Rebecca L. Mitchell, Rebecca L. Kern, Marvin R. Natowicz (2008) Mitochondrial Disease in Autism Spectrum Disorder Patients: A Cohort Analysis: PLoS ONE 3(11): e3815

78 Harumi Jyonouchi, Lee Geng, Deanna L Streck, and Gokce A Toruner (2012) Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): case study: J Neuroinflammation. 2012; 9: 4.

79 Sabrina Baieli, Lorenzo Pavone, Concetta Meli, Agata Fiumara, Mary Coleman: (2003) Autism and Phenylketonuria: Journal of Autism and Developmental Disorders: Vol. 33, No. 2, April 2003

80 Valerie W. Hu, Tewarit Sarachana, Kyung Soon Kim, AnhThu Nguyen, Shreya Kulkarni, Mara E. Steinberg, Truong Luu, Yinglei Lai, and Norman H. Lee: (2009) Gene Expression Profiling Differentiates Autism Case–Controls and Phenotypic Variants of Autism Spectrum Disorders: Evidence for Circadian Rhythm Dysfunction in Severe Autism: Autism Res. 2(2): 78–97. doi:10.1002/aur.73

81 OMIN#: Online Mendelian Inheritance in Man® http://omim.org/

82 Novarino G, El-Fishawy P, Kayserili H, Meguid NA, Scott EM, Schroth J, Silhavy JL, Kara M, Khalil RO, Ben-Omran T, Ercan-Sencicek AG, Hashish AF, Sanders SJ, Gupta AR, Hashem HS, Matern D, Gabriel S, Sweetman L, Rahimi Y, Harris RA, State MW, Gleeson JG.(2012) Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy: Science. 2012 Oct 19;338(6105):394-7

83 Richard E. Frye and Daniel A. Rossignol (2012) Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders: Pediatr Res. 2011 May; 69(5 Pt 2): 41R–47R

84 Diaz-Stransky A, Tierney E. (2012) Cognitive and behavioral aspects of Smith-Lemli-Opitz syndrome: Am J Med Genet C Semin Med Genet. 2012 Nov 15;160C(4):295-300.

85 Patrícia B. S. Celestino-Soper,Sara Violante, Emily L. Crawford, Rui Luo, Anath C. Lionel, Els Delaby, Guiqing C, Bekim Sadikovic, Kwanghyuk Lee, Charlene Lo, Kun Gao, Richard E. Person, Timothy J. Moss, Jennifer R. German, Ni Huang,i Marwan Shinawi, Diane Treadwell-Deering, Peter Szatmari, l Wendy Roberts, Bridget Fernandezn Richard J. Schroer, Roger E. Stevenson, Joseph D. Buxbaum, Catalina Betancur, Stephen W. Scherer, Stephan J. Sanders Daniel H. Geschwind, James S. Sutcliffe, Matthew E. Hurles, Ronald . A. Wanders, Chad A. Shaw, Suzanne M. Leal, Edwin H. Cook, Jr., Robin P. Goin-Kochel, Frédéric M. Vaz, and Arthur L. Beaudeta: (2012) A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism: Proc Natl Acad Sci U S A. 2012 May 22; 109(21): 7974–7981

86 Johannes Häberle, Nathalie Boddaert, Alberto Burlina, Anupam Chakrapani, Marjorie Dixon, Martina Huemer, Daniela Karall, Diego Martinelli, Pablo Sanjurjo Crespo, René Santer, Aude Servais1 Vassili Valayannopoulos, Martin Lindner, Vicente Rubio, and Carlo Dionisi-Vici (2012) Suggested guidelines for the diagnosis and management of urea cycle disorders: Orphanet J Rare Dis. 2012; 7: 32.

87 Işιk Görker and Ümran Tüzün (2005) Autistic-like findings associated with a urea cycle disorder in a 4-year-old girl: J Psychiatry Neurosci. 2005 March; 30(2): 133–135.

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88 Kevin G. Becker and Stephen T. Schultz: (2010) Similarities in features of autism and asthma and a possible link to acetaminophen use: Med Hypotheses. 2010 January; 74(1): 7–11.

89 Cubala-Kucharska M.(2010) The review of most frequently occurring medical disorders related to aetiology of autism and the methods of treatment: Acta Neurobiol Exp (Wars). 2010;70(2):141-6.

90 Mary Randolph-Gips and Pramila Srinivasan (2012) Modeling autism: a systems biology approach: J Clin Bioinforma. 2012; 2: 17

91 Millington DS, Stevens RD.(2011) Acylcarnitines: analysis in plasma and whole blood using tandem mass spectrometry: Methods Mol Biol. 2011;708:55-72.

92 G Oliveira MD, L Diogo MD, M Grazina MSc, P Garcia MD, A Ataíde Psych, C Marques MSC CPsych, T Miguel, L Borges MD, A M Vicente PhD, C R Oliveira MD PhD: (2007) Mitochondrial dysfunction in autism spectrum disorders: a population-based study: Developmental Medicine & Child Neurology Volume 47, Issue 3, pages 185–189

93 James B Adams, Tapan Audhya, Sharon McDonough-Means, Robert A Rubin, David Quig, Elizabeth Geis, Eva Gehn, Melissa Loresto, Jessica Mitchell, Sharon Atwood, Suzanne Barnhouse and Wondra Lee: (2011) Nutritional and metabolic status of children with autism vs. neurotypical children, and the association with autism severity: Adams et al. Nutrition & Metabolism 2011, 8:34

94 Shaw W: (2010) Increased urinary excretion of a 3-(3- hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia spp. in the gastrointestinal

tract, in urine samples from patients with autism and schizophrenia: Nutr: 13(3):135-43.

