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Metabolic and mitochondrial disorders associated with epilepsy in children with autism spectrum disorder

  • Richard E. Frye
    Correspondence
    Autism Research Program, Arkansas Children's Hospital Research Institute, Slot 512-41B, 13 Children's Way, Little Rock, AR 72202, USA. Tel.: +1 501 364 4662; fax: +1 501 978 6483.
    Affiliations
    Autism Research Program, Arkansas Children's Hospital Research Institute, Little Rock, AR, USA
    Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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Open AccessPublished:November 04, 2014DOI:https://doi.org/10.1016/j.yebeh.2014.08.134

      Highlights

      • Many children with ASD have underlying metabolic conditions.
      • Metabolic disorders are also commonly associated with epilepsy.
      • Treating metabolic disorders may optimize seizure management.

      Abstract

      Autism spectrum disorder (ASD) affects a significant number of individuals in the United States, with the prevalence continuing to grow. A significant proportion of individuals with ASD have comorbid medical conditions such as epilepsy. In fact, treatment-resistant epilepsy appears to have a higher prevalence in children with ASD than in children without ASD, suggesting that current antiepileptic treatments may be suboptimal in controlling seizures in many individuals with ASD. Many individuals with ASD also appear to have underlying metabolic conditions. Metabolic conditions such as mitochondrial disease and dysfunction and abnormalities in cerebral folate metabolism may affect a substantial number of children with ASD, while other metabolic conditions that have been associated with ASD such as disorders of creatine, cholesterol, pyridoxine, biotin, carnitine, γ-aminobutyric acid, purine, pyrimidine, and amino acid metabolism and urea cycle disorders have also been associated with ASD without the prevalence clearly known. Interestingly, all of these metabolic conditions have been associated with epilepsy in children with ASD. The identification and treatment of these disorders could improve the underlying metabolic derangements and potentially improve behavior and seizure frequency and/or severity in these individuals. This paper provides an overview of these metabolic disorders in the context of ASD and discusses their characteristics, diagnostic testing, and treatment with concentration on mitochondrial disorders. To this end, this paper aims to help optimize the diagnosis and treatment of children with ASD and epilepsy.
      This article is part of a Special Issue entitled “Autism and Epilepsy”.

      Keywords

      1. Introduction

      Autism spectrum disorders (ASDs) are a group of behaviorally defined neurodevelopmental disorders with lifelong consequences. They are defined by impairments in communication and social interaction along with restrictive and repetitive behaviors [
      • APA
      Diagnostic and statistical manual of mental disorders.
      ]. Autism spectrum disorder is now estimated to affect 1 out of 68 individuals in the United States, with approximately four times more males than females being affected [
      Developmental Disabilities Monitoring Network Surveillance Year Principal I, Centers for Disease C, Prevention. Prevalence of autism spectrum disorder among children aged 8 years — autism and developmental disabilities monitoring network, 11 sites, United States, 2010.
      ]. Although ASD is behaviorally defined, children with ASD also have many co-occurring medical conditions such as gastrointestinal abnormalities [
      • Chaidez V.
      • Hansen R.L.
      • Hertz-Picciotto I.
      Gastrointestinal problems in children with autism, developmental delays or typical development.
      ], seizures and epilepsy [
      • Frye R.E.
      • Rossignol D.
      • Casanova M.F.
      • Brown G.L.
      • Martin V.
      • Edelson S.
      • et al.
      A review of traditional and novel treatments for seizures in autism spectrum disorder: findings from a systematic review and expert panel.
      ], attention deficits [
      • Taurines R.
      • Schwenck C.
      • Westerwald E.
      • Sachse M.
      • Siniatchkin M.
      • Freitag C.
      ADHD and autism: differential diagnosis or overlapping traits? A selective review.
      ], anxiety [
      • Sukhodolsky D.G.
      • Bloch M.H.
      • Panza K.E.
      • Reichow B.
      Cognitive–behavioral therapy for anxiety in children with high-functioning autism: a meta-analysis.
      ], and allergies [
      • Angelidou A.
      • Alysandratos K.D.
      • Asadi S.
      • Zhang B.
      • Francis K.
      • Vasiadi M.
      • et al.
      Brief report: “allergic symptoms” in children with autism spectrum disorders. More than meets the eye?.
      ], just to name a few.
      One of the most significant comorbidities associated with ASD that causes significant disability is epilepsy. A number of studies suggest that epilepsy affects a high proportion of individuals with ASD. Indeed, the reported prevalence of epilepsy in ASD ranges from 5% to 38%, which is clearly higher than the 1%–2% prevalence in the general childhood population [
      • Tuchman R.
      • Rapin I.
      Epilepsy in autism.
      ,
      • Deykin E.Y.
      • MacMahon B.
      The incidence of seizures among children with autistic symptoms.
      ,
      • Volkmar F.R.
      • Nelson D.S.
      Seizure disorders in autism.
      ,
      • Danielsson S.
      • Gillberg I.C.
      • Billstedt E.
      • Gillberg C.
      • Olsson I.
      Epilepsy in young adults with autism: a prospective population-based follow-up study of 120 individuals diagnosed in childhood.
      ,
      • Hara H.
      Autism and epilepsy: a retrospective follow-up study.
      ]. In addition, the prevalence of treatment-resistant epilepsy is believed to be higher in children with ASD than in the general childhood population [
      • Sansa G.
      • Carlson C.
      • Doyle W.
      • Weiner H.L.
      • Bluvstein J.
      • Barr W.
      • et al.
      Medically refractory epilepsy in autism.
      ]. Interestingly, recent reviews note shared cognitive symptoms in epilepsy and ASD, suggesting a common etiopathology [
      • Gilby K.L.
      • O'Brien T.J.
      Epilepsy, autism, and neurodevelopment: kindling a shared vulnerability?.
      ], especially when ASD coexists with intellectual disability [
      • Tuchman R.
      Autism and social cognition in epilepsy: implications for comprehensive epilepsy care.
      ].
      The great preponderance of ASD research has concentrated on genetic causes of ASD [
      • Rossignol D.A.
      • Frye R.E.
      A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures.
      ], despite the fact that inherited single gene and chromosomal defects are only found in the minority of ASD cases [
      • Schaefer G.B.
      • Mendelsohn N.J.
      • Professional P.
      • Guidelines C.
      Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions.
      ]. However, unlike idiopathic ASD, many genetic syndromes that have a high prevalence of ASD also frequently have a high incidence of epilepsy [
      • Tuchman R.
      • Hirtz D.
      • Mamounas L.A.
      NINDS epilepsy and autism spectrum disorders workshop report.
      ], and gene mutations associated with ASD are also frequently associated with epilepsy [
      • Murdoch J.D.
      • State M.W.
      Recent developments in the genetics of autism spectrum disorders.
      ]. Epilepsy also frequently co-occurs with ASD in individuals who manifest metabolic abnormalities such as abnormalities in mitochondrial metabolism [
      • Rossignol D.A.
      • Frye R.E.
      A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures.
      ] as well as abnormalities in the regulation of essential metabolites such as folate [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ,
      • Rossignol D.
      • Frye R.E.
      Folate receptor alpha autoimmunity and cerebral folate deficiency in autism spectrum disorders.
      ], cholesterol [
      • Tierney E.
      • Bukelis I.
      • Thompson R.E.
      • Ahmed K.
      • Aneja A.
      • Kratz L.
      • et al.
      Abnormalities of cholesterol metabolism in autism spectrum disorders.
      ], and branched-chain amino acid [
      • Novarino G.
      • El-Fishawy P.
      • Kayserili H.
      • Meguid N.A.
      • Scott E.M.
      • Schroth J.
      • et al.
      Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy.
      ]. One interesting aspect of metabolic disorders in relation to ASD is that some children with ASD have clear classic inborn-inherited errors of metabolism, while perhaps more have metabolic abnormalities that do not have a clear relationship to known inherited genetic abnormalities.
      Several reviews have described some of the classic inborn-inherited errors of metabolism that are associated with ASD [
      • Schaefer G.B.
      • Mendelsohn N.J.
      • Professional P.
      • Guidelines C.
      Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions.
      ,
      • Zecavati N.
      • Spence S.J.
      Neurometabolic disorders and dysfunction in autism spectrum disorders.
      ]. Other reviews have taken a broader view by including metabolic disorders that do not necessarily have a clear genetic basis [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ,
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ,
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Frye R.E.
      • Rossignol D.
      Metabolic disorders and abnormalities associated with autism spectrum disorder.
      ]. However, these reviews have not concentrated on metabolic abnormalities associated with ASD with respect to epilepsy. The fact that metabolic disorders are associated with ASD suggests that, in some individuals, ASD symptoms may arise from systemic abnormalities rather than from abnormalities specifically localized to the brain.
      Identifying the metabolic abnormalities associated with ASD, especially for individuals with ASD who have comorbid epilepsy, is important as clarifying the underlying comorbid condition that may be causing both the epilepsy and ASD can potentially lead to optimizing treatment options in order to improve outcomes for individuals with ASD as well as their families. In addition, since many metabolic pathways are well understood, identifying metabolic defects can lead to augmenting standard epilepsy treatment with known or novel treatments [
      • Frye R.E.
      • Rossignol D.
      • Casanova M.F.
      • Brown G.L.
      • Martin V.
      • Edelson S.
      • et al.
      A review of traditional and novel treatments for seizures in autism spectrum disorder: findings from a systematic review and expert panel.
      ]. Furthermore, by understanding metabolic and genetic biomarkers that can identify these disorders, it might be possible to detect these disorders early in life, even prenatally, so treatment can be started at the earliest possible time, potentially before ASD symptoms or epilepsy develops, in order to improve long-term outcome.
      Given the fact that metabolic disorders may be amenable to treatment, it is of paramount importance that physicians are aware of the clinical features that are indicative of a metabolic disorder and appropriately investigate patients with suggestive presentations. However, unlike many diseases, metabolic disorders may not have particular classic presentations, so basing a diagnostic strategy on the search for one or two specific key symptoms is inappropriate. For example, patients with mitochondrial disorders can present with a variety of primary manifestations including neurologic, muscular, multisystemic, or psychiatric. Thus, it is essential for the clinician to become sensitive to the variety of potential symptoms and the patterns to which the symptoms manifest. This is of significant importance when patients manifest primary psychiatric symptomatology as such disorders are classically diagnosed based on symptoms rather than on biochemical, metabolic, or neuroimaging evaluations. Identifying an individual with a psychiatric disorder with underlying metabolic abnormalities can significantly positively alter therapeutic management [
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ,
      • Anglin R.E.
      • Garside S.L.
      • Tarnopolsky M.A.
      • Mazurek M.F.
      • Rosebush P.I.
      The psychiatric manifestations of mitochondrial disorders: a case and review of the literature.
      ].
      This article reviews the metabolic syndromes that are associated with individuals with ASD and comorbid epilepsy. As many of these syndromes are rather rare, the more rare syndromes are discussed briefly. However, mitochondrial disease will be discussed in detail as it is of particular interest in children with ASD since it is being increasingly recognized as a cause of epilepsy in individuals with ASD [
      • Frye R.E.
      • Rossignol D.
      • Casanova M.F.
      • Brown G.L.
      • Martin V.
      • Edelson S.
      • et al.
      A review of traditional and novel treatments for seizures in autism spectrum disorder: findings from a systematic review and expert panel.
      ,
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ] and those without ASD [
      • Kang H.C.
      • Lee Y.M.
      • Kim H.D.
      Mitochondrial disease and epilepsy.
      ,
      • Kunz W.S.
      The role of mitochondria in epileptogenesis.
      ,
      • Kudin A.P.
      • Zsurka G.
      • Elger C.E.
      • Kunz W.S.
      Mitochondrial involvement in temporal lobe epilepsy.
      ] and novel treatments are being developed for mitochondrial disease which may improve therapeutic options [
      • Ghanizadeh A.
      • Berk M.
      • Farrashbandi H.
      • Alavi Shoushtari A.
      • Villagonzalo K.A.
      Targeting the mitochondrial electron transport chain in autism, a systematic review and synthesis of a novel therapeutic approach.
      ,
      • Kerr D.S.
      Review of clinical trials for mitochondrial disorders: 1997–2012.
      ]. To help better understand some of the metabolic abnormalities underlying epilepsy and ASD, we will discuss the animal models of ASD that manifest epilepsy and metabolic abnormalities. Finally, to facilitate the clinical application of the information presented in the article, we discuss an approach to diagnosing metabolic abnormalities in individuals with ASD and epilepsy.

