Original article 171

Aberrations in metabolic pathway and altered susceptibility to Naushad Shaik Mohammada, Jamal Md Nurul Jaina, Krishna Prasad Chintakindia, Ram Prakash Singha, Usha Naikb and Radha Rama Devi Akellaa

Objective To investigate whether genetic polymorphisms frequency was similar in both the groups (53.3 vs. are the underlying causes for aberrations in folate pathway 53.6%) and hence individually not associated with that was reported in autistic children. any risk. However, this allele was found to act additively in the presence of MTHFR 677T allele as evidenced by Basic methods A total of 138 children diagnosed as 8.11-fold (95% CI: 2.84–22.92) risk associated with autistic based on Diagnostic and Statistical Manual MTHFR 677CT + TT/1298AC + CC genotypes cumulatively. of Mental Disorders, fourth edition criteria and Autism Behavior Checklist scoring and 138 age and sex matched Conclusion MTHFR C677T is a risk factor, whereas children who are nonautistic were tested for five genetic MTRR A66G and SHMT C1420T polymorphisms polymorphisms, that is, cytosolic serine hydroxyl methyl reduce risk for autism. MTHFR A1298C acts additively in transferase (SHMT1 C1420T), methylene tetrahydrofolate increasing the risk for autism. Psychiatr Genet 19:171–176 reductase (MTHFR C677T and MTHFR A1298C), c 2009 Wolters Kluwer Health | Lippincott Williams & synthase reductase (MTRR A66G), Wilkins. methionine synthase (MS A2756G) using PCR-restriction fragment length polymorphism methods. Fisher’s exact Psychiatric Genetics 2009, 19:171–176 test and logistic regression analysis were used for statistical analyses. Keywords: autism, methionine synthase reductase, methylene tetrahydrofolate reductase, serine hydroxymethyl transferase, single nucleotide polymorphisms, susceptibility Results MTHFR 677T-allele frequency was found to be higher in autistic children compared with nonautistic aCenter for DNA Fingerprinting and Diagnostics and bInstitute of Child Health, children (16.3 vs. 6.5%) with 2.79-fold increased risk Niloufer Hospital, Hyderabad, India for autism [95% confidence interval (CI): 1.58–4.93]. The Correspondence to Dr Radha Rama Devi Akella, MD, Consultant Medical frequencies of MTRR 66A allele (12.7 vs. 21.0%) and SHMT Geneticist, Diagnostic Division, Center for DNA Fingerprinting and Diagnostics 1420T allele (27.9 vs. 45.3%) were lower in autistic group (CDFD) 7-18, Nacharam, Hyderabad-500076, Andhra Pradesh, India compared with nonautistic group with odds ratios 0.55 Tel: + 91 40 27171462; e-mail: [email protected] (95% CI: 0.35–0.86) and 0.44 (95% CI: 0.31–0.62), Received 27 May 2008 Revised 12 December 2008 respectively, indicating reduced risk. MTHFR 1298C-allele Accepted 12 March 2009

Introduction dictated by a polygenic mode of inheritance as well as Autism (MIM:209850), one of the pervasive develop- being modulated by environmental exposures. mental disorders, manifests before 3 years of age and is characterized by impaired communication, social Moretti et al. (2005) have observed low cerebral methyl interaction, and creative or imaginative play. Clinically tetrahydrofolate in a 6-year-old girl with developmental the diagnosis of autism is based on Diagnostic and Statistical delay, psychomotor regression, seizures, mental retardation, Manual of Mental Disorders and International Classification of and autistic features, and marked improvement in motor Diseases, 10th Revision criteria. Folstein and Piven (1991) skills by folinic acid supplementation. James et al. (2004) have highlighted the role of genetic factors, the prenatal have observed low methionine, low S-adenosyl methio- and perinatal environment in the etiology of autism. nine (SAM)/S-adenosyl (SAH) ratio, low Bailey et al. (1995) have shown that autism cysteine, and low levels, and no alteration was predominantly genetically determined, with high in homocysteine levels in autism. They also showed prevalence among monozygotic twins compared with beneficial effects of supplementation with folic acid/ dizygotic twins. A common genetic basis is assumed but folinic acid, betaine, and in autistic as various forms or degrees of severity of autism have children. These studies indicate that defects in the folate been observed in a single family, susceptibility is probably pathway may play an important role in the pathophysiology