95 James B Adams, Tapan Audhya, Sharon McDonough-Means, Robert A Rubin, David Quig, Elizabeth Geis, Eva Gehn, Melissa Loresto, Jessica Mitchell, Sharon Atwood, Suzanne Barnhouse, and Wondra Lee (2011) Nutritional and metabolic status of children with autism vs. neurotypical children, and the association with autism severity: Nutr Metab (Lond). 2011; 8: 34

96 Aneja A, Tierney E. (2008) Autism: the role of cholesterol in treatment: Int Rev Psychiatry. 2008 Apr;20(2):165-70

97 James B Adams, Tapan Audhya, Sharon McDonough-Means, Robert A Rubin4 David Quig, Elizabeth Geis, Eva Gehn,1 Melissa Loresto,1 Jessica Mitchell, Sharon Atwood, Suzanne Barnhouse,and Wondra Lee: (2011) Effect of a vitamin/mineral supplement on children and adults with autism: BMC Pediatr. 2011; 11: 111

98 Renee Dufault, Roseanne Schnoll, Walter J Lukiw, Blaise LeBlanc, Charles Cornett, Lyn Patrick, David Wallinga, Steven G Gilbert, and Raquel Crider (2009) Mercury exposure, nutritional deficiencies and metabolic disruptions may affect learning in children: Behav Brain Funct. 2009; 5: 44.

99 Eleonor BLAUROCK-BUSCH; Omnia R. AMIN; Hani H. DESSOKI;Thanaa RABAH: (2012) Toxic Metals and Essential Elements in Hair and Severity of Symptoms among Children with Autism: Robenstr 20, D-912217,

100 S Jill James, Stepan Melnyk, George Fuchs, Tyra Reid, Stefanie Jernigan, Oleksandra Pavliv, Amanda Hubanks, and David W Gaylor (2009)

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Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism: Am J Clin Nutr. 2009 January; 89(1): 425–430. 101 Stacey Cornish and Lewis Mehl-Madrona: (2008) The Role of Vitamins and Minerals in Psychiatry: Integr Med Insights. 2008; 3: 33– 42.

102 S Jill James, Stepan Melnyk, George Fuchs, Tyra Reid, Stefanie Jernigan, Oleksandra Pavliv, Amanda Hubanks, and David W Gaylor (2008) Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism: Am J Clin Nutr January 2009 vol. 89 no. 1 425-430

103 Gehan A Mostafa and Laila Y AL-Ayadh: (2012) Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: Relation to autoimmunity: J Neuroinflammation doi: 10.1186/1742- 2094-9-201

104 Stephen Bent,Kiah Bertoglio, Paul Ashwood, Alan Bostrom, and Robert L. Hendren: (2011) A Pilot Randomized Controlled Trial of Omega-3 Fatty Acids for Autism Spectrum Disorder: J Autism Dev Disord. 2011 May; 41(5): 545–554

105 Afaf K El-Ansary, Abir G Ben Bacha, and Layla Y Al- Ayahdi: (2011) Plasma fatty acids as diagnostic markers in autistic patients from Saudi Arabia: Lipids Health Dis. 2011; 10: 62

106 Craig J. Newschaffer,Lisa A. Croen,Julie Daniels,Ellen Giarelli,udith K. Grether,Susan E. Levy, David S. Mandell,Lisa A. Miller,Jennifer Pinto- Martin,Judy Reaven,Ann M. Reynolds,Catherine E. Rice,Diana Schendel,and Gayle C. Windham: (2007) The Epidemiologyof Autism

Spectrum Disorders: J Child Neurol. Annu. Rev. Public Health 2007. 28:21.1–21.24

107 Cynthia A. Molloy, Ardythe L. Morrow, Jareen Meinzen-Derr, Geraldine Dawson, Raphael Bernier, Michelle Dunn, Susan L. Hyman, William M. McMahon, Julie Goudie-Nice, Susan Hepburn, Nancy Minshew, Sally Rogers, Marian Sigman, M. Anne Spence, Helen Tager-Flusberg, Fred R. Volkmar, and Catherine Lord: (2006) Familial Autoimmune Thyroid Disease as a Risk Factor for Regression in Children with Autism Spectrum Disorder: A CPEA Study: Journal of Autism and Developmental Disorders DOI 10.1007/s10803-005-0071-0

108 Sloane J. Freeman, MD, Wendy Roberts, MD and Denis Daneman, MD (2005) Type 1 Diabetes and Autism Is there a link?: DIABETES CARE, VOLUME 28, NUMBER 4, APRIL 2005

109 Paul Ashwood, Christina Kwong, Robin Hansen, Irva Hertz- Picciotto, Lisa Croen, Paula Krakowiak, Wynn Walker, Isaac N. Pessah, Judy Van de Water (2008) Brief Report: Plasma Leptin Levels are Elevated in Autism: Association with Early Onset Phenotype?: J Autism Dev Disord (2008) 38:169–175

110 A. K. BALTACI and R. MOGULKOC, Department of Physiology, Meram Medical School, Selcuk University, Konya, Turkey (2007) PINEALECTOMY AND MELATONIN ADMINISTRATION IN RATS: THEIR EFFECTS ON PLASMA LEPTIN LEVELS AND RELATIONSHIP WITH ZINC: Acta Biologica Hungarica 58 (4), pp. 335–343 (2007)

111 Mills JL, Hediger ML, Molloy CA, Chrousos GP, Manning-Courtney P, Yu KF, Brasington M, England LJ.(2007) Elevated levels of growth- related hormones in autism and autism spectrum disorder: Clin Endocrinol (Oxf). 2007 Aug;67(2):230-7. Epub 2007 Jun 4.