      1.1 Metabolic disorder associated with epilepsy in autism spectrum disorder

      Table 1 outlines the metabolic disorders associated with ASD and comorbid epilepsy. This table organizes the metabolic disorders into several categories, including disorders of energy, cholesterol, vitamin, γ-aminobutyric acid (GABA), purine, pyrimidine, and amino acid metabolism and urea cycle disorders. The prominent symptoms for each disorder, not including ASD and epilepsy, along with the diagnostic tests used to identify the disorder are outlined. Each disorder will be reviewed within its category below.
      Table 1Metabolic disorders associated with epilepsy and autism spectrum disorder.
      DisorderClinical featuresDiagnostic testing
      Disorders of energy metabolism
      Mitochondrial diseaseDevelopmental regression, gross motor delay, fatigability, ataxia, and gastrointestinal abnormalities• Fasting serum lactate, pyruvate, acylcarnitine, amino acids, and urine organic acids
      Creatine metabolism disorderDevelopmental regression, mental retardation, dyskinesia, and family history of x-linked mental retardation• Magnetic resonance spectroscopy
      • Urine and serum creatine and guanidinoacetic acid
      Disorders of cholesterol metabolism
      Smith–Lemli–Opitz syndromeLow birth weight, failure to thrive, poor feeding, eczema, and congenital structural abnormalities of the heart, gastrointestinal tract, genitalia, kidney, limbs, face, and brain• Blood 7-dehydrocholesterol and cholesterol
      • DHCR7 sequencing
      Disorders of cofactor (vitamin) metabolism
      Cerebral folate deficiencyAtaxia, pyramidal signs, acquired microcephaly, dyskinesias, and visual and hearing loss• Folate receptor alpha autoantibody
      • Cerebrospinal fluid 5-methyltetrahydrofolate
      Pyridoxine-dependent and pyridoxine-responsive seizuresMental retardation, breath-holding, aerophagia, and self-injurious behavior• Pyridoxine trial
      • Plasma and cerebrospinal fluid pipecolic acid
      • Urine α-aminoadipic semialdehyde
      ALDH7A1 sequencing
      Biotinidase deficiencyDevelopmental delays, seborrheic dermatitis, alopecia, feeding difficulties, vomiting, diarrhea, brain atrophy, and ataxia• Biotinidase activity
      • BTD gene sequencing
      Carnitine biosynthesis deficiencyNondysmorphic male–male siblings with autism spectrum disorder• Plasma and/or urine 6-N-trimethyllysine, 3-hydroxy-6-N-trimethyllysine, and γ-butyrobetaine.
      Disorders of γ-aminobutyric acid metabolism
      Succinic semialdehyde dehydrogenase deficiencyGlobal developmental delay, myoclonus, hallucinations, ataxia, choreoathetosis, and dystonia• Urine gamma-hydroxybutyric acid
      Disorders of pyrimidine and purine metabolism
      Adenylosuccinate lyase deficiencyGlobal developmental delay, microcephaly, distinct facies, growth retardation, mental retardation, cerebellar vermis hypoplasia, brain atrophy, excessive laughter, and extreme happiness• Urine and/or cerebrospinal fluid succinyladenosine
      Nucleotidase-associated PDDHyperactivity, compulsiveness, speech abnormalities, ataxia, abnormal gait, and frequent infections• Urine uridine
      Hyperuricosuric autismAltered sensory awareness, ataxia, and fine motor deficits• 24-hour urine urate
      Phosphoribosylpyrophosphate synthetase deficiencyDevelopmental delay and ataxia• Urine uric and orotic acids
      • Complete blood count
      Disorders of amino acid metabolism
      PhenylketonuriaGlobal developmental delay, mental retardation, microcephaly, spasticity, ataxia, poor growth, poor skin pigmentation, and aggressive behavior• Serum phenylalanine
      Branched-chain ketoacid dehydrogenase kinase deficiencyIntellectual disability and consanguinity• Plasma and cerebrospinal fluid branched-chain amino acids
      Altered tryptophan metabolismNo specific features besides autism spectrum disorder• Reduced cellular generation of nicotinamide adenine dinucleotide
      Urea cycle disorders
      Urea cycle disorderProtein intolerance, temperature instability, ataxia, episodic somnolence and lethargy, cyclic vomiting, and psychosis• Plasma ammonia and amino acids
      • Urinary orotic acid

      1.1.1 Disorders of energy metabolism

      Several disorders affecting energy metabolism have been documented in ASD, including mitochondrial disorders and creatine deficiency syndromes. The prevalence of mitochondrial abnormalities in ASD appears to be unusually high compared with that of typically developing individuals [
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ], and mitochondrial abnormalities have been implicated in epilepsy in individuals without ASD, especially in individuals with treatment-resistant [
      • Kunz W.S.
      The role of mitochondria in epileptogenesis.
      ] and temporal lobe [
      • Kudin A.P.
      • Zsurka G.
      • Elger C.E.
      • Kunz W.S.
      Mitochondrial involvement in temporal lobe epilepsy.
      ] epilepsies. Mitochondrial abnormalities in relationship to ASD will be discussed in more detail in a separate section below, while creatine deficiency syndromes are discussed immediately below.
      Creatine is synthesized in the liver and kidney and is transported through the blood to high-energy demand tissues, such as the brain and skeletal muscle, where it is actively transported into the tissue against a large concentration gradient by the sodium/chloride-dependent transporter known as CrT1 which is coded by the SLC6A8 gene. Once in tissues, creatine is phosphorylated by creatine kinase to phosphocreatine, the main energy storage molecule of the cell, using adenosine triphosphate (ATP). Without creatine, phosphocreatine cannot be produced, and cells will become rapidly depleted in energy.
      Disorders of creatine metabolism have been reported in children with ASD and epilepsy [
      • Poo-Arguelles P.
      • Arias A.
      • Vilaseca M.A.
      • Ribes A.
      • Artuch R.
      • Sans-Fito A.
      • et al.
      X-linked creatine transporter deficiency in two patients with severe mental retardation and autism.
      ,
      • Longo N.
      • Ardon O.
      • Vanzo R.
      • Schwartz E.
      • Pasquali M.
      Disorders of creatine transport and metabolism.
      ]. Three inborn disorders of creatine metabolism, collectively known as the creatine deficiency syndromes, have been described since 1994. Two disorders involve deficiencies in enzymes responsible for creatine production, arginine:glycine amidinotransferase (AGAT), and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT), while the third disorder involves a deficiency in the creatine transporter.
      The general presentation of children with disorders of creatine metabolism includes developmental delay, regression, ASD features, mental retardation, receptive and expressive language disorders, dyskinesia, and seizures [
      • Schulze A.
      Creatine deficiency syndromes.
      ]. The severity of the symptoms depends on the specific disorder. Individuals with GAMT deficiency are the most severely affected, with almost invariable development of ASD and seizures, severe delays in language, and magnetic resonance imaging (MRI) abnormalities. Individuals with creatine transporter disorder demonstrate a milder phenotype, while children with AGAT deficiency demonstrate the mildest phenotype [
      • Schulze A.
      Creatine deficiency syndromes.
      ]. Creatine transporter deficiency is an X-linked recessive disorder, so a family history of X-linked mental retardation is supportive of the diagnosis. Several reports suggest that creatine deficiency disorders can be treated with high-dose creatine monohydrate and a diet containing specific amino acids [
      • van Karnebeek C.D.
      • Stockler S.
      Treatable inborn errors of metabolism causing intellectual disability: a systematic literature review.
      ,
      • Anselm I.A.
      • Alkuraya F.S.
      • Salomons G.S.
      • Jakobs C.
      • Fulton A.B.
      • Mazumdar M.
      • et al.
      X-linked creatine transporter defect: a report on two unrelated boys with a severe clinical phenotype.
      ].

      1.1.2 Disorders of cholesterol metabolism

      Smith–Lemli–Opitz syndrome (SLOS) is a congenital disorder caused by mutations in both DHCR7 genes, the genes that encode the Δ-7-dehydrocholesterol reductase enzyme, a precursor step for the production of cholesterol. Metabolically, children with SLOS demonstrate elevated concentrations of 7-dehydrocholesterol and reduced cholesterol concentrations in the blood. Interestingly, 50%–75% of children with this disorder meet the criteria for ASD [
      • Sikora D.M.
      • Pettit-Kekel K.
      • Penfield J.
      • Merkens L.S.
      • Steiner R.D.
      The near universal presence of autism spectrum disorders in children with Smith–Lemli–Opitz syndrome.
      ,
      • Bukelis I.
      • Porter F.D.
      • Zimmerman A.W.
      • Tierney E.
      Smith–Lemli–Opitz syndrome and autism spectrum disorder.
      ]. This disorder is characterized by low birth weight, failure to thrive, poor feeding, eczema, seizures, and congenital structural abnormalities of the heart, gastrointestinal tract, genitalia, kidney, limbs, face, and brain [
      • Sikora D.M.
      • Pettit-Kekel K.
      • Penfield J.
      • Merkens L.S.
      • Steiner R.D.
      The near universal presence of autism spectrum disorders in children with Smith–Lemli–Opitz syndrome.
      ,
      • Bukelis I.
      • Porter F.D.
      • Zimmerman A.W.
      • Tierney E.
      Smith–Lemli–Opitz syndrome and autism spectrum disorder.
      ]. Treatment with cholesterol supplementation in children with SLOS has been reported to improve ASD and associated behavioral symptoms in a case report [
      • Martin A.
      • Koenig K.
      • Scahill L.
      • Tierney E.
      • Porter F.D.
      • Nwokoro N.A.
      Smith–Lemli–Opitz syndrome.
      ], case series [
      • Elias E.R.
      • Irons M.B.
      • Hurley A.D.
      • Tint G.S.
      • Salen G.
      Clinical effects of cholesterol supplementation in six patients with the Smith–Lemli–Opitz syndrome (SLOS).
      ], and prospective cohorts [
      • Irons M.
      • Elias E.R.
      • Abuelo D.
      • Bull M.J.
      • Greene C.L.
      • Johnson V.P.
      • et al.
      Treatment of Smith–Lemli–Opitz syndrome: results of a multicenter trial.
      ,
      • Nwokoro N.A.
      • Mulvihill J.J.
      Cholesterol and bile acid replacement therapy in children and adults with Smith–Lemli–Opitz (SLO/RSH) syndrome.
      ], especially in young children [
      • Aneja A.
      • Tierney E.
      Autism: the role of cholesterol in treatment.
      ], but the effectiveness of such treatment in seizure control has not been studied. This disorder can be diagnosed by measuring 7-dehydrocholesterol and cholesterol levels or by DHCR7 sequencing. It is important not to rely on cholesterol levels alone to diagnose SLOS as depressed levels of cholesterol are rather common in children with ASD who do not have SLOS [
      • Tierney E.
      • Bukelis I.
      • Thompson R.E.
      • Ahmed K.
      • Aneja A.
      • Kratz L.
      • et al.
      Abnormalities of cholesterol metabolism in autism spectrum disorders.
      ].

