Summary of Science Demonstrating the Harmful Nature of Mercury in Vaccines
Cellular Research
THIMEROSAL INDUCES TH2 RESPONSES VIA INFLUENCING CYTOKINE SECRETION BY HUMAN DENDRITIC CELLS
Agrawal A, Kaushal P, Agrawal S, Gollapudi S, Gupta S. J Leukoc Biol. 2007 Feb;81(2):474-82.
Agrawal documented that thimerosal exercised TH2-promoting effects through modulation of functions of human dendritic cells (DC) by inhibition of LPS induced proinflammatory cytokines TNF-alpha, IL-6, and IL-12p70 resulting in an increase TH2 (IL-5, IL-13 and decreased TH1 (IFN-gamma). Thimerosal exposure of DC led to depletion of intracellular glutathione (GSH) and the addition of exogenous GSH to DC abolished the TH2 promoting effect of thimerosal. (Note James has documented that children with autism have low levels of plasma glutathione)
MITOCHONDRIAL DYSFUNCTION, IMPAIRED OXIDATIVE-REDUCTION ACTIVITY, DEGENERATION, AND DEATH IN HUMAN NEURONAL AND FETAL CELLS INDUCED BY LOW-LEVEL EXPOSURE TO THIMEROSAL AND OTHER METAL COMPOUNDS
D.A. Geier et al.
Toxicological & Environmental Chemistry. 2009, 1–15, iFirst
Thimerosal (ethylmercurithiosalicylic acid), an ethylmercury (EtHg)-releasing compound (49.55% mercury (Hg)), was used in a range of medical products for more than 70 years. Of particular recent concern, routine administering of Thimerosal-containing biologics/childhood vaccines have become significant sources of Hg exposure for some fetuses/infants. This study was undertaken to investigate cellular damage among in vitro human neuronal (SH-SY-5Y neuroblastoma and 1321N1 astrocytoma) and fetal (nontransformed) model systems using cell vitality assays and microscope-based digital image capture techniques to assess potential damage induced by Thimerosal and other metal compounds (aluminum (Al) sulfate, lead (Pb)(II) acetate, methylmercury (MeHg) hydroxide, and mercury (Hg)(II) chloride) where the cation was reported to exert adverse effects on developing cells. Thimerosal-associated cellular damage was also evaluated for similarity to pathophysiological findings observed in patients diagnosed with autistic disorders (ADs). Thimerosal-induced cellular damage as evidenced by concentration- and time-dependent mitochondrial damage, reduced oxidative–reduction activity, cellular degeneration, and cell death in the in vitro human neuronal and fetal model systems studied. Thimerosal at low nanomolar (nM) concentrations induced significant cellular toxicity in human neuronal and fetal cells. Thimerosal-induced cytoxicity is similar to that observed in AD pathophysiologic studies. Thimerosal was found to be significantly more toxic than the other metal compounds examined. Future studies need to be conducted to evaluate additional mechanisms underlying Thimerosal-induced cellular damage and assess potential co-exposures to other compounds that may increase or decrease Thimerosal-mediated toxicity.
THIMEROSAL INDUCES APOPTOSIS IN A NUEROBLASTOMA MODEL VIA THE CJUN N-TERMINAL KINASE PATHWAY
Herdman ML, Marcelo A, Huang Y, Niles RM, Dhar S, Kiningham KK. Toxicol Sci. 2006 Jul;92(1):246-53.
Herdman notes that cJun N-terminase kinase (JNK)-signaling pathway activation has been implicated in neuronal apoptosis. Herdman investigated the role that the JNK pathway plays in neurotoxicity caused by thimerosal. SK-N-SH cells treated with thimerosal (0-10 microM) showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microM thimerosal treatment at 2 and 4 h.. To assess which components are essential to apoptosis, cells were treated with a cell-permeable JNK inhibitor and the downstream effectors of apoptosis were analyzed. Results indicate that thimerosal-induced neurotoxicity occurs through the JNK-signaling pathway, independent of cJun activation, leading to apoptotic cell death.
UNCOUPLING OF ATP-MEDIATED CALCIUM SIGNALING AND DYSREGULATION INTERLEUKIN-6 SECRETION IN DENDRITIC CELLS BY NANAMOLAR THIMEROSAL
Goth SR, Chu RA, Gregg JP, Cherednichenko G, Pessah IN. Environ Health Perspect. 2006 Jul;114(7):1083-91.
Goth investigated adenosine triphosphate (ATP) mediated Ca2+ responses in dendritic cells (responsible for initiating primary immune responses) exposed briefly to nanamolar concentrations (100nM, 5 min) of thimerosal and found that dendritic cells were exquisitely sensitive to thimerosal resulting in uncoupling of the positive and negative regulation of Ca2 + signals.
THIMEROSAL INDUCES NEURONAL CELL DEATH BY CAUSING CYTOCHROME C AND APOPTOSIS-INDUCING FACTOR RELEASE FROM MITOCHONDRIA.
Yel L, Brown LE, Su K, Gollapudi S, Gupta S. Int J Mol Med. 2005 Dec;16(6):971-7.
Yel demonstrated that thimerosal, at nanamolar concentrations, induced neuronal cell death through the mitochondrial pathway. The thimerosal induced apoptosis was associated with depolarization of mitochondrial membranes, generation of reactive oxygen species and release of cytochrome c and apoptosis-inducing factor, suggesting that thimerosal cause apoptosis in neuroblastoma cells by altering the mitochondrial microenvironment.