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112 Williams BL, Hornig M, Buie T, Bauman ML, Cho Paik M, Wick I, Bennett A, Jabado O, Hirschberg DL, Lipkin WI. (2011) Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances: PLoS One. 2011;6(9):e24585

113 P D'Eufemia, M Celli, R Finocchiaro, L Pacifico, L Viozzi, M Zaccagnini, E Cardi, O Giardini: (2008) Abnormal intestinal permeability in children with autism: DOI: 10.1111/j.1651- 2227.1996.tb14220.x

114 James B Adams, Leah J Johansen, Linda D Powell, David Quig, and Robert A Rubin: (2011) Gastrointestinal flora and gastrointestinal status in children with autism -- comparisons to typical children and correlation with autism severity: BMC Gastroenterol. 2011; 11: 22.

115 Penelope AE Main, Manya T Angley, Catherine E O'Doherty, Philip Thomas, and Michael Fenech (2012) The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis: Nutr Metab (Lond). 2012; 9: 35

117 Yusra A Al-Yafee, Laila Y Al- Ayadhi, Samina H Haq, and Afaf K El- Ansary (2011) Novel metabolic biomarkers related to sulfur- dependent detoxification pathways in autistic patients of Saudi Arabia: BMC Neurol. 2011; 11: 139

118 James SJ, Rose S, Melnyk S, Jernigan S, Blossom S, Pavliv O, Gaylor DW. (2009) Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism: FASEB J. 2009 Aug;23(8):2374-83. Epub 2009 Mar 23

119 Al-Salehi SM, Ghaziuddin M: (2008) G6PD deficiency in autism: a case-series from Saudi Arabia : Eur Child Adolesc Psychiatry.2009 Apr;18(4):227-30. Epub 2008 Sep 22.

120 Yubin Zhang, Donghong Gao, Valerie J. Bolivar, and David A. Lawrence (2011) Induction of Autoimmunity to Brain Antigens by Developmental Mercury Exposure: Toxicol Sci. 2011 February; 119(2): 270–280.

121 Connolly AM, Chez MG, Pestronk A, Arnold ST, Mehta S, Deuel RK. (1999) Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders: J Pediatr. 1999 May;134(5):607-13.

122 Vijendra K. Singh Sheren X. Lin Elizabeth Newell Courtney Nelson (2001) Abnormal Measles-Mumps-Rubella Antibodies and CNS Autoimmunity in Children with Autism: J Biomed Sci 2002;9:359–364

123 Reda M., Fahmy H.(2007) B lymphocyte Antigen D8/ 17 in Egyptian Children with Autism: Current Psychiatry Vol. 14 No.1 March 2007

124 Gehan A. Mostafaa, Abeer A. Shehab (2010) The link of C4B null allele to autism and to a family history of autoimmunity in Egyptian autistic children: Journal of Neuroimmunology Volume 223, Issues 1–2, June 2010, Pages 115–119

125 Laila Y AL-Ayadhi and Gehan A Mostafa (2011) Low plasma progranulin levels in children with autism: J Neuroinflammation. 2011; 8: 111.

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126 Gehan A Mostafaand Laila Y AL-Ayadhi (2011) The possible link between the elevated serum levels of neurokinin A and anti- ribosomal P protein antibodies in children with autism: J Neuroinflammation. 2011; 8: 180.

127 Angelidou A, Alysandratos KD, Asadi S, Zhang B, Francis K, Vasiadi M, Kalogeromitros D, Theoharides TC.(2011) Brief report: "allergic symptoms" in children with Autism Spectrum Disorders. More than meets the eye?: J Autism Dev Disord. 2011 Nov;41(11):1579-85.

128 Martí LF. (2010) Effectiveness of nutritional interventions on the functioning of children with ADHD and/or ASD. An updated review of research evidence: Bol Asoc Med P R. 2010 Oct- Dec;102(4):31-42

129 Pennesi CM, Klein LC. (2012) Effectiveness of the gluten-free, casein-free diet for children diagnosed with autism spectrum disorder: based on parental report.: Nutr Neurosci. 2012 Mar;15(2):85- 91

130 Afaf K El-Ansary, Abir G Ben Bacha, and Laila Y Al-Ayadhi (2011) Proinflammatory and proapoptotic markers in relation to mono and di-cations in plasma of autistic patients from Saudi Arabia: J Neuroinflammation. 2011; 8: 142.

131 Lintas C, Altieri L, Lombardi F, Sacco R, Persico AM (2010) Association of autism with polyomavirus infection in postmortem brains: J Neurovirol. 2010 Mar;16(2):141-9

132 Christine Winter, Teri J. Reutiman, Timothy D. Folsom, Reinhard Sohr, Rainer J. Wolf,d, S. Hossein Fatemi and Georg Juckel (2008) Dopamine and serotonin levels following prenatal viral infection in

mouse - implications for psychiatric disorders such as schizophrenia and autism: Eur Neuropsychopharmacol. 2008 October; 18(10): 712–716

133 Antonio M Persico (2010) Polyomaviruses and autism: more than simple association?: Journal of NeuroVirology, 16: 332–333, 2010

134 Carlo Contini, Silva Seraceni, Rosario Cultrera, Massimiliano Castellazzi, Enrico Granieri, and Enrico Fainardi (2010) Chlamydophila pneumoniae Infection and Its Role in Neurological Disorders: Interdiscip Perspect Infect Dis. 2010; 2010: 273573.

135 Jason Tan, Christine H. Smith, MB BS, and Ran D. Goldman, MD FRCPC (2012) Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections: Can Fam Physician. 2012 September; 58(9): 957–959

136 Aristo Vojdani, Frank Hebroni, Yaniv Raphael, Jonathan Erde and Bernard Raxlen (2007) Novel Diagnosis of Lyme Disease: Potential for CAM Intervention: eCAM 2009;6(3)283–295

137 Ashwood, Krakowiak, Hertz-Picciotto, Hansen, Isaac N. Pessah, Judy Van de Water (2011) Associations of impaired behaviors with elevated plasma chemokines in autism spectrum disorders: Journal of Neuroimmunology, Volume 232, Issue 1 , Pages 196-199, March 2011

138 Shahrzad Asadi and Theoharis C Theoharides (2012) Corticotropin-releasing hormone and extracellular mitochondria augment IgE-stimulated human mast-cell vascular endothelial growth factor release, which is inhibited by luteolin: J Neuroinflammation. 2012; 9: 85.