      1.1.3 Disorders of vitamin metabolism

      Disorders of vitamin metabolism that have been reported in children with ASD and epilepsy include disorders of folate, pyridoxine, biotin, and carnitine metabolism, each of which will be reviewed below.
      Several folate metabolism abnormalities have been linked to ASD. Polymorphisms in key folate pathway enzymes such as methylenetetrahydrofolate reductase [
      • Frustaci A.
      • Neri M.
      • Cesario A.
      • Adams J.B.
      • Domenici E.
      • Dalla Bernardina B.
      • et al.
      Oxidative stress-related biomarkers in autism: systematic review and meta-analyses.
      ,
      • Boris M.
      • Goldblatt A.
      • Galanko J.
      • James S.J.
      Association of MTHFR gene variants with autism.
      ,
      • Mohammad N.S.
      • Jain J.M.
      • Chintakindi K.P.
      • Singh R.P.
      • Naik U.
      • Akella R.R.
      Aberrations in folate metabolic pathway and altered susceptibility to autism.
      ,
      • Guo T.
      • Chen H.
      • Liu B.
      • Ji W.
      • Yang C.
      Methylenetetrahydrofolate reductase polymorphisms C677T and risk of autism in the Chinese Han population.
      ,
      • Schmidt R.J.
      • Tancredi D.J.
      • Ozonoff S.
      • Hansen R.L.
      • Hartiala J.
      • Allayee H.
      • et al.
      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.
      ,
      • Liu X.
      • Solehdin F.
      • Cohen I.L.
      • Gonzalez M.G.
      • Jenkins E.C.
      • Lewis M.E.
      • et al.
      Population- and family-based studies associate the MTHFR gene with idiopathic autism in simplex families.
      ,
      • Goin-Kochel R.P.
      • Porter A.E.
      • Peters S.U.
      • Shinawi M.
      • Sahoo T.
      • Beaudet A.L.
      The MTHFR 677C→T polymorphism and behaviors in children with autism: exploratory genotype–phenotype correlations.
      ,
      • Pasca S.P.
      • Dronca E.
      • Kaucsar T.
      • Craciun E.C.
      • Endreffy E.
      • Ferencz B.K.
      • et al.
      One carbon metabolism disturbances and the C677T MTHFR gene polymorphism in children with autism spectrum disorders.
      ,
      • James S.J.
      • Melnyk S.
      • Jernigan S.
      • Cleves M.A.
      • Halsted C.H.
      • Wong D.H.
      • et al.
      Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism.
      ,
      • Pu D.
      • Shen Y.
      • Wu J.
      Association between MTHFR gene polymorphisms and the risk of autism spectrum disorders: a meta-analysis.
      ], dihydrofolate reductase [
      • Adams M.
      • Lucock M.
      • Stuart J.
      • Fardell S.
      • Baker K.
      • Ng X.
      Preliminary evidence for involvement of the folate gene polymorphism 19 bp deletion-DHFR in occurrence of autism.
      ], and the reduced folate carrier [
      • James S.J.
      • Melnyk S.
      • Jernigan S.
      • Cleves M.A.
      • Halsted C.H.
      • Wong D.H.
      • et al.
      Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism.
      ] have been associated with ASD but have not specifically been associated with epilepsy in individuals with ASD. However, children with cerebral folate deficiency (CFD) are commonly diagnosed with epilepsy and/or ASD [
      • Ramaekers V.T.
      • Rothenberg S.P.
      • Sequeira J.M.
      • Opladen T.
      • Blau N.
      • Quadros E.V.
      • et al.
      Autoantibodies to folate receptors in the cerebral folate deficiency syndrome.
      ,
      • Ramaekers V.T.
      • Blau N.
      • Sequeira J.M.
      • Nassogne M.C.
      • Quadros E.V.
      Folate receptor autoimmunity and cerebral folate deficiency in low-functioning autism with neurological deficits.
      ,
      • Ramaekers V.T.
      • Blau N.
      Cerebral folate deficiency.
      ]. Folate is transported across the blood–brain barrier by an energy-dependent receptor-mediated system that utilizes the folate receptor alpha (FRα) [
      • Wollack J.B.
      • Makori B.
      • Ahlawat S.
      • Koneru R.
      • Picinich S.C.
      • Smith A.
      • et al.
      Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model.
      ]. Autoantibodies that can bind to the FRα and greatly impair its function have been linked to CFD [
      • Ramaekers V.T.
      • Rothenberg S.P.
      • Sequeira J.M.
      • Opladen T.
      • Blau N.
      • Quadros E.V.
      • et al.
      Autoantibodies to folate receptors in the cerebral folate deficiency syndrome.
      ]. Since this transport system is energy-dependent, a wide variety of mitochondrial diseases [
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Allen R.J.
      • DiMauro S.
      • Coulter D.L.
      • Papadimitriou A.
      • Rothenberg S.P.
      Kearns–Sayre syndrome with reduced plasma and cerebrospinal fluid folate.
      ,
      • Pineda M.
      • Ormazabal A.
      • Lopez-Gallardo E.
      • Nascimento A.
      • Solano A.
      • Herrero M.D.
      • et al.
      Cerebral folate deficiency and leukoencephalopathy caused by a mitochondrial DNA deletion.
      ,
      • Ramaekers V.T.
      • Weis J.
      • Sequeira J.M.
      • Quadros E.V.
      • Blau N.
      Mitochondrial complex I encephalomyopathy and cerebral 5-methyltetrahydrofolate deficiency.
      ,
      • Hasselmann O.
      • Blau N.
      • Ramaekers V.T.
      • Quadros E.V.
      • Sequeira J.M.
      • Weissert M.
      Cerebral folate deficiency and CNS inflammatory markers in Alpers disease.
      ,
      • Perez-Duenas B.
      • Ormazabal A.
      • Toma C.
      • Torrico B.
      • Cormand B.
      • Serrano M.
      • et al.
      Cerebral folate deficiency syndromes in childhood: clinical, analytical, and etiologic aspects.
      ,
      • Garcia-Cazorla A.
      • Quadros E.V.
      • Nascimento A.
      • Garcia-Silva M.T.
      • Briones P.
      • Montoya J.
      • et al.
      Mitochondrial diseases associated with cerebral folate deficiency.
      ,
      • Shoffner J.
      • Hyams L.
      • Langley G.N.
      • Cossette S.
      • Mylacraine L.
      • Dale J.
      • et al.
      Fever plus mitochondrial disease could be risk factors for autistic regression.
      ] and novel forms of mitochondrial dysfunction related to ASD [
      • Frye R.E.
      • Naviaux R.K.
      Autistic disorder with complex IV overactivity: a new mitochondrial syndrome.
      ] have been associated with CFD. Recently, Frye et al. [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ] reported that 75% of 93 children with ASD were positive for one of the two FRα autoantibodies. The high rate of FRα autoantibody positivity was confirmed by a recent study from Belgium that measured the FRα blocking autoantibody. This study found that 47% of 75 children with ASD were positive for the FRα blocking autoantibody compared with 3% of 30 controls with developmental delays but not autism [
      • Ramaekers V.T.
      • Quadros E.V.
      • Sequeira J.M.
      Role of folate receptor autoantibodies in infantile autism.
      ]. Many children with ASD and CFD have marked improvement in clinical status when treated with folinic acid — a reduced form of folate that can cross the blood–brain barrier using the reduced folate carrier rather than the FRα transport system [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ,
      • Ramaekers V.T.
      • Rothenberg S.P.
      • Sequeira J.M.
      • Opladen T.
      • Blau N.
      • Quadros E.V.
      • et al.
      Autoantibodies to folate receptors in the cerebral folate deficiency syndrome.
      ,
      • Ramaekers V.T.
      • Blau N.
      • Sequeira J.M.
      • Nassogne M.C.
      • Quadros E.V.
      Folate receptor autoimmunity and cerebral folate deficiency in low-functioning autism with neurological deficits.
      ,
      • Moretti P.
      • Sahoo T.
      • Hyland K.
      • Bottiglieri T.
      • Peters S.
      • del Gaudio D.
      • et al.
      Cerebral folate deficiency with developmental delay, autism, and response to folinic acid.
      ]. Folate receptor alpha autoantibody testing can be performed clinically, but a lumbar puncture to measure the cerebrospinal fluid concentration of 5-methyltetrahydrofolate is the gold standard diagnostic test for CFD.
      Pyridoxine and its primary biologically active form pyridoxal-5-phosphate play major roles in the proper function of over 60 enzymes. Pyridoxal-5-phosphate is a cofactor for glutamic acid decarboxylase, the enzyme that metabolizes glutamic acid to GABA. Usually, pyridoxine-dependent seizures and pyridoxine-responsive seizures present as intractable seizures in the first months of life and are defined by their clinical response to pyridoxine therapy [
      • Basura G.J.
      • Hagland S.P.
      • Wiltse A.M.
      • Gospe Jr., S.M.
      Clinical features and the management of pyridoxine-dependent and pyridoxine-responsive seizures: review of 63 North American cases submitted to a patient registry.
      ,
      • Gupta V.K.
      • Mishra D.
      • Mathur I.
      • Singh K.K.
      Pyridoxine-dependent seizures: a case report and a critical review of the literature.
      ]. The majority of pyridoxine-dependent seizures appear to result from a deficiency in the enzyme α-aminoadipic semialdehyde dehydrogenase associated with mutations in the ALDH7A1 (antiquitin) gene [
      • Mills P.B.
      • Struys E.
      • Jakobs C.
      • Plecko B.
      • Baxter P.
      • Baumgartner M.
      • et al.
      Mutations in antiquitin in individuals with pyridoxine-dependent seizures.
      ,
      • Plecko B.
      • Paul K.
      • Paschke E.
      • Stoeckler-Ipsiroglu S.
      • Struys E.
      • Jakobs C.
      • et al.
      Biochemical and molecular characterization of 18 patients with pyridoxine-dependent epilepsy and mutations of the antiquitin (ALDH7A1) gene.
      ]. These mutations result in the excess production of Δ1-piperideine-6-carboxylate, a compound which complexes with and depletes pyridoxal-5-phosphate [
      • Mills P.B.
      • Struys E.
      • Jakobs C.
      • Plecko B.
      • Baxter P.
      • Baumgartner M.
      • et al.
      Mutations in antiquitin in individuals with pyridoxine-dependent seizures.
      ]. Pyridoxal-5-phosphate depletion reduces glutamic acid decarboxylase activity, resulting in a reduction in GABA synthesis [
      • Mills P.B.
      • Struys E.
      • Jakobs C.
      • Plecko B.
      • Baxter P.
      • Baumgartner M.
      • et al.
      Mutations in antiquitin in individuals with pyridoxine-dependent seizures.
      ,
      • Gospe S.M.
      Pyridoxine-dependent seizures: findings from recent studies pose new questions.
      ,
      • Gospe Jr., S.M.
      • Olin K.L.
      • Keen C.L.
      Reduced GABA synthesis in pyridoxine-dependent seizures.
      ]. Although early-onset intractable tonic–clonic seizures are the usual presentation, late-onset seizures [
      • Bankier A.
      • Turner M.
      • Hopkins I.J.
      Pyridoxine dependent seizures—a wider clinical spectrum.
      ,
      • Coker S.B.
      Postneonatal vitamin B6-dependent epilepsy.
      ,
      • Goutieres F.
      • Aicardi J.
      Atypical presentations of pyridoxine-dependent seizures: a treatable cause of intractable epilepsy in infants.
      ] and other seizure types [
      • Haenggeli C.A.
      • Girardin E.
      • Paunier L.
      Pyridoxine-dependent seizures, clinical and therapeutic aspects.
      ,
      • Krishnamoorthy K.S.
      Pyridoxine-dependency seizure: report of a rare presentation.
      ,
      • Mikati M.A.
      • Trevathan E.
      • Krishnamoorthy K.S.
      • Lombroso C.T.
      Pyridoxine-dependent epilepsy: EEG investigations and long-term follow-up.
      ] have been described. It has been suggested that pyridoxine-responsive seizures may be a clinical entity distinct from pyridoxine-dependent seizures [
      • Baxter P.
      Epidemiology of pyridoxine dependent and pyridoxine responsive seizures in the UK.
      ]. In children with ASD, several studies have reported significant improvement in behavior and cognition attributable to combined therapy with magnesium and pyridoxine [
      • Martineau J.
      • Barthelemy C.
      • Garreau B.
      • Lelord G.
      Vitamin B6, magnesium, and combined B6-Mg: therapeutic effects in childhood autism.
      ,
      • Mousain-Bosc M.
      • Roche M.
      • Polge A.
      • Pradal-Prat D.
      • Rapin J.
      • Bali J.P.
      Improvement of neurobehavioral disorders in children supplemented with magnesium-vitamin B6. II. Pervasive developmental disorder-autism.
      ,
      • Lelord G.
      • Callaway E.
      • Muh J.P.
      Clinical and biological effects of high doses of vitamin B6 and magnesium on autistic children.
      ,
      • Lelord G.
      • Muh J.P.
      • Barthelemy C.
      • Martineau J.
      • Garreau B.
      • Callaway E.
      Effects of pyridoxine and magnesium on autistic symptoms—initial observations.
      ], but others have not been able to document such a response [
      • Nye C.
      • Brice A.
      Combined vitamin B6–magnesium treatment in autism spectrum disorder.
      ,
      • Rossignol D.A.
      Novel and emerging treatments for autism spectrum disorders: a systematic review.
      ]. There has been one case report of ASD associated with severe mental retardation, aerophagia, breath-holding, self-injury, and pyridoxine-dependent seizures [
      • Burd L.
      • Stenehjem A.
      • Franceschini L.A.
      • Kerbeshian J.
      A 15-year follow-up of a boy with pyridoxine (vitamin B6)-dependent seizures with autism, breath holding, and severe mental retardation.
      ]. According to the report, pyridoxine improved seizures but did not improve ASD features. When suspected, pyridoxine-dependent seizures can be diagnosed by measuring plasma and/or cerebrospinal fluid concentration of pipecolic acid, measuring urine concentrations of α-aminoadipic semialdehyde, or sequencing the ALDH7A1 gene. A pyridoxine trial can also be useful clinically.
      Biotinidase deficiency is caused by mutations in both BTD genes which results in a deficiency of the biotinidase enzyme, an enzyme that is needed to recycle biotin, an essential cofactor for several carboxylase enzymes. Typical onset is early in life, between the 1st and 24th months of life. Symptoms include seizures, developmental delays, skin rash, seborrheic dermatitis, alopecia, feeding difficulties, vomiting, diarrhea, brain atrophy, and ataxia. Metabolic testing can demonstrate elevated blood lactate and ammonia as well as abnormal urine organic acids including elevations in β-hydroxyisovalerate, β-methylcrotonylglycine, β–hydroxypropionate, and methylcitrate. Standard treatment is 10 mg of daily biotin. The one reported child with ASD and partial biotinidase deficiency did not respond to treatment, although it was believed that ASD was prevented in his younger brother who also manifested symptoms of partial biotinidase deficiency [
      • Zaffanello M.
      • Zamboni G.
      • Fontana E.
      • Zoccante L.
      • Tato L.
      A case of partial biotinidase deficiency associated with autism.
      ]. Biotinidase deficiency is characterized by neurodevelopment problems and seborrheic dermatitis in children. Biotinidase activity can be tested clinically, and any suspicious case should be confirmed by BTD gene sequencing.
      Recently, a defect in the carnitine biosynthesis pathway was described in seven children with ASD, one of whom also had epilepsy [
      • Celestino-Soper P.B.
      • Violante S.
      • Crawford E.L.
      • Luo R.
      • Lionel A.C.
      • Delaby E.
      • et al.
      A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism.
      ]. This defect in exon 2 of the X chromosome TMLHE gene encodes the first enzyme in the carnitine biosynthesis pathway, specifically 6-N-trimethyllysine dioxygenase. Interestingly, this genetic change was not more common in children with ASD than in control children overall but was more common in probands from families with male–male multiplex ASD compared with controls, suggesting that this was a risk factor for these families rather than a causative metabolic disease. Carnitine deficiency appears to be common in the wider populations with ASD, but children with ASD and carnitine deficiency have not been well characterized regarding their ASD and medical symptoms such as epilepsy [
      • Filipek P.A.
      • Juranek J.
      • Nguyen M.T.
      • Cummings C.
      • Gargus J.J.
      Relative carnitine deficiency in autism.
      ]. Given the lack of carnitine genes found to be directly related to ASD, some authors have hypothesized that carnitine deficiency in ASD may be secondary to mitochondrial disease or dysfunction [
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ,
      • Palmieri L.
      • Persico A.M.
      Mitochondrial dysfunction in autism spectrum disorders: cause or effect?.
      ], while others have suggested that carnitine metabolism abnormalities may be related to the overproduction of short-chain fatty acids resulting from imbalances in enteric bacteria [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ,
      • Macfabe D.F.
      Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders.
      ].