0955-8829 c 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/YPG.0b013e32832cebd2 172 Psychiatric Genetics 2009, Vol 19 No 4 of autism. These observations have resulted in consider- wide ethnic and population level variations in the able interest in the study of polymorphisms in genes distribution of these polymorphisms. Polymorphisms in involved in the folate pathway as risk factors for autism. other loci involved in folate such as reduced folate carrier (80G-A), transcobalamin II (776G-C), - We have hypothesized that low 5-methyl tetrahydro- catechol-O-methyltransferase (472G A), MTHFR - - folate could be because of alterations in the activities of (677C T and 1298A C), and glutathione-S-transferase two enzymes, that is, serine hydroxymethyl transferase (M1) have been studied by James et al. (2006) and (SHMT1, MIM:182144) and methylene tetrahydro- significant gene–gene interactions were found. Given the folate reductase (MTHFR, MIM:607093) because the interconnected nature of the folate pathway, it is possible former catalyzes the reversible conversion of serine that particular combinations of genetic variants may and tetrahydrofolate to glycine and 5,10-methylene underlie autism susceptibility. tetrahydrofolate and the latter catalyzes the flavin adenine dinucleotide-dependent conversion of 5,10- Here, we examine the associations between five folate methylene tetrahydrofolate to 5-methyltetrahydrofolate. pathway genetic polymorphisms, that is, SHMT1 Low methionine and low SAM/SAH ratio could be C1420T, MTHFR C677T, MTHFR A1298C, MTRR because of alterations in the activities of two enzymes, A66G, and methionine synthase (MS A2756G) and the that is, 5-methyltetrahydrofolate homocysteine methyl susceptibility to autism in a cohort of autistic children transferase (MTR, MIM:156570) and methionine and nonautistic children from South India. synthase reductase (MTRR, MIM:602568) because the former catalyzes the remethylation of homocysteine to Methods methionine in presence of 5-methyltetrahydrofolate as Participants cosubstrate and methylcobalamin as cofactor and the The study population consisted of autistic children latter is responsible for regeneration of MTR enzyme, (n = 138), nonautistic control children (n = 138). Re- which is inactivated because of oxidation of its cofactor cruitment took place during the period of 2001–2006. cob(I)alamin to cob(II)alamin, by reductive A multidisciplinary team comprising of psychiatrists, in which SAM is used as methyl donor. clinical psychologists, speech therapists, and a psychiatric social worker evaluated the cases. The diagnosis of Among these four genes, two genes, that is, SHMT and autism was made in a two-tier system. Initially all the MTHFR encode possible rate-limiting enzymes within participants were evaluated for DSM-IV criteria. All the pathway. Increase in cSHMT expression increases the participants who met the criteria were subjected flux of one carbon metabolism through thymidylate to Autism Behavior Checklist (ABC) scoring based on synthesis and inhibits homocysteine remethylation by 57 questions. Parents were asked to answer all the competing with MTHFR for 5,10-methylene tetrahydro- questions. The scores for five different domains namely folate substrate (Herbig et al., 2002). Changes in MTHFR sensory stimuli, relating, body and object use, language, activity may tilt the balance of one-carbon metabolism and social self-help were obtained based on the parental in favor of DNA synthesis at the expense of methyl interview. Children who scored greater than 68 were supply (i.e. SAM) for methylation reactions; a suboptimal enrolled as cases and control children had ABC scoring methyl supply can lead to aberrant DNA methylation less than 21. In addition, children were assessed on (Xu et al., 2007). Among the several polymorphisms in these language function using Receptive Expressive, Emergent four crucial genes, five polymorphisms, that is, SHMT Language Scale and developmental assessment (using C1420T (L474F, rs1979277), MTHFR C677T (A222V, different tests including the Gessells Developmental rs1801133), MTHFR A1298C (E429A, rs1801131), MTR Test, the Raven’s Coloured Matrices, the Vineland Social A2756G (D919G, rs1805087), and MTRR A66G (I22M, Maturity test, and the Binet Kamat test for general rs1801394) are widely distributed throughout the globe, Mental Ability). The developmental quotient (DQ) in synonymous and except one (MTHFR A1298C), the autistic group ranged from 30 to 80 and most of all others are functionally relevant. The reported them have mild-to-moderate developmental delay. The frequencies of minor alleles for MTHFR C677T, mean age of cases was 4.4 ± 1.7 years (age group ranging MTHFR A1298C, MTR A2756G, and MTRR A66G from 2 to 10 years). In the study group, there were 120 polymorphisms in this population were 10, 40.7, (Radha males and 18 females. Rama Devi et al., 2004) 26.4, and 30.1% (Naushad et al., 2008), respectively. The frequency of SHMT C1420T Out of the 150 normal controls who were evaluated for polymorphism was not yet reported in this population. DSM-IV criteria and DQ and found to be nonautistic (have less than 2 two positive symptoms) and have Boris et al. (2004) have reported that C677T and A1298C DQ greater than 80, 138 were selected by matching polymorphisms in MTHFR are risk factors for autism in with cases with reference to age, sex, ethnicity, and Caucasian population. Schneider et al. (1998) reported geographical area to avoid population stratification error. Autism and aberrations in folate pathway Mohammad et al. 173