139 Asimenia Angelidou, Konstantinos Francis, Magdalini Vasiadi, Konstantinos-Dionysios Alysandrato, Bodi Zhang, Athanasios

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Theoharides, Lefteris Lykouras, Kyriaki Sideri, Dimitrios Kalogeromitros, and Theoharis C Theoharides (2010) Neurotensin is increased in serum of young children with autistic disorder: J Neuroinflammation. 2010; 7: 48.

140 Antoinette R. Bailey, Brian N. Giunta, Demian Obregon, William V. Nikolic, Jun Tian, Cyndy D. Sanberg, Danielle T. Sutton, and Jun Tan (2008) Peripheral biomarkers in Autism: secreted amyloid precursor protein-α as a probable key player in early diagnosis: Int J Clin Exp Med. 2008; 1(4): 338–344.

141 Jerzy Wegiel, Janusz Frackowiak, Bozena Mazur-Kolecka, N. Carolyn Schanen, Edwin H. Cook, Jr., Marian Sigman,W. Ted Brown, Izabela Kuchna, Jarek Wegiel, Krzysztof Nowicki, Humi Imaki, Shuang Yong Ma, Abha Chauhan, Ved Chauhan, David L. Miller, Pankaj D. Mehta, Michael Flory, Ira . Cohen, Eric London, Barry Reisberg, Mony J. de Leon, and Thomas Wisniewski (2012) Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders: PLoS One. 2012; 7(5): e35414.

142 Laila Y Al- Ayadhi, Abir G Ben Bacha, Malak Kotb, and Afaf K El- Ansary (2012) A novel study on amyloid β peptide 40, 42 and 40/42 ratio in Saudi autistics: Behav Brain Funct. 2012; 8: 4.

143 Rodney R. Dietert, Janice M. Dietert, and Jamie C. Dewitt (2011) Environmental risk factors for autism: Emerg Health Threats J. 2011; 4: 10.3402/ehtj.v4i0.7111

144 Eleonor BLAUROCK-BUSCH, Omnia R. AMIN, Hani H. DESSOKI, and Thanaa RABAH (2012) Toxic Metals and Essential Elements in Hair and Severity of Symptoms among Children with Autism: Maedica (Buchar). 2012 January; 7(1): 38–48

145 Matthew Garrecht and David W. Austin (2011) The plausibility of a role for mercury in the etiology of autism: a cellular perspective: Toxicol Environ Chem. 2011 May-Jul; 93(5-6): 1251–1273.

146 Raymond F. Palmer, Steven Blanchard, Zachary Stein, David Mandell, Claudia Miller (2006) Environmental mercury release, special education rates, and autism disorder: an ecological study of Texas: Health & Place 12 (2006) 203–209

147 James S. Woods, Sarah E. Armel, Denise I. Fulton, Jason Allen, Kristine Wessels, P. Lynne Simmonds, Doreen Granpeesheh, Elizabeth Mumper, J. Jeffrey Bradstreet, Diana Echeverria, Nicholas J. Heyer, and James P.K. Rooney (2010) Urinary Porphyrin Excretion in Neurotypical and Autistic Children: Environ Health Perspect. 2010 October; 118(10): 1450–1457.

148 Al-Farsi YM, Waly MI, Al-Sharbati MM, Al-Shafaee MA, Al-Farsi OA, Al-Khaduri MM, Gupta I, Ouhtit A, Al-Adawi S, Al-Said MF, Deth RC.(2012) Levels of Heavy Metals and Essential Minerals in Hair Samples of Children with Autism in Oman: a Case-Control Study: Biol Trace Elem Res. 2012 Nov 28. [Epub ahead of print]

149 Eleonor Blaurock-Busch, Omnia R. Amin, Hani H. Dessoki, Thanaa Rabah (2012) Toxic Metals and Essential Elements in Hair and Severity of Symptoms among Children with Autism: Mædica J Clin Med 2012; 7 (1): 38-48

150 Shaw CA, Petrik MS.(2009) Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration: J Inorg Biochem. 2009 Nov;103(11):1555-62. Epub 2009 Aug 20

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151 Eleonor BLAUROCK-BUSCH, Omnia R. AMIN, and Thanaa RABAH (2011) Heavy Metals and Trace Elements in Hair and Urine of a Sample of Arab Children with Autistic Spectrum Disorder: Maedica (Buchar). 2011 October; 6(4): 247–257.

152 Matthew Garrecht and David W. Austin (2011) The plausibility of a role for mercury in the etiology of autism: a cellular perspective: Toxicol Environ Chem. 2011 May-Jul; 93(5-6): 1251–1273

153 Kuhn M, Grave S, Bransfield R, Harris S. (2012) Long term antibiotic therapy may be an effective treatment for children co- morbid with Lyme disease and autism spectrum disorder: Med Hypotheses. 2012 May;78(5):606-15. Epub 2012 Feb 22

154 Renee Dufault, Walter J Lukiw, Raquel Crider, Roseanne Schnoll, David Wallinga, and Richard Deth (2012) A macroepigenetic approach to identify factors responsible for the autism epidemic in the United States: Clin Epigenetics. 2012; 4(1): 6

155 Martyn A. Sharpe, Andrew D. Livingston, and David S. Baskin (2012) Thimerosal-Derived Ethylmercury Is a Mitochondrial Toxin in Human Astrocytes: Possible Role of Fenton Chemistry inthe Oxidation and Breakage of mtDNA: Hindawi Publishing Corporation Journal of Toxicology Volume 2012, Article ID 373678, 12 pages doi:10.1155/2012/373678

156 Isaac N. Pessah, Richard F. Seegal, Pamela J. Lein, Janine LaSalle, Benjamin K. Yee, Judy Van De Water, and Robert F. Berman: (2008) Immunologic and Neurodevelopmental Susceptibilities of Autism: eurotoxicology. 2008 May; 29(3): 531–544.