      1.1.4 Disorders of γ-aminobutyric acid metabolism

      First described in 1981, succinic semialdehyde dehydrogenase deficiency is a rare disorder of GABA metabolism that results from a mutation in both ALDH5A1 genes [
      • Jakobs C.
      • Bojasch M.
      • Monch E.
      • Rating D.
      • Siemes H.
      • Hanefeld F.
      Urinary excretion of gamma-hydroxybutyric acid in a patient with neurological abnormalities. The probability of a new inborn error of metabolism.
      ]. Since the enzyme is partially responsible for the degradation of GABA, brain GABA levels are elevated, and GABA is degraded by an alternative pathway that produces γ-hydroxybutyric acid. Elevated γ-hydroxybutyric acid results in many of the neurological manifestations of this disorder. Positron emission tomography studies suggest that elevated GABA levels downregulate brain GABAA receptors [
      • Pearl P.L.
      • Gibson K.M.
      • Quezado Z.
      • Dustin I.
      • Taylor J.
      • Trzcinski S.
      • et al.
      Decreased GABA-A binding on FMZ-PET in succinic semialdehyde dehydrogenase deficiency.
      ]. Symptom onset commonly occurs before 1 year of age with global developmental delay, hypotonia, hyporeflexia, ASD features, seizures, ataxia, choreoathetosis, dystonia, myoclonus, strabismus, nystagmus, retinitis, disc pallor, and oculomotor apraxia [
      • Knerr I.
      • Gibson K.M.
      • Jakobs C.
      • Pearl P.L.
      Neuropsychiatric morbidity in adolescent and adult succinic semialdehyde dehydrogenase deficiency patients.
      ,
      • Pearl P.L.
      • Gibson K.M.
      Clinical aspects of the disorders of GABA metabolism in children.
      ]. Magnetic resonance imaging can demonstrate increased T2 signal in the basal ganglia, subcortical white matter, brainstem, and cerebellum [
      • Pearl P.L.
      • Novotny E.J.
      • Acosta M.T.
      • Jakobs C.
      • Gibson K.M.
      Succinic semialdehyde dehydrogenase deficiency in children and adults.
      ,
      • Pearl P.L.
      • Gibson K.M.
      • Acosta M.T.
      • Vezina L.G.
      • Theodore W.H.
      • Rogawski M.A.
      • et al.
      Clinical spectrum of succinic semialdehyde dehydrogenase deficiency.
      ]. Metabolically, the urine, serum, and cerebrospinal fluid may demonstrate an elevation in 4-hydroxybutyric acid, but this highly volatile compound can be difficult to measure. Sequencing the ALDH5A1 gene can confirm the diagnosis.

      1.1.5 Disorders of pyrimidine and purine metabolism

      Children with ASD and comorbid seizures have been described to have disorders of purine and pyrimidine metabolism. These include both classic inborn-inherited disorders of metabolism, such as adenylosuccinate lyase deficiency and phosphoribosylpyrophosphate synthetase deficiency, as well as novel disorders of purine metabolism that do not have a clearly genetic basis.
      Adenylosuccinate lyase deficiency is a rare disorder of de novo purine synthesis that results in the accumulation of succinyl purines [
      • Jurecka A.
      • Zikanova M.
      • Tylki-Szymanska A.
      • Krijt J.
      • Bogdanska A.
      • Gradowska W.
      • et al.
      Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency.
      ,
      • Spiegel E.K.
      • Colman R.F.
      • Patterson D.
      Adenylosuccinate lyase deficiency.
      ]. Patients have a unique behavioral phenotype including excessive laughter, a very happy disposition, and stereotyped movements mimicking Angelman syndrome [
      • Gitiaux C.
      • Ceballos-Picot I.
      • Marie S.
      • Valayannopoulos V.
      • Rio M.
      • Verrieres S.
      • et al.
      Misleading behavioural phenotype with adenylosuccinate lyase deficiency.
      ]. Patients show a variable combination of mental retardation, epilepsy, ASD features, and cerebellar vermis hypoplasia [
      • Jurecka A.
      • Zikanova M.
      • Tylki-Szymanska A.
      • Krijt J.
      • Bogdanska A.
      • Gradowska W.
      • et al.
      Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency.
      ,
      • Spiegel E.K.
      • Colman R.F.
      • Patterson D.
      Adenylosuccinate lyase deficiency.
      ]. This disorder can be diagnosed using the Bratton–Marshall assay to measure succinylaminoimidazole carboxamide riboside and succinyladenosine concentration in the urine and/or cerebrospinal fluid.
      One patient with developmental delay and ataxia as well as seizures and ASD features has been described with phosphoribosylpyrophosphate synthetase deficiency [
      • Page T.
      Metabolic approaches to the treatment of autism spectrum disorders.
      ]. Metabolic abnormalities included decreased uric acid and increased orotic acid excretion and megaloblastic anemia and erythrocyte phosphoribosylpyrophosphate synthetase activity that was 10% of normal. Treatment with adrenocorticotrophic hormone reportedly increased erythrocyte phosphoribosylpyrophosphate synthetase activity and improved behavior and seizures.
      Others have described some novel abnormalities in the purine pathway. In 1993, hyperuricosuric autism was described in which 24-hour urine urate was above the normal range and half of the patients had comorbid seizures [
      • Page T.
      Metabolic approaches to the treatment of autism spectrum disorders.
      ]. Although it was believed that the metabolic basis was likely to be an abnormality of purine nucleotide interconversion, no abnormality in enzyme activity could be detected in fibroblasts, and no genetic basis could be identified. A low-purine diet and allopurinol may improve behavior and seizures in these patients.
      In 1997, Page et al. [
      • Page T.
      • Yu A.
      • Fontanesi J.
      • Nyhan W.L.
      Developmental disorder associated with increased cellular nucleotidase activity.
      ] described four patients with ASD, seizures, immune system abnormalities, and a decrease in urinary urate. This disorder was later termed nucleotidase-associated pervasive developmental disorder [
      • Page T.
      Metabolic approaches to the treatment of autism spectrum disorders.
      ]. Fibroblasts from the patients demonstrated decreased incorporation of uridine into nucleotides and an increase in cytosolic-5′-nucleotidase activity, suggesting an increase in nucleotide catabolic activity. All patients treated with oral pyrimidine nucleoside or nucleotide compounds showed a remarkable improvement in speech, behavior, and seizure activity. No genetic basis was found for this disorder.

      1.1.6 Disorders of amino acid metabolism

      Disorders in the metabolism of phenylalanine, branched-chain amino acids, and tryptophan have been described in children with ASD and comorbid epilepsy and will be reviewed in this section.
      Phenylketonuria (PKU), for the most part, has been eliminated in the developed world. Phenylketonuria is an autosomal recessive inborn error of phenylalanine metabolism resulting from deficiency of phenylalanine hydroxylase secondary to a mutation in the PAH gene on chromosome 12q23.2. Newborn screening programs identify children with PKU at birth, allowing the implementation of specific dietary intervention. With good adherence to diet, children born with PKU can be expected to lead a normal life [
      • Williams R.A.
      • Mamotte C.D.
      • Burnett J.R.
      Phenylketonuria: an inborn error of phenylalanine metabolism.
      ]. However, children with PKU who go untreated or who do not adhere to the diet adequately may demonstrate poor growth, poor skin pigmentation, microcephaly, seizures, spasticity, ataxia, aggressive behavior, hyperactivity, ASD features, global developmental delay, and/or severe intellectual impairment. For example, Baieli et al. [
      • Baieli S.
      • Pavone L.
      • Meli C.
      • Fiumara A.
      • Coleman M.
      Autism and phenylketonuria.
      ] found that no children with classic PKU identified as neonates met the criteria for ASD, whereas 6% of those with late diagnosed classic PKU were identified with ASD. The prevalence of seizures and epilepsy is dependent on metabolic control [
      • Martynyuk A.E.
      • Ucar D.A.
      • Yang D.D.
      • Norman W.M.
      • Carney P.R.
      • Dennis D.M.
      • et al.
      Epilepsy in phenylketonuria: a complex dependence on serum phenylalanine levels.
      ]. Gross et al. [
      • Gross P.T.
      • Berlow S.
      • Schuett V.E.
      • Fariello R.G.
      EEG in phenylketonuria. Attempt to establish clinical importance of EEG changes.
      ] demonstrated that children who started treatment later were more likely to have seizures and mental retardation.
      Recently an inactivating mutation in the branched-chain ketoacid dehydrogenase kinase was described to be associated with autism, epilepsy, and intellectual disability in three families with two children each who were products of first-cousin consanguinity [
      • Novarino G.
      • El-Fishawy P.
      • Kayserili H.
      • Meguid N.A.
      • Scott E.M.
      • Schroth J.
      • et al.
      Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy.
      ]. In this disorder, phosphorylation-mediated inactivation of branched-chain ketoacid dehydrogenase is deficient, leading to abnormally low levels of branched-chain amino acids. Behavioral and neurological deficiencies were reversed in a mouse model of this disorder but not in the patients.
      Using Biolog (Hayward, CA) phenotype microarrays, a recent study demonstrated a reduced production of nicotinamide adenine dinucleotide in cell lines derived from patients with syndromic ASD and from those with nonsyndromic ASD when tryptophan was provided as a sole carbon source, suggesting an abnormality in tryptophan metabolism [
      • Boccuto L.
      • Chen C.F.
      • Pittman A.R.
      • Skinner C.D.
      • McCartney H.J.
      • Jones K.
      • et al.
      Decreased tryptophan metabolism in patients with autism spectrum disorders.
      ]. Further studies of patients with ASD demonstrated a reduction in the expression of several tryptophan pathway enzymes, including TPH2, a gene that has been linked to both ASD [
      • Egawa J.
      • Watanabe Y.
      • Endo T.
      • Someya T.
      Association of rs2129575 in the tryptophan hydroxylase 2 gene with clinical phenotypes of autism spectrum disorders.
      ,
      • Yang S.Y.
      • Yoo H.J.
      • Cho I.H.
      • Park M.
      • Kim S.A.
      Association with tryptophan hydroxylase 2 gene polymorphisms and autism spectrum disorders in Korean families.
      ] and epilepsy [
      • Bragatti J.A.
      • Bandeira I.C.
      • de Carvalho A.M.
      • Abujamra A.L.
      • Leistner-Segal S.
      • Bianchin M.M.
      Tryptophan hydroxylase 2 (TPH2) gene polymorphisms and psychiatric comorbidities in temporal lobe epilepsy.
      ]. Altered central tryptophan metabolism has also been identified in a mouse model of progressive myoclonic epilepsy [
      • Arbatova J.
      • D'Amato E.
      • Vaarmann A.
      • Zharkovsky A.
      • Reeben M.
      Reduced serotonin and 3-hydroxyanthranilic acid levels in serum of cystatin B-deficient mice, a model system for progressive myoclonus epilepsy.
      ,
      • Vaarmann A.
      • Kaasik A.
      • Zharkovsky A.
      Altered tryptophan metabolism in the brain of cystatin B-deficient mice: a model system for progressive myoclonus epilepsy.
      ]. The exact biological mechanism by which tryptophan is linked to both ASD and epilepsy is not clear, but there are many possibilities since it is an essential precursor of many neurotransmitters including serotonin, kynurenic acid, and quinolinic acid, and the critical mitochondrial energy carrier nicotinamide adenine dinucleotide.