All measures were taken to match each case with a interaction for the trend test. The expected frequency of control from same ethnic (Indo-Aryans or Mongoloids or genotypes is an application of the Hardy–Weinberg law. Dravidians or Tribals), linguistic (based on regional Maximum likelihood estimates for the expectation were language), and religious (Hindu or Muslim or Christian calculated and tested against the observed counts using or other) group. The mean age of controls was 4.4 ± 1.6 a standard likelihood ratio test (against w2). Maximum years. There were 120 male controls and 18 female likelihoods were also calculated for cases given the controls. Consent was obtained from parents or guardian Hardy– Weinberg equilibrium of the controls. All analyses of each participant before enrollment, after informing were performed using software from the web page goals and objectives of this study. Ethical clearance for statpages.org this study was obtained from the bioethical committee of Center for DNA Fingerprinting and Diagnostics. All Results the participants were recruited only after excluding Autistic children had mean overall ABC score of 80.86, the possibility of any comorbid disorder after a panel whereas control children had mean ABC score of 13.71 of investigations involving metabolic screening, chromo- (P < 0.0001) (Table 1). somal analysis, and Fragile X syndrome. All the investiga- tions were carried out at Center for DNA Fingerprinting MTHFR 677T-variant allele frequencies in cases and and Diagnostics, a genetic research center. controls were 16.3 and 6.5%, respectively. This variant allele was associated with 2.79-fold (95% CI: 1.58–4.93) Genetic analyses risk for autism and this risk was dose dependent DNA was extracted from whole blood samples using (Ptrend < 0.0001). MTHFR 1298C-variant allele fre- standard techniques. Five genetic polymorphisms in four quencies in cases and controls were 53.3 and 53.6%, folate pathway genes, that is, MTHFR 677 C-T, respectively, and this polymorphism was not associated MTHFR 1298 A-C, MTRR 66 A-G, MTR 2756 A- with any risk for autism independently. MTRR G, and SHMT 1420 C-T were examined by 66A-variant allele frequencies in cases and controls were PCR-restriction fragment length polymorphism. (Frost 12.7 and 21.0%, respectively. This variant allele was et al., 1995; Van der Put et al., 1998; Wilson et al., 1999; associated with 1.82-fold (OR: 0.55, 95% CI: 0.35–0.86) Heil et al., 2001; Skibola et al., 2002). Mutational analysis reduced risk for autism. SHMT 1420T-variant allele was performed using coded samples and the analyst was frequencies in cases and controls were 27.9 and 45.3%. not aware whether the sample is a case or control. DNA This variant allele was associated with 2.27-fold (OR: quantity was kept uniform in all samples so that uniform 0.44, 95% CI: 0.31–0.62) reduced risk for autism. The intensities could be obtained under identical conditions. reduction in the risk associated with MTRR and SHMT All restriction enzymes were checked for their quality minor alleles was dose dependent (Ptrend < 0.0005). by digesting the plasmids with known restriction sites. MTR 2756G-variant allele frequencies were 31.2 and Every PCR was accompanied with three controls: (i) a 27.5%, respectively and this polymorphism was not negative control without any genomic DNA, (ii) a negative associated with any significant risk (Tables 2 and 3). control with genomic DNA that does not digest with the Cosegregation of MTHFR 677Tand 1298C variant alleles particular restriction enzyme, and (iii) a positive control was associated with 8.11-fold increased risk for autism with genomic DNA that digests completely with the (95% CI: 2.84–22.92) when compared with MTHFR particular restriction enzyme. All samples were tested in 677CC/1298AA genotype (wild) (Table 4). duplicate to avoid genotyping errors. Discussion Statistical analyses Previous studies have reported that perturbations in Univariate analysis was performed first categorically folate metabolic pathway and polymorphisms at positions and then by treating the number of minor alleles as C677T and A1298C of MTHFR gene are associated a continuous variable to increase the power to detect with increased susceptibility to autism. Three putative trends (referred to as a trend test). For the categorical analyses, we report the individual odds ratios (ORs) and Table 1 ABC scoring in cases and controls confidence intervals (CIs) of the nonwild-type (wild-type is baseline), as well as the overall likelihood ratio test (w2) ABC scoring for different domains Cases Controls P value of association. For the trend test, we report only the P value of the trend (association) test. In addition, we Sensory stimuli 13.72 (2.93) Relating 20.20 (3.33) also examined potential statistical interactions between Body and object use 16.28 (3.96) genes. Again, both categorical and continuous (number of Language 16.79 (3.51) Social self-help 13.85 (2.50) minor alleles) analyses were performed, and, as in the Overall ABC score 80.86 (8.58) 13.71 (2.38) < 0.0001 univariate analyses, we report the ORs for the categorical The ABC scoring was expressed in mean (SD) format. Statistical analysis was analyses (stratified by polymorphisms in each gene) and based on Fisher’s exact t-test. the likelihood ratio test of interaction, but only the test of ABC, Autism Behavior Checklist. 174 Psychiatric Genetics 2009, Vol 19 No 4