157 Irva Hertz-Picciotto, Åke Bergman, Britta Fängström, Melissa Rose, Paula Krakowiak, Isaac Pessah, Robin Hansen, and Deborah H Bennett (2011) Polybrominated diphenyl ethers in relation to autism and developmental delay: a case-control study: Environ Health. 2011; 10: 1.

158 Janie F. Shelton, Irva Hertz-Picciotto, and Isaac N. Pessah (2012) Tipping the Balance of Autism Risk: Potential Mechanisms Linking Pesticides and Autism: Environ Health Perspect. 2012 July; 120(7): 944– 951.

159 Mostafa I. Waly, Mady Hornig, Malav Trivedi, Nathaniel Hodgson, Radhika Kini, Akio Ohta, and Richard Deth: (2012) Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism: Autism Res Treat. 2012; 2012: 190930

160 Rebecca J. Schmidt, Robin L. Hansen, Jaana Hartiala, Hooman Allayee, Linda C. Schmidt, Daniel J. Tancredi, Flora Tassone, and Irva Hertz-Picciotto: (2011) Prenatal vitamins, one-carbon metabolism gene variants, and risk for autism: Epidemiology. 22(4): 476–485.

161 Judith S. Nijmeijer, Catharina A. Hartman, Nanda N.J. Rommelse, Marieke E. Altink, Cathelijne J.M. Buschgens, Ellen A. Fliers, Barbara Franke, Ruud B. Minderaa,1 Johan Ormel, Joseph A. Sergeant, Frank C. Verhulst, Jan K. Buitelaar, and Pieter J. Hoekstra (2010) Perinatal risk factors interacting with catechol O-methyl transferase and the serotonin transporter gene predict ASD symptoms in children with ADHD: Journal of Child Psychology and Psychiatry 51:11 (2010), pp 1242–1250

162 Andrew W. Zimmerman, Susan L. Connors, Karla J. Matteson, Li- Ching Lee, Harvey S. Singer, Julian A. Castaneda, David A. Pearce (2007)

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Maternal antibrain antibodies in autism: Brain, Behavior, and Immunity 21 (2007) 351–357

163 Johnson WG, Buyske S, Mars AE, Sreenath M, Stenroos ES, Williams TA, Stein R, Lambert GH.(2009) HLA-DR4 as a risk allele for autism acting in mothers of probands possibly during pregnancy: Arch Pediatr Adolesc Med. 2009 Jun;163(6):542-6.

164 Krakowiak P, Walker CK, Bremer AA, Baker AS, Ozonoff S, Hansen RL, Hertz-Picciotto I.(2012) Maternal metabolic conditions and risk for autism and other neurodevelopmental disorders: Pediatrics. 2012 May;129(5):e1121-8. Epub 2012 Apr 9.

165 Rebecca J. Schmidt, Robin L. Hansen, Jaana Hartiala, Hooman Allayee, Linda C. Schmidt, Daniel J. Tancredi, Flora Tassone, and Irva Hertz-Picciotto: (2011) Prenatal vitamins, one-carbon metabolism gene variants, and risk for autism: Epidemiology. 22(4): 476–485.

166 Sharon K. Sagiv, PhD, MPH; Sally W. Thurston, PhD; David C. Bellinger, PhD, MS; Chitra Amarasiriwardena, PhD; Susan A. Korrick, MD, MPH: (2012) Prenatal Exposure to Mercury and Fish Consumption During Pregnancy and Attention-Deficit/Hyperactivity Disorder– Related Behavior in Children: Arch Pediatr Adolesc Med. 2012;():1-9. doi:10.1001/archpediatrics.2012.1286.

167 Adrienne S. Ettinger, ScD, MPH, Anne Guthrie Wengrovitz, MPH, Centers for Disease Control and Prevention, National Center for Environmental Health/Agency for Toxic Substances and Disease Registry, Christopher Portier, PhD, Director, Healthy Homes and Lead Poisoning Prevention Branch, Mary Jean Brown, ScD, RN (2010) GUIDELINES FOR THE IDENTIFICATION AND MANAGEMENT OF LEAD EXPOSURE IN

PREGNANT AND LACTATING WOMEN: U.S. Department of Health and Human Services Atlanta, GA: CS2016857

168 J. B. Adams, C. E. Holloway, F. George, D. Quig: (2006) Analyses of toxic metals and essential minerals in the hair of arizona children with autism and associated conditions, and their mothers: Biological Trace Element Research: June 2006, Volume 110, Issue 3, pp 193-209

169 Schmidt RJ, Tancredi DJ, Ozonoff S, Hansen RL, Hartiala J, Allayee H, Schmidt LC, Tassone F, Hertz-Picciotto I. (2012) Maternal periconceptional folic acid intake and risk of autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks from Genetics and Environment) case-control study: Am J Clin Nutr. 2012 Jul;96(1):80-9

170 William B. Grant and Connie M. Soles: (2009) Epidemiologic evidence for supporting the role of maternal vitamin D deficiency as a risk factor for the development of infantile autism: [Dermato- Endocrinology 1:4, 223-228; July/August 2009]