      1.1.7 Urea cycle disorders

      The urea cycle disposes of nitrogenous waste derived from the breakdown of protein. The key sign of a urea cycle disorder is a large elevation in blood ammonia after a high-protein meal or during times of illness or physiological stress. Symptoms can range from decreased appetite to cyclical vomiting to lethargy or, in severe cases, coma. In some cases, patients may self-select low-protein diets to minimize symptoms. Psychosis, seizures, and pyramidal signs develop over time. Two cases of children with urea cycle disorders, one with ornithine transcarbamylase deficiency and arginase deficiency [
      • Gorker I.
      • Tuzun U.
      Autistic-like findings associated with a urea cycle disorder in a 4-year-old girl.
      ] and another with carbamyl phosphate synthetase deficiency [
      • Serrano M.
      • Martins C.
      • Perez-Duenas B.
      • Gomez-Lopez L.
      • Murgui E.
      • Fons C.
      • et al.
      Neuropsychiatric manifestations in late-onset urea cycle disorder patients.
      ], have been reported. Treatment focused on reducing ammonia through a low-protein diet and ammonia binders and supplementation with specific amino acids and various vitamin supplements [
      • Braissant O.
      Current concepts in the pathogenesis of urea cycle disorders.
      ]. Improvement in ASD symptoms has been reported with treatment.