Table 2 Genotype and allele frequency distribution in cases and controls

Genotype distribution in cases/controls

Minor alleles in cases/controls Gene/SNP 0 1 2 Ptrend (frequency in cases/controls) OR (95% CI) P value

MTHFR CC CT TT T C677T 98/120 35/18 5/0 < 0.0001 45/18 (16.3/6.5) 2.79 (1.58–4.93) 0.0005 MTHFR AA AC CC C A1298C 35/48 59/32 44/58 0.95 147/148 (53.3/53.6) 0.99 (0.71–1.38) 1.00 MTRR GG AG AA A A66G 105/87 31/44 2/7 < 0.01 35/58 (12.7/21.0) 0.55 (0.35–0.86) 0.02 SHMT CC CT TT T C1420T 61/13 77/125 0/0 < 0.0001 77/125 (27.9/45.3) 0.44 (0.31–0.62) 0.0001 MTR AA AG GG G A2756G 68/73 54/54 16/11 0.36 86/76 (31.2/27.5) 1.19 (0.83–1.72) 0.40

Ptrend was based on logistic regression analysis. P value was based on Fisher’s exact t-test. CI, confidence interval; MTR, methionine synthase; MTHFR, methylene tetrahydrofolate reductase; MTRR, methionine synthase reductase; OR, odds ratio; SHMT, serine hydroxyl methyltransferase; SNP, single nucleotide polymorphism.