171 N.Q. Liu, M. Hewison (2011) Vitamin D, the placenta and pregnancy: Archives of Biochemistry and Biophysics, Volume 523, Issue 1, 1 July 2012, Pages 37–47

172 Rebecca J. Schmidt, Robin L. Hansen, Jaana Hartiala, Hooman Allayee, Linda C. Schmidt, Daniel J. Tancredi, Flora Tassone,and Irva Hertz-Picciotto (2011) Prenatal vitamins, one-carbon metabolism gene variants, and risk for autism: Epidemiology. 2011 July; 22(4): 476–485

173 Todd A. Jusko, Anneclaire J. De Roos, Stephen M. Schwartz, B. Paige Lawrence4 Lubica Palkovicova, Tomas Nemessanyi, Beata Drobna, Anna

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Fabisikova, Anton Kocan, Eva Jahnova, Terrance J. Kavanagh, Tomas Trnovec, and Irva Hertz-Picciotto (2011) Maternal and early postnatal polychlorinated biphenyl exposure in relation to total serum immunoglobulin concentrations in 6-month-old infants: J Immunotoxicol. 2011 JAN-MAR; 8(1): 95–100.

174 Bouchard MF, Chevrier J, Harley KG, Kogut K, Vedar M, Calderon N, Trujillo C, Johnson C, Bradman A, Barr DB, Eskenazi B.(2011) Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children.: Environ Health Perspect. 2011 Aug;119(8):1189-95. Epub 2011 Apr 13.

175 Jean El-Cheikh, Sabine Fürst, Francois Casalonga, Roberto Crocchiolo, Luca Castagna, Angela Granata, Claire Oudin, Catherine Faucher, Pierre Berger, Anthony Sarran, and Didier Blaise (2012) JC Virus Leuko-Encephalopathy in Reduced Intensity Conditioning Cord Blood Transplant Recipient with a Review of the Literature: Mediterr J Hematol Infect Dis. 2012; 4(1): e2012043

176 Martin LA, Ashwood P, Braunschweig D, Cabanlit M, Van de Water J, Amaral DG.(2008) Stereotypies and hyperactivity in rhesus monkeys exposed to IgG from mothers of children with autism.: Brain Behav Immun. 2008 Aug;22(6):806-16. Epub 2008 Feb 8.

177 S Jill James, Stepan Melnyk, George Fuchs, Tyra Reid, Stefanie Jernigan, Oleksandra Pavliv, Amanda Hubanks, and David W Gaylor (2008 ) Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism: Am J Clin Nutr January 2009 vol. 89 no. 1 425-430

178 Mostafa GA, Shehab AA.(2011) The link of C4B null allele to autism and to a family history of autoimmunity in Egyptian autistic children: J Neuroimmunol. 2010 Jun;223(1-2):115-9. Epub 2010 May 10.

179 Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, Gudjonsson SA, Sigurdsson A, Jonasdottir A, Jonasdottir A, Wong WS, Sigurdsson G, Walters GB, Steinberg S, Helgason H, Thorleifsson G, Gudbjartsson DF, Helgason A, Magnusson OT, Thorsteinsdottir U, Stefansson K.(2012) Rate of de novo mutations and the importance of father's age to disease risk.: Nature. 2012 Aug 23;488(7412):471-5. doi: 10.1038/nature11396.

180 Carla Lintas, Francesco Guidi, Barbara Manzi, Antonio Mancini, Paolo Curatolo, and Antonio M. Persico (2011) Lack of Infection with XMRV or Other MLV-Related Viruses in Blood, Post-Mortem Brains and Paternal Gametes of Autistic Individuals: PLoS One. 2011; 6(2): e16609

181 Sally Ozonoff, PhD, Gregory S. Young, PhD, Alice Carter, PhD, Daniel Messinger, PhD, Nurit Yirmiya, PhD, Lonnie Zwaigenbaum, MD, Susan Bryson, PhD,fLeslie J. Carver, PhD, John N. Constantino, MD, Karen Dobkins, PhD, Ted Hutman, PhD, Jana M. Iverson, PhD, Rebecca Landa, PhD, Sally J. Rogers, PhD, Marian Sigman, PhD, and Wendy L. Stone, PhD (2011) Recurrence Risk for Autism Spectrum Disorders: A Baby Siblings Research Consortium Study: Pediatrics. 2011 September; 128(3): e488–e495.

182 John N. Constantino, MD, Yi Zhang, MS, Thomas Frazier, PhD, Anna M. Abbacchi, MS, and Paul Law, MD, MPH (2010) Sibling recurrence and the genetic epidemiology of autism: Am J Psychiatry. 2010 November; 167(11): 1349–1356.

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Addendum II

Epigenetics and Clinical Origen of Behaviors to

Optimizing Health of ASD Patients Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Research and Clinical Research Direction Pharmacology, Boston University Director of Research, Athena Biomedical Institute

Kazuko Grace A Paradigm Shift in Athena Biomedical Institute [email protected] Diagnosing and Treating ASD patients:

617-500-5980 Autism is a Treatable Medical www.athenabiomedicalinstitute.org and Metabolic Disease with Behavioral Components

Prepared Statement: Congressional Autism Hearing November 29, 2012 Last updated January 31, 2013

Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute [email protected]

Kazuko Grace Athena Biomedical Institute

www.athenabiomedicalinstitute.org

Epigenetics and Clinical Origen of Behaviors to Optimizing Health of ASD Patients:

Research and Clinical Research Direction

Addendum II

Cassandra L. Smith, and Kazuko Grace Boston University and Athena Biomedical Institute

Abstract: Autism remains a complex disorder that resists the best efforts of dedicated clinicians, researchers, educators and families seeking cause(s), robust diagnostic criteria and most importantly effective treatments and preventive measures. Clinical research is directed towards understanding cause and effect relationship and the established scientifically proven therapeutic treatments.i Our goal is to develop state-of -the-art diagnostic assessment protocols and individualized treatment regimes for patients with autism and related disorders that are consistent with available but admittedly complex data. The best effort must include input from a variety of individuals including patients and caregivers who are excluded generally from medical and scientific discussion to improve diagnosing, treating and managing patients. This aspect is especially problematic in neurobehavioral diseases. The expert group needs to include computer scientist and system biologists who are expert at handling and analyzing complex datasets.