      1.2 Mitochondrial dysfunction associated with epilepsy in autism spectrum disorder

      A recent meta-analysis found that 5% of children with ASD met the criteria for classic mitochondrial disease, while as many as 30% of children with ASD may manifest mitochondrial dysfunction [
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. Other studies suggested that 30% to 50% of children with ASD have biomarkers consistent with mitochondrial dysfunction [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ,
      • Frye R.E.
      Biomarker of abnormal energy metabolism in children with autism spectrum disorder.
      ], and the prevalence of abnormal mitochondrial function in immune cells derived from children with ASD is exceedingly high [
      • Giulivi C.
      • Zhang Y.F.
      • Omanska-Klusek A.
      • Ross-Inta C.
      • Wong S.
      • Hertz-Picciotto I.
      • et al.
      Mitochondrial dysfunction in autism.
      ,
      • Napoli E.
      • Wong S.
      • Hertz-Picciotto I.
      • Giulivi C.
      Deficits in bioenergetics and impaired immune response in granulocytes from children with autism.
      ]. Mitochondrial dysfunction has been demonstrated in the postmortem ASD brain [
      • Rose S.
      • Melnyk S.
      • Pavliv O.
      • Bai S.
      • Nick T.G.
      • Frye R.E.
      • et al.
      Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain.
      ,
      • Chauhan A.
      • Gu F.
      • Essa M.M.
      • Wegiel J.
      • Kaur K.
      • Brown W.T.
      • et al.
      Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism.
      ,
      • Tang G.
      • Gutierrez Rios P.
      • Kuo S.H.
      • Akman H.O.
      • Rosoklija G.
      • Tanji K.
      • et al.
      Mitochondrial abnormalities in temporal lobe of autistic brain.
      ,
      • Anitha A.
      • Nakamura K.
      • Thanseem I.
      • Yamada K.
      • Iwayama Y.
      • Toyota T.
      • et al.
      Brain region-specific altered expression and association of mitochondria-related genes in autism.
      ,
      • Anitha A.
      • Nakamura K.
      • Thanseem I.
      • Matsuzaki H.
      • Miyachi T.
      • Tsujii M.
      • et al.
      Downregulation of the expression of mitochondrial electron transport complex genes in autism brains.
      ,
      • Rossignol D.A.
      • Frye R.E.
      Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism.
      ] and in animal models of ASD [
      • Kriaucionis S.
      • Paterson A.
      • Curtis J.
      • Guy J.
      • Macleod N.
      • Bird A.
      Gene expression analysis exposes mitochondrial abnormalities in a mouse model of Rett syndrome.
      ]. Novel types of mitochondrial dysfunction have been described in children with ASD [
      • Frye R.E.
      • Naviaux R.K.
      Autistic disorder with complex IV overactivity: a new mitochondrial syndrome.
      ,
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ,
      • Graf W.D.
      • Marin-Garcia J.
      • Gao H.G.
      • Pizzo S.
      • Naviaux R.K.
      • Markusic D.
      • et al.
      Autism associated with the mitochondrial DNA G8363A transfer RNA(Lys) mutation.
      ] and in cell lines derived from children with ASD [
      • Rose S.
      • Frye R.E.
      • Slattery J.
      • Wynne R.
      • Tippett M.
      • Pavliv O.
      • et al.
      Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort.
      ,
      • Rose S.
      • Frye R.E.
      • Slattery J.
      • Wynne R.
      • Tippett M.
      • Melnyk S.
      • et al.
      Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines.
      ]. Several studies suggest that children with ASD and mitochondrial dysfunction have more severe behavioral and cognitive disabilities compared with children with ASD without mitochondrial dysfunction [
      • Minshew N.J.
      • Goldstein G.
      • Dombrowski S.M.
      • Panchalingam K.
      • Pettegrew J.W.
      A preliminary 31P MRS study of autism: evidence for undersynthesis and increased degradation of brain membranes.
      ,
      • Mostafa G.A.
      • El-Gamal H.A.
      • El-Wakkad A.S.E.
      • El-Shorbagy O.E.
      • Hamza M.M.
      Polyunsaturated fatty acids, carnitine and lactate as biological markers of brain energy in autistic children.
      ,
      • Frye R.E.
      • Delatorre R.
      • Taylor H.
      • Slattery J.
      • Melnyk S.
      • Chowdhury N.
      • et al.
      Redox metabolism abnormalities in autistic children associated with mitochondrial disease.
      ]. Interestingly, a recent review of all of the known published cases of mitochondrial disease and ASD demonstrated that only about 25% have a known genetic mutation that can account for their mitochondrial dysfunction [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ].
      A meta-analysis found that, overall, 41% of children with ASD and documented mitochondrial disease are reported to have seizures [
      • Rossignol D.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. This perhaps should not be surprising as mitochondrial dysfunction has been implicated in seizures and epilepsy, especially in therapy-resistant [
      • Kunz W.S.
      The role of mitochondria in epileptogenesis.
      ] and temporal lobe [
      • Kudin A.P.
      • Zsurka G.
      • Elger C.E.
      • Kunz W.S.
      Mitochondrial involvement in temporal lobe epilepsy.
      ] epilepsies. The association between ASD and seizures in children with mitochondrial disease was first described as part of the HEADD (hypotonia, epilepsy, autism, and developmental delay) syndrome [
      • Fillano J.J.
      • Goldenthal M.J.
      • Rhodes C.H.
      • Marin-Garcia J.
      Mitochondrial dysfunction in patients with hypotonia, epilepsy, autism, and developmental delay: HEADD syndrome.
      ]. These cases demonstrated deficiencies in complexes I, III, and IV or large-scale mitochondrial deoxyribonucleic acid (DNA) deletions. Further case reports and series have linked seizure disorders in ASD with complex deficiencies, particularly complex III deficiencies. For example, seizures have been reported in individuals with complex III deficiency [
      • Filipek P.A.
      • Juranek J.
      • Smith M.
      • Mays L.Z.
      • Ramos E.R.
      • Bocian M.
      • et al.
      Mitochondrial dysfunction in autistic patients with 15q inverted duplication.
      ] and combined complex I + III deficiency [
      • Poling J.S.
      • Frye R.E.
      • Shoffner J.
      • Zimmerman A.W.
      Developmental regression and mitochondrial dysfunction in a child with autism.
      ]. Since the electron transport chain (ETC) is the final common pathway for producing the common cellular energy carrier, adenosine triphosphate (ATP), deficiencies in ETC function can have profound effects on the production of energy at the cellular level. Interestingly, mitochondrial dysfunction has been reported in many genetic syndromes associated with ASD and epilepsy. For example, mitochondrial dysfunction has been reported in Rett syndrome [
      • Grosser E.
      • Hirt U.
      • Janc O.A.
      • Menzfeld C.
      • Fischer M.
      • Kempkes B.
      • et al.
      Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.
      ,
      • Gibson J.H.
      • Slobedman B.
      • NH K.
      • Williamson S.L.
      • Minchenko D.
      • El-Osta A.
      • et al.
      Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain.
      ,
      • Condie J.
      • Goldstein J.
      • Wainwright M.S.
      Acquired microcephaly, regression of milestones, mitochondrial dysfunction, and episodic rigidity in a 46, XY male with a de novo MECP2 gene mutation.
      ], PTEN haploinsufficiency [
      • Napoli E.
      • Ross-Inta C.
      • Wong S.
      • Hung C.
      • Fujisawa Y.
      • Sakaguchi D.
      • et al.
      Mitochondrial dysfunction in Pten haplo-insufficient mice with social deficits and repetitive behavior: interplay between Pten and p53.
      ], Phelan–McDermid syndrome [
      • Frye R.E.
      Mitochondrial disease in 22q13 duplication syndrome.
      ], 15q11-q13 duplication syndrome [
      • Filipek P.A.
      • Juranek J.
      • Smith M.
      • Mays L.Z.
      • Ramos E.R.
      • Bocian M.
      • et al.
      Mitochondrial dysfunction in autistic patients with 15q inverted duplication.
      ,
      • Frye R.E.
      15q11.2-13 duplication, mitochondrial dysfunction, and developmental disorders.
      ], Angelman syndrome [
      • Su H.
      • Fan W.
      • Coskun P.E.
      • Vesa J.
      • Gold J.A.
      • Jiang Y.H.
      • et al.
      Mitochondrial dysfunction in CA1 hippocampal neurons of the UBE3A deficient mouse model for Angelman syndrome.
      ], Septo-optic dysplasia [
      • Schuelke M.
      • Krude H.
      • Finckh B.
      • Mayatepek E.
      • Janssen A.
      • Schmelz M.
      • et al.
      Septo-optic dysplasia associated with a new mitochondrial cytochrome b mutation.
      ], and Down syndrome [
      • Pagano G.
      • Castello G.
      Oxidative stress and mitochondrial dysfunction in Down syndrome.
      ,
      • Pallardo F.V.
      • Lloret A.
      • Lebel M.
      • d'Ischia M.
      • Cogger V.C.
      • Le Couteur D.G.
      • et al.
      Mitochondrial dysfunction in some oxidative stress-related genetic diseases: ataxia–telangiectasia, Down syndrome, Fanconi anaemia and Werner syndrome.
      ]. Thus, mitochondrial dysfunction may underlie the phenotype of ASD with epilepsy, regardless of the underlying cause.
      Some of the novel forms of mitochondrial dysfunction associated with ASD have a high prevalence of epilepsy. Eighty percent of the five children described with complex IV overactivity manifested either generalized seizures or subclinical epileptiform discharges on overnight electroencephalogram [
      • Frye R.E.
      • Naviaux R.K.
      Autistic disorder with complex IV overactivity: a new mitochondrial syndrome.
      ], and the case with complex I overactivity had complex partial seizures and frequent bursts of predominantly left-sided multispike–wave discharges on electroencephalogram [
      • Graf W.D.
      • Marin-Garcia J.
      • Gao H.G.
      • Pizzo S.
      • Naviaux R.K.
      • Markusic D.
      • et al.
      Autism associated with the mitochondrial DNA G8363A transfer RNA(Lys) mutation.
      ]. Another unique form of mitochondrial dysfunction in ASD appears to be associated with an abnormal increase in a particular pattern of short and long acylcarnitines [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ]. This pattern of metabolic abnormalities parallels a rodent model of ASD in which these same metabolic abnormalities are induced by propionic acid [
      • Macfabe D.F.
      Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders.
      ]. Detailed study of children with this biomarker reveals a unique change in ETC and citric acid cycle function consistent with excess metabolic flux of propionic acid [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ]. Theoretically, propionic acid can be overproduced by the overrepresented species of Clostridia found in the gastrointestinal tract of children with ASD [
      • De Angelis M.
      • Piccolo M.
      • Vannini L.
      • Siragusa S.
      • De Giacomo A.
      • Serrazzanetti D.I.
      • et al.
      Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified.
      ,
      • Macfabe D.
      Autism: metabolism, mitochondria, and the microbiome.
      ]. Given that propionic acid is a short-chain fatty acid that can complex with l-carnitine, increased propionic acid production could also explain the relatively common carnitine deficiency documented in children with ASD [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ,
      • Macfabe D.F.
      Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders.
      ,
      • Macfabe D.
      Autism: metabolism, mitochondria, and the microbiome.
      ]. The propionic rodent model of ASD demonstrates epileptiform-like spikes in the hippocampus, neocortex, and basal ganglia, with discharges in the basal ganglia associated with measurable behavioral abnormalities [
      • Macfabe D.F.
      Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders.
      ]. However, in the published case series of children with ASD, only 27% demonstrated abnormalities on overnight video electroencephalogram, and only 10% reported a history of seizures [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ]. Clearly, further research is needed to further elucidate the correspondence between novel forms of mitochondrial dysfunction and epilepsy in ASD.
      Abnormalities in mitochondrial function can lead to abnormal development in brain circuits, resulting in both neurodevelopmental disorders and epilepsy through several mechanisms. Mitochondria are ubiquitous organelles that are essential for almost every tissue in the body, especially the brain. Abnormalities in mitochondrial biomarkers found peripherally have also been found in the brains of individuals with ASD [
      • Rossignol D.A.
      • Frye R.E.
      Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism.
      ]. Thus, it is very likely that changes in mitochondrial function in the brain affect neural transmission and function in children with ASD. This could occur through several mechanisms. Neural synapses are areas of high energy consumption [
      • Ames III, A.
      CNS energy metabolism as related to function.
      ] and are especially dependent on mitochondrial function [
      • Mattson M.P.
      • Liu D.
      Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders.
      ]. Mitochondria are concentrated in the dendritic and axonal termini where they play an important role in ATP production, calcium homeostasis, and synaptic plasticity [
      • Chen H.
      • Chan D.C.
      Mitochondrial dynamics—fusion, fission, movement, and mitophagy—in neurodegenerative diseases.
      ,
      • Li Z.
      • Okamoto K.
      • Hayashi Y.
      • Sheng M.
      The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses.
      ]. Mitochondrial dysfunction can compromise neurons with high firing rates, such as GABAergic interneurons [
      • Anderson M.P.
      • Hooker B.S.
      • Herbert M.R.
      Bridging from cells to cognition in autism pathophysiology: biological pathways to defective brain function and plasticity.
      ]. Reduced GABAergic transmission may be relevant in ASD as GABA neurons are essential for the generation and synchronization of high-frequency gamma rhythms — rhythms essential for high-level cortical processing of sensory information and ‘binding’ that have been shown to be abnormal in children with ASD [
      • Orekhova E.V.
      • Stroganova T.A.
      • Nygren G.
      • Tsetlin M.M.
      • Posikera I.N.
      • Gillberg C.
      • et al.
      Excess of high frequency electroencephalogram oscillations in boys with autism.
      ]. The age range when autistic regression typically occurs corresponds to a time when there is an overabundance of cortical excitatory glutamatergic neurotransmitters and receptors [
      • Herlenius E.
      • Lagercrantz H.
      Development of neurotransmitter systems during critical periods.
      ,
      • Huttenlocher P.R.
      • Dabholkar A.S.
      Regional differences in synaptogenesis in human cerebral cortex.
      ] and rapid brain growth [
      • Groeschel S.
      • Vollmer B.
      • King M.D.
      • Connelly A.
      Developmental changes in cerebral grey and white matter volume from infancy to adulthood.
      ]. Thus, this is a developmental window in which mitochondrial energy production is pivotal. Given the importance of glutamate and GABA transmission in the development and progression of epilepsy, these same imbalances of glutamate and GABA neurotransmission could result in seizures and epilepsy in children with ASD [
      • Rowley N.M.
      • Madsen K.K.
      • Schousboe A.
      • Steve White H.
      Glutamate and GABA synthesis, release, transport and metabolism as targets for seizure control.
      ,
      • Lason W.
      • Chlebicka M.
      • Rejdak K.
      Research advances in basic mechanisms of seizures and antiepileptic drug action.
      ].
      Mitochondrial dysfunction is well known to result in increased levels of reactive oxygen species. Reactive species can damage neural tissue and interfere with neural transmission. Glutathione is the principal intrinsic molecule that protects cells against reactive species. Given that glutathione levels in the brain are about 50-fold lower than those in peripheral tissues, such as hepatocytes, neurons may be especially sensitive to increases in reactive oxygen species [
      • Pastore A.
      • Federici G.
      • Bertini E.
      • Piemonte F.
      Analysis of glutathione: implication in redox and detoxification.
      ]. Recent studies have suggested that oxidative stress may be involved in the development of epilepsy [
      • Aguiar C.C.
      • Almeida A.B.
      • Araujo P.V.
      • de Abreu R.N.
      • Chaves E.M.
      • do Vale O.C.
      • et al.
      Oxidative stress and epilepsy: literature review.
      ]. In fact, several studies have discussed the use of antioxidants as a treatment for epilepsy [
      • Grosso C.
      • Valentao P.
      • Ferreres F.
      • Andrade P.B.
      The use of flavonoids in central nervous system disorders.
      ,
      • Azam F.
      • Prasad M.V.
      • Thangavel N.
      Targeting oxidative stress component in the therapeutics of epilepsy.
      ], and several groups have hypothesized that one of the mechanisms for seizure control with the ketogenic diet is through the improvement of oxidative stress [
      • Milder J.
      • Patel M.
      Modulation of oxidative stress and mitochondrial function by the ketogenic diet.
      ,
      • Scheck A.C.
      • Abdelwahab M.G.
      • Fenton K.E.
      • Stafford P.
      The ketogenic diet for the treatment of glioma: insights from genetic profiling.
      ]. Interestingly, studies have demonstrated the connection between reactive oxygen species and mitochondrial dysfunction in brain tissue from individuals with ASD [
      • Rose S.
      • Melnyk S.
      • Pavliv O.
      • Bai S.
      • Nick T.G.
      • Frye R.E.
      • et al.
      Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain.
      ]. This may be another mechanism in which mitochondrial dysfunction and disease can lead to the development of epilepsy in children with ASD.
      Immune dysfunction has been implicated in the development of epilepsy [
      • Xu D.
      • Miller S.D.
      • Koh S.
      Immune mechanisms in epileptogenesis.
      ], and evidence of cellular and humoral immune dysfunction has been implicated in ASD [
      • Rossignol D.A.
      • Frye R.E.
      Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism.
      ,
      • Gesundheit B.
      • Rosenzweig J.P.
      • Naor D.
      • Lerer B.
      • Zachor D.A.
      • Prochazka V.
      • et al.
      Immunological and autoimmune considerations of autism spectrum disorders.
      ]. In fact, the same autoantibodies to neural antigens have been linked to both autism and epilepsy [
      • Connolly A.M.
      • Chez M.G.
      • Pestronk A.
      • Arnold S.T.
      • Mehta S.
      • Deuel R.K.
      Serum autoantibodies to brain in Landau–Kleffner variant, autism, and other neurologic disorders.
      ,
      • Connolly A.M.
      • Chez M.
      • Streif E.M.
      • Keeling R.M.
      • Golumbek P.T.
      • Kwon J.M.
      • et al.
      Brain-derived neurotrophic factor and autoantibodies to neural antigens in sera of children with autistic spectrum disorders, Landau–Kleffner syndrome, and epilepsy.
      ]. This is significant as mitochondrial dysfunction in ASD has been repeatedly reported in immune cells [
      • Napoli E.
      • Wong S.
      • Hertz-Picciotto I.
      • Giulivi C.
      Deficits in bioenergetics and impaired immune response in granulocytes from children with autism.
      ,
      • Rose S.
      • Frye R.E.
      • Slattery J.
      • Wynne R.
      • Tippett M.
      • Pavliv O.
      • et al.
      Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort.
      ,
      • Napoli E.
      • Ross-Inta C.
      • Wong S.
      • Hung C.
      • Fujisawa Y.
      • Sakaguchi D.
      • et al.
      Mitochondrial dysfunction in Pten haplo-insufficient mice with social deficits and repetitive behavior: interplay between Pten and p53.
      ] with such mitochondrial dysfunction linked to abnormalities in immune cell function [
      • Napoli E.
      • Wong S.
      • Hertz-Picciotto I.
      • Giulivi C.
      Deficits in bioenergetics and impaired immune response in granulocytes from children with autism.
      ]. In addition, abnormalities in microglia have been linked to abnormal brain development in Rett syndrome [
      • Derecki N.C.
      • Cronk J.C.
      • Lu Z.
      • Xu E.
      • Abbott S.B.
      • Guyenet P.G.
      • et al.
      Wild-type microglia arrest pathology in a mouse model of Rett syndrome.
      ], and studies suggest that the immune system may be essential for synaptic pruning during development [
      • Boulanger L.M.
      • Shatz C.J.
      Immune signalling in neural development, synaptic plasticity and disease.
      ]. Thus, abnormalities in immune cell function as a result of mitochondrial dysfunction may also result in seizures in children with ASD.
      Another physiological abnormality that is becoming increasingly recognized in both ASD and epilepsy is the dysregulation of calcium [
      • Gargus J.J.
      Genetic calcium signaling abnormalities in the central nervous system: seizures, migraine, and autism.
      ,
      • Schmunk G.
      • Gargus J.J.
      Channelopathy pathogenesis in autism spectrum disorders.
      ]. Its relationship to ASD and epilepsy is best characterized by the novel genetic disorder Timothy syndrome which includes arrhythmias, particular dysmorphology, congenital heart disease, immune deficiency, hypoglycemia, ASD, and seizures [
      • Splawski I.
      • Timothy K.W.
      • Sharpe L.M.
      • Decher N.
      • Kumar P.
      • Bloise R.
      • et al.
      Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism.
      ]. Calcium regulation is important not only in the regulation of the cell membrane potential, where its dysregulation in cardiomyocytes causes arrhythmias, but also within the cell where it regulates enzymes and transcription factors. Most interesting is the connection between mitochondrial function and calcium. Mitochondria have a significant role in the buffering of cellular calcium having multiple calcium transporters. Many mitochondrial enzymes are regulated by calcium, resulting in cellular calcium flux having both short-term and long-term consequences on mitochondrial activity [
      • Rizzuto R.
      • Bernardi P.
      • Pozzan T.
      Mitochondria as all-round players of the calcium game.
      ]. Evidence suggests that calcium plays a significant role in regulation of cellular bioenergetics, production of reactive oxygen species, and induction of autophagy and apoptosis through its interaction with the mitochondria at endoplasmic reticulum-specific subdomains known as mitochondria-associated membranes [
      • Kaufman R.J.
      • Malhotra J.D.
      Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics.
      ,
      • van Vliet A.R.
      • Verfaillie T.
      • Agostinis P.
      New functions of mitochondria associated membranes in cellular signaling.
      ]. Thus, abnormalities in cellular calcium regulation can have significant effects on mitochondrial function and have led some authors to point out the possibility of mitochondrial dysfunction in neurological and neurodevelopmental disorders associated with abnormalities in calcium metabolism [
      • Schmunk G.
      • Gargus J.J.
      Channelopathy pathogenesis in autism spectrum disorders.
      ]. Thus, abnormalities in calcium metabolism related to ASD and epilepsy provide another link to mitochondrial dysfunction in ASD and epilepsy.
      Interestingly, treatments that are typically used for patients with mitochondrial disease have been shown to improve functioning in some children with ASD [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. Several studies, including two double-blind, placebo-controlled studies [
      • Geier D.A.
      • Kern J.K.
      • Davis G.
      • King P.G.
      • Adams J.B.
      • Young J.L.
      • et al.
      A prospective double-blind, randomized clinical trial of levocarnitine to treat autism spectrum disorders.
      ,
      • Fahmy S.F.
      • El-hamamsy M.H.
      • Zaki O.K.
      • Badary O.A.
      l-Carnitine supplementation improves the behavioral symptoms in autistic children.
      ], have reported improvements in core and associated ASD behaviors with l-carnitine treatment [
      • Filipek P.A.
      • Juranek J.
      • Smith M.
      • Mays L.Z.
      • Ramos E.R.
      • Bocian M.
      • et al.
      Mitochondrial dysfunction in autistic patients with 15q inverted duplication.
      ,
      • Poling J.S.
      • Frye R.E.
      • Shoffner J.
      • Zimmerman A.W.
      Developmental regression and mitochondrial dysfunction in a child with autism.
      ,
      • Geier D.A.
      • Kern J.K.
      • Davis G.
      • King P.G.
      • Adams J.B.
      • Young J.L.
      • et al.
      A prospective double-blind, randomized clinical trial of levocarnitine to treat autism spectrum disorders.
      ,
      • Fahmy S.F.
      • El-hamamsy M.H.
      • Zaki O.K.
      • Badary O.A.
      l-Carnitine supplementation improves the behavioral symptoms in autistic children.
      ,
      • Pastural E.
      • Ritchie S.
      • Lu Y.
      • Jin W.
      • Kavianpour A.
      • Khine Su-Myat K.
      • et al.
      Novel plasma phospholipid biomarkers of autism: mitochondrial dysfunction as a putative causative mechanism.
      ,
      • Gargus J.J.
      • Lerner M.A.
      Familial autism with primary carnitine deficiency, sudden death, hypotonia and hypochromic anemia.
      ,
      • Gargus J.J.
      • Imtiaz F.
      Mitochondrial energy-deficient endophenotype in autism.
      ,
      • Ezugha H.
      • Goldenthal M.
      • Valencia I.
      • Anderson C.E.
      • Legido A.
      • Marks H.
      5q14.3 deletion manifesting as mitochondrial disease and autism: case report.
      ]. Two double-blind, placebo-controlled studies using a multivitamin containing B vitamins, antioxidants, vitamin E, and coenzyme Q10 reported various improvements in ASD symptoms compared with placebo [
      • Adams J.B.
      • Holloway C.
      Pilot study of a moderate dose multivitamin/mineral supplement for children with autistic spectrum disorder.
      ,
      • Adams J.B.
      • Audhya T.
      • McDonough-Means S.
      • Rubin R.A.
      • Quig D.
      • Geis E.
      • et al.
      Effect of a vitamin/mineral supplement on children and adults with autism.
      ]. Several other antioxidants [
      • Rossignol D.A.
      Novel and emerging treatments for autism spectrum disorders: a systematic review.
      ], including vitamin C [
      • Dolske M.C.
      • Spollen J.
      • McKay S.
      • Lancashire E.
      • Tolbert L.
      A preliminary trial of ascorbic acid as supplemental therapy for autism.
      ], methylcobalamin [
      • Frye R.E.
      • Melnyk S.
      • Fuchs G.
      • Reid T.
      • Jernigan S.
      • Pavliv O.
      • et al.
      Effectiveness of methylcobalamin and folinic acid treatment on adaptive behavior in children with autistic disorder is related to glutathione redox status.
      ,
      • James S.J.
      • Melnyk S.
      • Fuchs G.
      • Reid T.
      • Jernigan S.
      • Pavliv O.
      • et al.
      Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism.
      ,
      • Nakano K.
      • Noda N.
      • Tachikawa E.
      • Urano M.A.N.
      • Takazawa M.
      • Nakayama T.
      • et al.
      A preliminary study of methylcobalamin therapy in autism.
      ], N-acetyl-L-cysteine [
      • Ghanizadeh A.
      • Derakhshan N.
      N-acetylcysteine for treatment of autism, a case report.
      ,
      • Ghanizadeh A.
      • Moghimi-Sarani E.
      A randomized double blind placebo controlled clinical trial of N-acetylcysteine added to risperidone for treating autistic disorders.
      ,
      • Hardan A.Y.
      • Fung L.K.
      • Libove R.A.
      • Obukhanych T.V.
      • Nair S.
      • Herzenberg L.A.
      • et al.
      A randomized controlled pilot trial of oral N-acetylcysteine in children with autism.
      ], ubiquinol [
      • Gvozdjakova A.
      • Kucharska J.
      • Ostatnikova D.
      • Babinska K.
      • Nakladal D.
      • Crane F.L.
      Ubiquinol improves symptoms in children with autism.
      ], and carnosine [
      • Chez M.G.
      • Buchanan C.P.
      • Aimonovitch M.C.
      • Becker M.
      • Schaefer K.
      • Black C.
      • et al.
      Double-blind, placebo-controlled study of l-carnosine supplementation in children with autistic spectrum disorders.
      ], have also been reported to demonstrate significant improvements in ASD behaviors. However, the effect of these treatments on seizures or epilepsy in these children was not investigated in these studies.