Table 3 Combined effects of genotypes based on number of risk 1298C variant allele although not associated with any alleles or number of protective alleles independent risk, it was found to act additively in No. of inflating the risk for autism in combination with MTHFR alleles Cases/controls OR (95% CI) 677T variant allele. Apart from increased prevalence of Risk compound heterozygous MTHFR (MTHFR 677CT/ 0 98/120 1.00 (reference) 1298AC) genotype in autistic children, rare MTHFR 1 35/18 2.38 (1.28–4.43) 2 5/0 Infinity (1.57–infinity) combinations such as triple (MTHFR 677CT/1298CC) Ptrend < 0.0001 and quadruple (MTHFR 677TT/1298CC) were also Protective observed in cases. The overall risk associated with 0 23/2 1.00 (reference) 1 52/57 0.08 (0.02–0.32) cosegregation of these two variant alleles was 8.11-fold 2 10/23 0.038 (0.008–0.18) when compared with wild genotype. Two minor alleles, 3 53/82 0.056 (0.014–0.23) 40/0NA that is, MTRR 66A and SHMT 1420T were found to Ptrend < 0.0005 have negative association with autism. These two variant Statistical analysis was based on logistic regression analysis. alleles were associated with 1.82-fold and 2.27-fold CI, confidence interval; OR, odds ratio. reduced risk for autism. The risk conferred by MTHFR 677T allele and the protection conferred by MTRR 66A and SHMT 1420T alleles were dose dependent. Table 4 Impact of combined genotypes of MTHFR C677T and MTHFR A1298C on autism risk Polymorphisms in MTHFR do affect the amino acid

Combined genotypes Cases/controls OR (95% CI) sequence; the C677T encodes Ala222Val, which is thought to be functionally relevant, whereas A1298C MTHFR C677T MTHFR A1298C CC AA 26/34 1.00 (reference) encodes Glu429Ala, which is thought not to be function- CC AC + CC 72/86 1.40 (1.07–1.84) ally relevant because these polymorphisms lie in cata- CT + TT AA 9/13 0.91 (0.34–2.40) CT + TT AC + CC 31/5 8.11 (2.84–22.92) lytic and regulatory domains of the enzyme, respectively. The risk associated with MTHFR 677T allele could be Statistical analysis was based on Fisher’s exact t-test. CI, confidence interval; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio. because of formation of thermolabile variant of MTHFR enzyme that has enhanced propensity to dissociate into inactive monomers from active dimer form with subsequent loss in flavin adenine dinucleotide-binding metabolic biomarkers in autistic children, that is, low capacity as shown by Yamada et al. (2001). The reduction methyl tetrahydrofolate, low methionine, and low SAM/ in the specific activity is positively correlated to low SAH ratio, have triggered the need to screen for four candi- 5-methyltetrahydrofolate levels. Van der Put et al. (1998) date genes, that is, SHMT, MTHFR, MTR, and MTRR, has shown significant reduction in the specific activity which in turn are crucial for 5,10-methylene tetrahydrofo- of MTHFR enzyme in compound heterozygotes sup- late, 5-methyl tetrahydrofolate, methionine, and cobalamin porting the additive role of A1298C polymorphism in I synthesis, respectively. We postulated that any functional presence of C677T polymorphism. Our finding replicates defect in these loci could have a possible role in autism. the findings of Boris et al. (2004) who have reported 3.46-fold increased risk for autism in MTHFR 677 CT and TT geno- In this study, a positive association was observed between types and much elevated risk in participants with compound MTHFR 677T variant allele and autism risk. MTHFR heterozygous genotype (cases vs. controls: 25 vs. 15%). Autism and aberrations in folate pathway Mohammad et al. 175