(Addendum II) Epigenetics and Clinical Origen of Behaviors to Optimizing Health of ASD Patients Research and Clinical Research Direction

Introduction: This document is being provided as a starting point for discussion for how to improve the lives of autism patients and their families. We hope the discussion will include the collective experiences of clinicians, staff, patients and families. Towards this end, we encourage the Oversight Committee to support:

1. The formation of a group of working committees composed not only of researchers and clinicians but also parents, staff, and patients to tackle the demanding task of formulating new approaches (diagnosis, treatment and management) to autism based on what might seem to be a bewildering array of research results. This community effort requires the participation of computer scientists, and system biologist used to handling large datasets. The results of each of groups should be made publically available.

2. The development of a publically available database with various research and clinical results from individual studies from anonymized patients.

3. The development of a reference set of anonymized patient samples that will be publically available, and where individuals wanting to analyze the samples agree to testing the entire reference set and to put results into the public domain.

Today, autism is defined primarily by behaviors symptoms. Current diagnostic strategies are observational in focus, but include: extensive medical history and physical exam, non-specific evaluations such as intelligence, language and achievement testing. Specific autism inventories and questionnaires that are age specific, such as the Autism Diagnostic Observation Scales (ADOS), Autism Diagnostic Inventory (ADI) and others. Diagnosis depends on the observational and clinical skills of the examiner.

Medical testing such as MRI or CT scans, EEG’s, blood and urine tests, and genetic tests are done primarily to rule in, or out, any contributing medical disorders. Diagnosis is based on pooling all of the acquired information resulting in a diagnostic impression. Similar diagnostic strategies are used for virtually all mental health and developmental disorders.

1) What causes autism and the increase incidence of disease?

Autism has many underlying etiologies difficulties. Autism is not a single disorder like Down's syndrome that is due to a single cause, chromosome 21 trisomy. Instead, autism is the pathological outcome of variety of assaults on the

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developing brain. Many different etiologies have been linked to an increase in autism including genetic predispositions, rare genetic diseases, infectious agents, nutrition and exposures to toxins.

Although research worldwide is identifying factors that contribute to autism, we need better integration of results in order to improve understanding of disease presentation in individuals. Likewise a better understanding and integration of factors linked to autism can reduce the increasing incidence of autism. Autism is linked to many genetic and environmental factors, as are other common diseases.

Much progress has been made in understanding the genetic liabilities with over one hundred genes linked to autism. However, these findings have had little affect on patient treatment or outcome. Further, changes in diagnostic criteria proposed in DMS-V are putting rare genetic diseases with precise causes under the umbrella of autism subtracting rather than increasing our knowledge of disease.

Research into toxicity mechanisms is ongoing although not to the same level of organization and intensity as genetics. Immune system abnormalities, infectious diseases, dietary problems, and adverse environmental exposures are all areas that are being studied as well. Many of these issues overlap factors linked to other common diseases that do not appear to be increasing.

2) How to improve diagnosis of autism?

Without knowing the causes, we are left with a diagnosis based on behavior, and this is fraught with inconsistencies, and is at best an inexact science. At present there is no useful set of markers to aid diagnosis, or screen families for risk factors for developing autism. Instead a robust diagnostic and predictive tool that combines testing of factors linked to autism needs to be established.

3) What treatments make a significant difference to autism patients?

Our current treatment strategies are focused on behavioral outcomes. The many and varied medical and metabolic aspects of disease need to be carefully and scientifically assessed for each patient and where necessary treated.

(Addendum II) Epigenetics and Clinical Origen of Behaviors to Optimizing Health of ASD Patients Research and Clinical Research Direction

Parents have instituted treatment regimes that are not scientific proven, but have been reported to improve the health and behavior of autism patients. All treatments should be monitored scientifically whether nutritional or otherwise non-conventional so that the best information is available for what treatments are most effective for which subsets of patients. This type of understanding only comes about through cooperation between clinicians, researchers, caregivers, families and patients.

Theranostics approach to autism: Here, we advocating for what some have called a theranostic medicine approach. This approach includes a portmanteau of diagnostic testing and the development of individualized treatment regimes. Research is tell us that making inroads into autism and other serious neurobehavioral disorders will require theranostic approaches, although clearly all patients will benefit from a change in attitude. ii

Ten million DNA differences between any two individual representing only 1% of the genome. However, we are all aware that there are significant differences in humans. These genetic differences lead to variations in obvious phenotypes such as height, skin color, and ethnic/racial characteristics and how we respond to medications and even foods. Two major areas of personalized medicine are pharmacogenomics and nutritional genomics.iii

Pharmacogenomics: One form of personalized medicine involves pharmacogenomics. This is a branch of pharmacology which deals with how inherited genetic variations influence the body’s response to medications by correlating single-nucleotide polymorphisms to a drug’s efficacy or toxicity. Just as genetic variation can determine hair color, there are genetic variants that determine how an individual metabolizes specific medicines. For example, mutations may cause certain drugs may stay in one person's body longer than usual lead to serious side effects. Alternatively, another mutation will make the same medication less potent in other individuals.