      1.3 Animal models of autism and epilepsy

      There are several models of ASD that also manifest epilepsy and metabolic abnormalities. As mentioned above, the propionic rodent model of ASD demonstrates mitochondrial, oxidative stress and lipid abnormalities as well as epileptiform-like discharges in the brain [
      • Macfabe D.F.
      Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders.
      ,
      • Macfabe D.
      Autism: metabolism, mitochondria, and the microbiome.
      ]. However, the majority of biochemical data from the rodent model are derived from adult animals exposed to proprionic acid by intraventicular injections, thereby limiting the generalizability and translatability of the data. Despite this limitation, there appears to be preliminary evidence of a corresponding ASD metabolic subgroup of children with similar manifestations [
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ]. Ongoing animal studies using prenatal and neonatal exposure to enteric metabolites demonstrated the induction of ASD-like behaviors, lending support for the validity of the adult animal model [
      • Foley K.A.
      • Ossenkopp K.P.
      • Kavaliers M.
      • Macfabe D.F.
      Pre- and neonatal exposure to lipopolysaccharide or the enteric metabolite, propionic acid, alters development and behavior in adolescent rats in a sexually dimorphic manner.
      ,
      • Foley K.A.
      • Macfabe D.F.
      • Vaz A.
      • Ossenkopp K.P.
      • Kavaliers M.
      Sexually dimorphic effects of prenatal exposure to propionic acid and lipopolysaccharide on social behavior in neonatal, adolescent, and adult rats: implications for autism spectrum disorders.
      ]. Several genetic models of ASD syndromes in which epilepsy is common have demonstrated mitochondrial dysfunction including Rett syndrome [
      • Grosser E.
      • Hirt U.
      • Janc O.A.
      • Menzfeld C.
      • Fischer M.
      • Kempkes B.
      • et al.
      Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.
      ], PTEN haploinsufficiency [
      • Napoli E.
      • Ross-Inta C.
      • Wong S.
      • Hung C.
      • Fujisawa Y.
      • Sakaguchi D.
      • et al.
      Mitochondrial dysfunction in Pten haplo-insufficient mice with social deficits and repetitive behavior: interplay between Pten and p53.
      ], and Angelman syndrome [
      • Su H.
      • Fan W.
      • Coskun P.E.
      • Vesa J.
      • Gold J.A.
      • Jiang Y.H.
      • et al.
      Mitochondrial dysfunction in CA1 hippocampal neurons of the UBE3A deficient mouse model for Angelman syndrome.
      ], while abnormalities in oxidative metabolism have been demonstrated in the Rett syndrome mouse model [
      • Grosser E.
      • Hirt U.
      • Janc O.A.
      • Menzfeld C.
      • Fischer M.
      • Kempkes B.
      • et al.
      Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.
      ]. Classic environmental exposure models of ASD have also been shown to involve mitochondrial dysfunction as ASD behaviors induced by prenatal valproic acid exposure have been shown to be associated with reduced mitochondrial respiration [
      • Ahn Y.
      • Narous M.
      • Tobias R.
      • Rho J.M.
      • Mychasiuk R.
      The ketogenic diet modifies social and metabolic alterations identified in the prenatal valproic acid model of autism spectrum disorder.
      ]. A new rat model of ASD and ADHD, which has a predisposition toward seizures, manifests aberrant lipid handling [
      • Gilby K.L.
      A new rat model for vulnerability to epilepsy and autism spectrum disorders.
      ]. Interestingly, ASD-like symptoms improved when the ketogenic diet, a metabolic diet with therapeutic effect for both epilepsy and mitochondrial disorders, was used therapeutically in several well-established mouse models of ASD that demonstrate comorbid seizure susceptibility, including the BTBR [
      • Ruskin D.N.
      • Svedova J.
      • Cote J.L.
      • Sandau U.
      • Rho J.M.
      • Kawamura Jr., M.
      • et al.
      Ketogenic diet improves core symptoms of autism in BTBR mice.
      ], EL [
      • Mantis J.G.
      • Meidenbauer J.J.
      • Zimick N.C.
      • Centeno N.A.
      • Seyfried T.N.
      Glucose reduces the anticonvulsant effects of the ketogenic diet in EL mice.
      ], and prenatal valproic acid exposure [
      • Ahn Y.
      • Narous M.
      • Tobias R.
      • Rho J.M.
      • Mychasiuk R.
      The ketogenic diet modifies social and metabolic alterations identified in the prenatal valproic acid model of autism spectrum disorder.
      ] mouse models. Indeed, understanding the metabolic abnormalities in these animal models and how they correspond to children with ASD not only will help us understand the pathophysiology in more detail but also will allow treatment to be optimized on the animal models before launching human clinical trials.