In this study, the protective role of MTRR 66A and Yi et al. (2000) have shown that impaired remethylation SHMT 1420T alleles was shown for the first time. There of homocysteine to methionine results in decreased are controversial reports on designating MTRR 66A synthesis of SAM, which further leads to altered allele or 66G allele as the putative mutant allele. Wilson methylation of DNA, proteins, and neurotransmitters. et al. (1999) have hypothesized 66G allele as the putative Abnormal DNA methylation at the 50 CpG island of mutant allele on the basis of an evolutionary conserved Angelman gene (UBE3A) was observed by Jiang et al. isoleucine codon (A allele). However, 66A allele is (2004) in an autistic brain indicating a possible role of considered as a minor allele in this study as well as in epigenetics in autism pathology. studies by Wilson et al. (1999) and Gaughan et al. (2001) based on allele frequency distribution. Wilson et al. (1999) Schatz et al. (1981) have shown that in-vivo elevation and Zhu et al. (2003) have reported association between of brain SAH was associated with decreased cate- 66G allele and risk for neural tube defects. However, no cholamine-O-methyl transferase activity leading to such association study has been reported to date on altered neurotransmitter synthesis. Chiang (1998) autism. Botto et al. (2003) have reported elevated plasma have reported that elevated SAH reduces membrane homocysteine in participants with MTHFR 677CC/ phosphatidylcholine synthesis resulting in altered MTRR 66GG genotype and increased frequency of membrane fluidity and signaling. A review paper by micronuclei in participants with MTHFR 677TT/MTRR Kidd (2002) suggests that in autistic children, reduced 66GG genotype than participants with MTRR 66AG or methylation and detoxification of heavy metals results 66AA genotypes. This indicates inverse relationship in increased oxidative damage. between impaired remethylation and MTRR 66A allele. As the activities of SHMT, MTHFR, MS, and MTRR are MTR A2756G genetic polymorphism was not asso- dependent on the availability of B6, folic acid/folinic ciated with any risk. Silaste et al. (2001) have observed acid, and B12, clinical interventions based on supple- no statistically significant differences in plasma homo- menting these cofactors were found to be beneficial in cysteine in AA, AG, and GG MTR genotypes. Our study treating autism. Rimland (1988) have reviewed 18 clinical on homocysteine in relation to MTR genotypes also did intervention studies and shown the beneficial effect of not show any statistically significant variation according supplementing along with magnesium, but to genotype (unpublished). This indicates that the it was not a controlled study of the treatment. How- remethylation process may not be affected significantly ever,whendouble-blindplacebo/Mg-vitaminB6treatment due to MTR polymorphism alone. Using purified recombi- crossover studies were performed, no benefits for the Mg- nant proteins, Yamada et al. (2006) have shown that MTRR B6 treatment were reported (Nye and Brice, 2005). maintains the activity of MTR at a stoichiometric ratio Lelord et al. (1982) have observed marked improvement of 1 : 1 by stabilizing apo-MTR, enhancing holo enzyme in dopamine metabolism and average evoked potentials formationincombinationwithmethylcobalaminand (sensory processing ability). serving as a molecular chaperone for MTR. The strengths of this study are: its sample size, multi- The protective role associated with SHMT 1420T allele locus analyses, and novelty in projecting the protective could be because of induction of futile folate cycle, role of MTRR 66 A and SHMT 1420 T alleles in autism. where SHMT and 5,10-methylenetetrahydrofolate synthe- Correlation of these genotypes with biochemical markers tase enzymes together seem to buffer the intracellular for remethylation such as SAM and SAH will be more concentration of 5-formylH4folate. Girgis et al. (1997) helpful in understanding the role of these single suggested that the relative activities of each enzyme nucleotide polymorphisms on remethylation. could influence intracellular 5-formylH4folate polygluta- mate levels and consequently a number of folate-dependent In summary, MTHFR 677C-T genetic polymorphism is synthetic activities. an independent risk factor for autism. The risk is inflated further in the presence of MTHFR 1298C variant allele. Waly et al. (2004) have conducted in-vitro studies Two minor alleles, that is, MTRR 66A and SHMT 1420T on neuroblastoma cells and observed significant reduction reduce the risk for autism in dose-dependent manner. of MTR activity on treatment with 1 mmol/l mercury Advances in molecular genetics have resulted in single- and 10 nm thimerosal. Geier and Geier (2004) have locus investigations being superceded by multilocus observed that heavy metal exposure and an increase analyses; however, interpretation of data is consequently in the number of required vaccines containing thimerosal complex. A significant challenge remains in translating as a preservative are significant risk factors for autism. the observations of association studies, such as those Genetic polymorphisms along with these environmental presented in this study, to a clinical setting where factors may adversely affect the remethylation and parents of autistic children are counseled with regard to contribute to autism. genotype-derived susceptibility. 176 Psychiatric Genetics 2009, Vol 19 No 4

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