An additional concern is the affect of xenobiotic metabolism on metabolic process indirectly through the production of oxidative stress. Xenobiotic metabolism will produce reactive oxygen species and induce oxidative stress directly. Further, xenobiotic metabolism will produce oxidative stress indirectly because this process itself requires energy, and may impair mitochondrial function. The primary source for energy metabolism in the cell is the mitochondria. Energy production will increase the level of reactive oxygen species. Excess activity of an enzyme like a P450 enzyme located within the mitochondrial membrane will interfere with energy production.

The treatment of patients is slowly changing. Today, before diagnosis and even a single dose of medication, a simple DNA test could reveal the medication

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and dose to be used in a particular patient. Personalized medicine will also help in adjusting pharmaceutical dose. Genetic testing can aid the design and application of better drugs in the pharmaceutical arena and decrease medical costs.

Most important, medicine will move into a new era where patients are no longer guinea pigs that are subjected to trial and error with toxic drugs.iv v This is especially important for patients with neurobehavioral disorders like schizophrenia that are subjected to one severe treatment regime after another. The outcome is eventual improvements in a minority, and increased resistance to drug treatment in a majority of patients.

“As the field advances, we expect to see more efficient clinical trials based on a more thorough understanding of the genetic basis of disease. We also anticipate that some previously failed medications will be recognized as safe and effective and will be approved for subgroups of patients with specific genetic markers.”

-Margaret Hamburg, M.D. Commissioner, U.S. Food and Drug Administration -Francis Collins, M.D., Ph.D. Director, National Institutes of Health (The Case for Personalized Medicine, 3rd Edition 2011)

Nutritional Genomics: Nutritional imbalances are observed in many disease including: aging, alcoholism/substance abuse, behavioral disorders, cancer, cardiovascular diseases, chronic fatigue, deafness, diabetes, immune disorders, macular degeneration, multiple sclerosis, neurological disorders, osteoporosis, Parkinson's, and stroke. The basic premise of nutritional genomics is that dietary recommendations based on our understanding of nutrient-gene interactions will be important for the management of complex chronic diseases.

For example, 5,10 Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme that directs folate (vitamin B12) obtained from food towards DNA and RNA synthesis, the synthesis of key metabolites: S-adenosyl methionine, homocysteine, cysteine, glutathione, and methylation of the dopamine receptor D4. S-adenosyl methionine is the second most used metabolite in the cell after ATP the major energy transducer produced in the mitochondria. Glutathione is the major intracellular antioxidant and increasing evidence links oxidative stress to autism. Homocysteine is the major metabolite shared by these pathways. In some cells, dietary choline can substitute for folate at least for the production of S-adenosyl methionine, homocysteine, and glutathione. These pathways

(Addendum II) Epigenetics and Clinical Origen of Behaviors to Optimizing Health of ASD Patients Research and Clinical Research Direction required other nutrients that must be obtained from the diet such as vitamin B6, B9, and methionine.

Genetic variations in the MTFHR (C677T and A677T) code for enzymes with lower levels of activity. Lower activity of the MTHFR enzyme as well as low levels of vitamins B12 and B6 leads to increased levels homocysteine linked to many diseases, including autism, schizophrenia, neural tube defect, cardiovascular disease, and cancer. Folate deficiencies during pregnancy are linked to increased incidence of schizophrenia and autism in offspring. Further, in some cases treatment of schizophrenia patients with folate improves psychotic symptoms. The idea that nutrition is important in neurobehavioral diseases was advocated by 50 years ago by Linus Pauling the only individual to receive two undivided Nobel prizes.

Summary: Research examining a reference set of individuals with a large variety of biochemical, and medical, and molecular tests should enable improvements in patient's health. This is a combination of research and clinical research approach, and requires the input of system biology approaches to integrate large and complex data sets.

Clinicians have many years of training and experience in medicine and sometimes research. Researchers have vast knowledge in basic and in some cases applied science but generally do not treat patients. As important as these experts are, caregivers, patients and families have personal experiences with autism, and can make important observations from their own experiences that often provide direction for treatment and research strategies.

The treatment of neurobehavioral disorders is difficult and involves knowledge across many fields including psychology, behavior, education, nutrition, research, medicine, genetics, physical/occupational therapy, and computer science. There is no one individual who is or can be an in all these areas. Instead, respective expertise must be shared in an environment that is conducive to trust and mutual respect. Then, we can make inroads into the development of best practices for the evaluation and treatment of individuals struggling with consequences of autism.

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References:

i B. M. Lester, C. J. Marsit, E. Conradt, C. Bromer and J. F. Padbury (2012) Behavioral epigenetics and the developmental origins of child mental health disorders: Journal of Developmental Origins of Health and Disease (DOHaD) pp 1-14

ii Davide Brambilla(2012) Polymeric nanoparticles as original theranostic approach for alzheimer‟s disease: Lundi 18 Juin 2012, 12:47:13

iii W. Gregory Feero, M.D., Ph.D., Editor, Alan E. Guttmacher, M.D., Editor (2012) Genomic Medicine — An Updated Primer: N Engl J Med 2010; 362:2001-2011

iv President's Council of Advisors on Science and Technology (2008) Priorities for Personalized Medicine : PRESIDENT’S COUNCIL OF ADVISORS ON SCIENCE AND TECHNOLOGY

v The Care for Personalized Medicine, Third Edition (2009) Personalized Medicine Coalition

(Addendum II) Epigenetics and Clinical Origen of Behaviors to Optimizing Health of ASD Patients Research and Clinical Research Direction

Developing a New Diagnostic and Treatment Paradigm for Autism

Autism is a Treatable Medical and Metabolic Disease with Behavioral Components

Prepared Statement of Dr. Cassandra L. Smith Professor, Biomedical Engineer, Biology and Experimental Therapeutics and Pharmacology, Boston University Director of Research, Athena Biomedical Institute

Kazuko Grace Athena Biomedical Institute www.athenabiomedicalinstitute.org Original: November 29th, 2012, Last updated January 31, 2013

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