Table 4.

Association between folate/folate metabolites, gene polymorphism in folate and Autism*.

Authors, country, and year of publication	Study design and subjects	Case definition	Outcome measure	Results
Boris et al. USA, 2004 [69]	Case control study   Cases:   168 Caucasian children 148 (84.5%) males 26 (5.5%) females Controls:   5389 Caucasians	73.8% autism, 26.3% PDD diagnosed by neurologist, psychiatrist, or neuropsychologist. DSM IV criteria used for diagnosis	Frequency of MTHFR alleles 677C→T 1298A→C in cases and controls	Significantly increased (P < 0.0001) frequencies of homozygous mutation 677CT allele (23% in cases versus 11% in controls) and heterozygous 677CT allele (56% in cases versus 41% in controls). Overall increased risk of ASD associated with common mutations affecting the folate/methylation cycle.
James et al. USA 2006 [63]	Case control study   Cases:   80 Caucasian children from autism clinics 89% males 11% females Mean age 7.3 ± 3.2 yrs. Controls:   73 unrelated healthy Caucasian from a metabolic study Mean age: 10.8 ± 4.1 yrs.	Autism diagnoses: by independent specialists DSM IV or ADOS or CARS criteria	Plasma levels of folate metabolites: Methionine, Homocysteine Cystathionine, Cysteine SAM, SAH Glutathione Allele frequency: RFC 80>A TCN2 776G>C COMT 472G>A MTHFR 667>T	Plasma methionine and SAM/SAH ratio was significantly lower in autistic cases as compared to age matched controls (an indicator of lower methylation capacity). Antioxidant capacity as indicated by plasma levels of cysteine and glutathione was significantly decreased in cases. Significant increase in odds ratio, allele frequency, and genotype distribution among autistic cases were found for RFC-1 80A>G, TCN2 776C>G, and COMT 472G>A genes. An increase in frequency of MTHFR 667C>T reached border line significance in autistic cases. Increased vulnerability to oxidative stress related to folate metabolism may contribute to development of autism.
Ramaekers et al. Belgium 2007 [62]	Case control study   Cases:   25 children with early-onset low functioning autism Controls:   100 healthy age matched controls	Diagnosis established by DSM IV criteria and ADOS in conjunction with ADI around 3 years of age	Serum folate, cerebrospinal (CSF) folate, CSF 5MTHF folate receptor (FR) autoantibodies	There was no significant difference in serum folate levels between autistic cases and controls. The mean CSF level of MTHF was significantly lower than controls (27.3 nmol/L compared to 82.0 nmol/L). Autistic cases with low CSF MTFR had autoantibodies of blocking type against human FR. The reduced CSF folate in autistic cases was associated with FR autoantibodies blocking the folate binding site.
Schmidt et al. USA, 2011 [61]	CHARGE population based case control study   Cases:   284 autistic and 141 children with ASD between ages 2–5 years Controls:   278 children with typical development	Diagnoses confirmed by: Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule Generic	Consumption of prenatal multivitamins, nutrient specific vitamins at any time during the period of 3 months before conception through pregnancy Maternal, paternal, and child samples were genotyped for MTHFR 667C>T MTHFR A1298C COMT 472G>A MTRR A66G TCN2	Use of prenatal vitamins during 3 months before and first month of pregnancy was associated with reduced risk of autism. (OR = 0.62, 95%. C.I. = 0.42–0.93). No association observed for vitamins intake during months 2 through 9 of pregnancy. Significant interactions were observed for autism between lack of perinatal maternal prenatal vitamin intake and both maternal MTFHR 667TT and CBSrs 234715GT+TT genotypes. With combined OR = 4.5, C.I. = 1.4–14.6 and 2.6 (1.2–5.4), respectively.
Schmidt et al. USA, 2012 [58]	CHARGE case control study   Cases:   429 children with ASD Controls:   278 children with typical development. Cases and controls were frequency matched to age and catchment area distribution of autism cases with a 4 : 1 male-to-female ratio	The diagnoses of autism cases were confirmed by the Autism Diagnostic Interview-Revised (ADI-R) and by ADOS. The two initially identified subgroups, ASD and Autism, were later collapsed and results presented for combined ASD groups	Maternal folic acid intake was calculated from data on intake of multivitamins and prenatal vitamin including folic acid specific vitamins, cereal, and supplements during the index period (3 months before and throughout pregnancy) Maternal and cases MTHFR 667C>T was genotyped	The mean (±SEM) folic acid intake was significantly greater for typical development children than for mothers of children with ASD (P < 0.01) in first month of pregnancy. A mean folic acid intake of ≥600 µg versus <600 µg was associated with reduced risk of ASD (AOR = 0.62, 95% C.I. = 0.42–0.92) The association between folic acid and reduced ASD risk was strongest for mothers and children with MTHR 667C>T variant genotype. Periconceptional folic acid may reduce ASD risk in those with inefficient folate metabolism.
Suren et al. Norway, 2013 [53]	Population based prospective study   The study sample of 85,175 children born in 2002–2008 was derived from Primary Norwegian Mother and Child Cohort study (MoBa), with the mean age of 6.4 years Exposure of interest was use of folic acid from 4 weeks before to 8 weeks after the start of pregnancy	Cases of ASD confirmed by linkages to the Norwegian Patient registry, capturing data for all children diagnosed with ASD by Norwegian health services	Specialist confirmed diagnoses of ASD	270 children who were in the study sample were diagnosed with ASD at the end of the study. In children whose mother took folic acid, 0.10% had ASD as compared to 0.21% in those unexposed to folic acid. The AOR for autistic children of folic acid users was 0.61, 95% C.I. = 0.41–0.90 as compared to folic acid nonusers. There was no association with Asperger syndrome or PDD-NOs, but power was limited. Use of prenatal folic acid supplements around the time of conception was associated with a significantly lower risk of ASD in the MoBa cohort.

*Enzymes and their alleles:

MTHFR: 5,10-methylenetetrahydrofolate reductase

COMT: catechol-O-methyltransferase

MTR: methyltetrahydrofolate homocysteine methyl transferase

TCN2: transcobalamin II

CBS: cystathionine β-synthase.