      1.4 Diagnostic approach to metabolic disease

      There are several factors that make the diagnosis of metabolic disease difficult, most prominently the heterogeneity of the presentation of metabolic disorders. While many traditional diseases are diagnosed by symptoms that are specific to the disease, it is unusual for metabolic diseases to have specific symptoms. As an example, we will consider mitochondrial disorders. Adult patients with mitochondrial disorders often show obvious gross motor issues, including easy fatigability, myopathies, or cardiomyopathies, while muscular manifestations are seen in a minority of patients with pediatric mitochondrial disease [
      • Munnich A.
      • Rotig A.
      • Chretien D.
      • Cormier V.
      • Bourgeron T.
      • Bonnefont J.P.
      • et al.
      Clinical presentation of mitochondrial disorders in childhood.
      ], and it is actually unusual for children with mitochondrial disease to present with characteristic hallmarks [
      • Nissenkorn A.
      • Zeharia A.
      • Lev D.
      • Watemberg N.
      • Fattal-Valevski A.
      • Barash V.
      • et al.
      Neurologic presentations of mitochondrial disorders.
      ]. Indeed, in the pediatric population, classic biochemical markers such as lactic acidosis and muscle tissue histology such as ragged red fibers are not commonly seen with mitochondrial disease caused by well-established genetic mutations [
      • Zeviani M.
      • Di Donato S.
      Mitochondrial disorders.
      ]. This makes the diagnosis of metabolic disease rather different from many classic diseases and requires a shift in the diagnostic strategy. Indeed, many times, diagnosis is based on a diagnostic criterion that indicates the probability of a metabolic disorder or a collection of symptoms and biomarkers that are only suggestive of a metabolic syndrome. In addition, many metabolic diseases associated with neurodevelopmental disorders do not appear to have a simply clear genetic fingerprint. In the case of mitochondrial disease, two genomes, the mitochondrial and the nuclear genome, as well as the interaction between the two genomes, need to be considered. In addition, the mitochondrion is sensitive to damage from extrinsic environmental factors as well as intrinsic factors that increase oxidative stress, thus raising the possibility of acquired mitochondrial dysfunction [
      • Rossignol D.A.
      • Frye R.E.
      A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures.
      ,
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ,
      • Rossignol D.A.
      • Frye R.E.
      Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism.
      ,
      • Chauhan A.
      • Chauhan V.
      Oxidative stress in autism.
      ,
      • Rossignol D.A.
      • Genuis S.J.
      • Frye R.E.
      Environmental toxicants and autism spectrum disorders: a systematic review.
      ].
      We have some evidence of a clinical phenotype of mitochondrial dysfunction in some children with ASD that may raise our index of suspicion in any particular patient. A recent meta-analysis that reviewed all of the cases of mitochondrial disease reported in children with ASD noted an unusually high prevalence of regression, gastrointestinal dysfunction, and motor delays, as well as seizures, in children with ASD and mitochondrial disease compared with the general population with ASD [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. Specific mitochondrial disease syndromes associated with ASD have certain clinical symptoms that could be helpful in diagnosing a mitochondrial disorder. The HEADD syndrome includes hypotonia and developmental delays [
      • Fillano J.J.
      • Goldenthal M.J.
      • Rhodes C.H.
      • Marin-Garcia J.
      Mitochondrial dysfunction in patients with hypotonia, epilepsy, autism, and developmental delay: HEADD syndrome.
      ]. However, since the prevalence of hypotonia was not found to differ between children with ASD and mitochondrial disease and the general population with ASD, hypotonia may not be a symptom specific enough to differentiate individuals with ASD with mitochondrial disease from those without mitochondrial disease [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. In a biomarker analysis, individuals with ASD who manifested the unique elevations in short and long acylcarnitine abnormalities showed a high rate of regression [
      • Frye R.E.
      Biomarker of abnormal energy metabolism in children with autism spectrum disorder.
      ], consistent with others who have demonstrated that the enteric gut bacteria that produce propionic acid appear to be associated with regressive-type ASD [
      • Sandler R.H.
      • Finegold S.M.
      • Bolte E.R.
      • Buchanan C.P.
      • Maxwell A.P.
      • Vaisanen M.L.
      • et al.
      Short-term benefit from oral vancomycin treatment of regressive-onset autism.
      ,
      • Finegold S.M.
      • Molitoris D.
      • Song Y.
      • Liu C.
      • Vaisanen M.L.
      • Bolte E.
      • et al.
      Gastrointestinal microflora studies in late-onset autism.
      ]. This preliminary biomarker analysis also found that the subset of children with an elevation in the alanine-to-lysine ratio during screening for metabolic disorders tended to have a diagnosis of epilepsy [
      • Frye R.E.
      Biomarker of abnormal energy metabolism in children with autism spectrum disorder.
      ]. All of the cases of children with complex I or IV overactivity and epilepsy or subclinical epileptiform discharges demonstrated normal early development followed by a substantial developmental regression with poor developmental recovery despite therapeutic interventions [
      • Frye R.E.
      • Naviaux R.K.
      Autistic disorder with complex IV overactivity: a new mitochondrial syndrome.
      ,
      • Graf W.D.
      • Marin-Garcia J.
      • Gao H.G.
      • Pizzo S.
      • Naviaux R.K.
      • Markusic D.
      • et al.
      Autism associated with the mitochondrial DNA G8363A transfer RNA(Lys) mutation.
      ]. Thus, reviewing the cases of mitochondrial dysfunction and epilepsy associated with ASD does underscore two phenotypes that appear to be associated with mitochondrial disease, either developmental delays from early in life, particularly motor delays, or abrupt regression with the onset of seizures. When these symptoms are found in a child with ASD and seizures, an investigation of mitochondrial disease is warranted.
      Given the fact that children with ASD can have a wide manifestation of symptoms, usually affecting many systems other than the brain, and that children with mitochondrial disease can have a wide variety of symptoms and biochemical findings [
      • Morava E.
      • van den Heuvel L.
      • Hol F.
      • de Vries M.C.
      • Hogeveen M.
      • Rodenburg R.J.
      • et al.
      Mitochondrial disease criteria: diagnostic applications in children.
      ,
      • Smeitink J.A.
      Mitochondrial disorders: clinical presentation and diagnostic dilemmas.
      ], several reviews have suggested that mitochondrial disease should be considered in all children with ASD [
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. Recently, several algorithms outlining the workup for mitochondrial disease in ASD have been published [
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Frye R.E.
      • Rossignol D.
      Metabolic disorders and abnormalities associated with autism spectrum disorder.
      ,
      • Frye R.E.
      • Melnyk S.
      • Macfabe D.F.
      Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder.
      ,
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ]. Most importantly, it has been pointed out that given the low prevalence of known genetic mutations associated with mitochondrial disease in ASD and the many novel forms of mitochondrial disorders associated with ASD, a mitochondrial disease diagnostic criterion based on symptoms and biochemical findings may be more useful in the diagnostic workup of children with ASD than a diagnostic criterion more specific to classic mitochondrial disease [
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Frye R.E.
      • Rossignol D.
      Metabolic disorders and abnormalities associated with autism spectrum disorder.
      ].
      Other metabolic disorders associated with ASD and epilepsy appear to have a much lower prevalence except for disorders that affect the folate pathway, particularly the association with FRα autoantibodies [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ]. Thus, in addition to mitochondrial disease and dysfunction, abnormalities of folate metabolism and FRα autoantibodies should be considered in all children with ASD [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ]. Children with negative findings given a workup for these disorders and/or those with treatment-resistant epilepsy should undergo further investigation for other metabolic disorders. Many of these disorders do not have specific clinical symptomatology to point toward, while some clinical clues may help for other disorders. For example, X-linked inheritance is seen in creatine transporter disorder and certain urea cycle disorders, and consanguinity has been noted in branched-chain ketoacid dehydrogenase kinase deficiency; recurrent episodes of vomiting and lethargy, particularly after a protein meal, are seen in urea cycle disorders; particular facial dysmorphology is common in adenylosuccinate lyase deficiency and Smith–Lemli–Opitz syndrome; movement disorders are seen in creatine metabolism disorder, CFD, and succinic semialdehyde dehydrogenase deficiency; and recurrent infections are seen in disorders of purine metabolism. Thus, it is difficult to develop a specific diagnostic algorithm to investigate these metabolic disorders systematically since diagnosis requires careful consideration of the individual patient to gather specific clues.

      2. Discussion

      We have reviewed all of the metabolic disorders that are associated with ASD in which epilepsy has been reported to be a comorbid condition. Interestingly, all of the metabolic diseases associated with ASD appear to include cases of children with comorbid epilepsy. Thus, epilepsy may be a common symptom of metabolic disorders and may be a clue that a metabolic disorder may be underlying the etiology of the neurodevelopmental abnormalities in children with epilepsy and ASD. It is also important to consider many of the metabolic disorders even when genetic disorders are diagnosed. Indeed, CFD can coexist with Rett syndrome [
      • Ramaekers V.T.
      • Sequeira J.M.
      • Artuch R.
      • Blau N.
      • Temudo T.
      • Ormazabal A.
      • et al.
      Folate receptor autoantibodies and spinal fluid 5-methyltetrahydrofolate deficiency in Rett syndrome.
      ], and mitochondrial dysfunction has been reported in a wide variety of genetic disorders associated with ASD [
      • Filipek P.A.
      • Juranek J.
      • Smith M.
      • Mays L.Z.
      • Ramos E.R.
      • Bocian M.
      • et al.
      Mitochondrial dysfunction in autistic patients with 15q inverted duplication.
      ,
      • Grosser E.
      • Hirt U.
      • Janc O.A.
      • Menzfeld C.
      • Fischer M.
      • Kempkes B.
      • et al.
      Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.
      ,
      • Gibson J.H.
      • Slobedman B.
      • NH K.
      • Williamson S.L.
      • Minchenko D.
      • El-Osta A.
      • et al.
      Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain.
      ,
      • Condie J.
      • Goldstein J.
      • Wainwright M.S.
      Acquired microcephaly, regression of milestones, mitochondrial dysfunction, and episodic rigidity in a 46, XY male with a de novo MECP2 gene mutation.
      ,
      • Napoli E.
      • Ross-Inta C.
      • Wong S.
      • Hung C.
      • Fujisawa Y.
      • Sakaguchi D.
      • et al.
      Mitochondrial dysfunction in Pten haplo-insufficient mice with social deficits and repetitive behavior: interplay between Pten and p53.
      ,
      • Frye R.E.
      Mitochondrial disease in 22q13 duplication syndrome.
      ,
      • Frye R.E.
      15q11.2-13 duplication, mitochondrial dysfunction, and developmental disorders.
      ,
      • Su H.
      • Fan W.
      • Coskun P.E.
      • Vesa J.
      • Gold J.A.
      • Jiang Y.H.
      • et al.
      Mitochondrial dysfunction in CA1 hippocampal neurons of the UBE3A deficient mouse model for Angelman syndrome.
      ,
      • Schuelke M.
      • Krude H.
      • Finckh B.
      • Mayatepek E.
      • Janssen A.
      • Schmelz M.
      • et al.
      Septo-optic dysplasia associated with a new mitochondrial cytochrome b mutation.
      ,
      • Pagano G.
      • Castello G.
      Oxidative stress and mitochondrial dysfunction in Down syndrome.
      ,
      • Pallardo F.V.
      • Lloret A.
      • Lebel M.
      • d'Ischia M.
      • Cogger V.C.
      • Le Couteur D.G.
      • et al.
      Mitochondrial dysfunction in some oxidative stress-related genetic diseases: ataxia–telangiectasia, Down syndrome, Fanconi anaemia and Werner syndrome.
      ]. In addition, CFD should be ruled out in the context of mitochondrial dysfunction as it has been shown to coexist with several mitochondrial diseases [
      • Frye R.E.
      • Rossignol D.A.
      Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders.
      ,
      • Allen R.J.
      • DiMauro S.
      • Coulter D.L.
      • Papadimitriou A.
      • Rothenberg S.P.
      Kearns–Sayre syndrome with reduced plasma and cerebrospinal fluid folate.
      ,
      • Pineda M.
      • Ormazabal A.
      • Lopez-Gallardo E.
      • Nascimento A.
      • Solano A.
      • Herrero M.D.
      • et al.
      Cerebral folate deficiency and leukoencephalopathy caused by a mitochondrial DNA deletion.
      ,
      • Ramaekers V.T.
      • Weis J.
      • Sequeira J.M.
      • Quadros E.V.
      • Blau N.
      Mitochondrial complex I encephalomyopathy and cerebral 5-methyltetrahydrofolate deficiency.
      ,
      • Hasselmann O.
      • Blau N.
      • Ramaekers V.T.
      • Quadros E.V.
      • Sequeira J.M.
      • Weissert M.
      Cerebral folate deficiency and CNS inflammatory markers in Alpers disease.
      ,
      • Perez-Duenas B.
      • Ormazabal A.
      • Toma C.
      • Torrico B.
      • Cormand B.
      • Serrano M.
      • et al.
      Cerebral folate deficiency syndromes in childhood: clinical, analytical, and etiologic aspects.
      ,
      • Garcia-Cazorla A.
      • Quadros E.V.
      • Nascimento A.
      • Garcia-Silva M.T.
      • Briones P.
      • Montoya J.
      • et al.
      Mitochondrial diseases associated with cerebral folate deficiency.
      ,
      • Shoffner J.
      • Hyams L.
      • Langley G.N.
      • Cossette S.
      • Mylacraine L.
      • Dale J.
      • et al.
      Fever plus mitochondrial disease could be risk factors for autistic regression.
      ,
      • Frye R.E.
      • Naviaux R.K.
      Autistic disorder with complex IV overactivity: a new mitochondrial syndrome.
      ].
      One advantage of investigating and diagnosing metabolic disorders is that treatments for many of these metabolic disorders are available [
      • Frye R.E.
      • Rossignol D.
      • Casanova M.F.
      • Brown G.L.
      • Martin V.
      • Edelson S.
      • et al.
      A review of traditional and novel treatments for seizures in autism spectrum disorder: findings from a systematic review and expert panel.
      ]. Preliminary studies suggest that there are a substantial number of children with ASD with these metabolic abnormalities. For example, mitochondrial dysfunction may be seen in 5% to 80% of children with ASD [
      • Rossignol D.A.
      • Frye R.E.
      Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis.
      ,
      • Frye R.E.
      Biomarker of abnormal energy metabolism in children with autism spectrum disorder.
      ,
      • Giulivi C.
      • Zhang Y.F.
      • Omanska-Klusek A.
      • Ross-Inta C.
      • Wong S.
      • Hertz-Picciotto I.
      • et al.
      Mitochondrial dysfunction in autism.
      ], and FRα autoantibodies may be found in 47% [
      • Ramaekers V.T.
      • Quadros E.V.
      • Sequeira J.M.
      Role of folate receptor autoantibodies in infantile autism.
      ] to 75% [
      • Frye R.E.
      • Sequeira J.M.
      • Quadros E.V.
      • James S.J.
      • Rossignol D.A.
      Cerebral folate receptor autoantibodies in autism spectrum disorder.
      ] of children with ASD. Clearly, further studies will be required to clarify the percentages of these subgroups. As several studies have suggested that treatment for these metabolic disorders can improve seizures, this is an important consideration in the diagnosis and treatment of epilepsy in children with ASD.
      Many of the metabolic diseases described have only been reported in case reports or case series, so the prevalence of these metabolic abnormalities in children with ASD and epilepsy is not fully known. Given the small number of patients described with some disorders, it is not always clear if epilepsy is strongly linked to the disorder or whether the epilepsy simply coexists with the underlying neural dysfunction that has resulted in the ASD features. Further studies will be needed to determine the exact relationship between certain metabolic disorders, ASD, and epilepsy. However, some disorders like mitochondrial disorders are clearly prevalent in children with ASD and epilepsy and, thus, deserve consideration during the diagnostic workup. Clearly, many children with ASD and their families may be able to benefit from treatments which are focused on addressing metabolic abnormalities.

      Conflict of Interest

      The authors declares no conflict of interest.

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