IN VITRO UPTAKE OF GLUTAMATE IN GLAST AND GLT-1 TRANSFECTED MUTANT CHO-K1 CELLS IS INHIBITED BY THE ETHYLMERCURY-CONTAINING PRESERVATIVE THIMEROSAL
Mutkus L, Aschner JL, Syversen T, Shanker G, Sonnewald U, Aschner M. Bio Trace Elem Res. 2005 Summer;105(1-3):71-86
Mutkus determined that thimerosal caused significant and selective changes in both glutamate transporter mRNA and protein expression in the CHO-K1 cell line. This study suggests that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis. Glutamate is a neurotransmitter and is necessary for proper brain functioning. Note: Yip (2007) documented decreased levels of glutamate in autistic cerebral brain tissue and Hornig ( 2004) noted altered glutamate receptors in thimerosal exposed mice.
THIMEROSAL INDUCES DNA BREAKS, CASPASE-3 ACTIVATION, MEMBRANE DAMAGE, AND CELL DEATH IN CULTURED HUMAN NEURONS AND FIBROBLASTS
Baskin DS, Ngo H, Didenko VV. Toxicological Sciences. 2003 Aug;74(2):361-8.
Baskin documented that thimerosal disrupts cell membranes, damages DNA and alters cell shape at concentrations only 4 times those expected from vaccines. Greater effects were seen as the length of time of exposure grew, suggesting that under real conditions the concentration needed for the observed alterations would be much lower. It has been documented in subsequent research that exposure of cells to nanomolar levels of thimerosal after 24 hours results in cell alterations.
MITOCHONDRIAL MEDIATED THIMEROSAL-INDUCED APOPTOSIS IN A HUMAN NEUROBLASTOMA CELL LINE (SK-N-SH)
Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK. Neurotoxicology. 2005 Jun;26(3):407-16.
Humphrey noted that after only short (2 hour) exposures to thimerosal at 5 micromolar concentrations in a human neuroblastoma cell line caused morphological changes including membrane alterations and cell shrinkage leading to cell death. Cytochrome C was shown to leak from the mitochondria followed by caspase 9 cleavage. These findings support deleterious effects on cellular cytoarchitecture and initiation of mitochondrial-mediated apoptosis induced by thimerosal.
THIMEROSAL NEUROTOXICITY IS ASSOCIATED WITH GLUTATHIONE DEPLETION: PROTECTION WITH GLUTATHIONE PRECURSORS
JAMES SJ, SLIKKER W 3RD, MELNYK S, NEW E, POGRIBNA M, JERNIGAN S. NEUROTOXICOLOGY. 2005 JAN;26(1):1-8.
James note that the viability of neuronal cell lines was decreased after just 3 hour exposure to 2.5 micromolar concentrations of thimerosal. Also noted was that cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosal in comparison to glioblastoma cells that contain higher levels of GSH. Furthermore, pretreatment with glutathione ethyl ester or NAC prevented cytotoxicity with exposure up to 15 micromolar thimerosal.
BIOCHEMICAL AND MOLECULAR BASIS OF THIMEROSAL-INDUCED APOPTOSIS IN T CELLS: A MAJOR ROLE OF MITOCHONDRIAL PATHWAY
Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S. Genes & Immunity. 2002 Aug;3(5):270-8.
Makani found thimerosal, in micromolar concentration, causes cell death (apoptosis) in immune cells (T cells). The data also suggested that the thimerosal induced apoptosis in T cells occurred via mitochondrial pathways by inducing oxidative stress and depletion of glutathione.
EFFECTS OF THIMEROSAL ON NGF SIGNAL TRANSDUCTION AND CELL DEATH IN NEUROBLASTOMA CELLS
Parran DK, Barker A, Ehrich M. Toxicological Sciences. 2005 Jul;86(1):132-40. Parran documented that thimerosal causes DNA fragmentation of neuronal cells and disrupts neuronal growth factor signaling at micromolar and even nanomolar concentrations. With and without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM (apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that thimerosal could alter NGF-induced signaling in neurotrophin-treated cells at concentrations lower than those responsible for cell death.
ACTIVATION OF METHIONINE SYNTHASE BY INSULIN-LIKE GROWTH FACTOR-1 AND DOPAMINE: A TARGET FOR EURODEVELOPMENTAL TOXINS AND THIMEROSAL
Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC. Molecular Psychiatry. 2004 Apr;9(4):358-70.
Waly noted that thimerosal inhibits critical DNA methylation and attentional pathways at nanomolar concentrations, leading to alterations in brain function. Thimerosal inhibited both IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated MS activity which can lead to alterations in brain function. A novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation was also identified.
THIMEROSAL INDUCES MICRONUCLEI IN THE CYTOCHALASIN B BLOCK MICRONUCLEUS TEST WITH HUMAN LYMPHOCYTES
Westphal GA, Asgari S, Schulz TG, Bünger J, Müller M, Hallier E. Archives of Toxicology. 2003 Jan; 77(1):50 – 55.
Significant induction of micronuclei was seen at concentrations of thimerosal between 0.05-0.5 µg/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 µg/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes (immune cells). These data raise some concern on the widespread use of thimerosal.
ZINC IONS CAUSE THE THIMEROSAL-INDUCED SIGNAL OF FLUORESCENT CALCIUM PROBES IN LYMPHOCYTES
Cell Calcium. 2008 Oct 31. [Epub ahead of print]
Haase H, Hebel S, Engelhardt G, Rink L.,
Institute of Immunology, RWTH Aachen University Hospital, Aachen, Germany.
Most fluorescent probes for the investigation of calcium signaling also detect zinc ions. Consequently, changes in the intracellular zinc concentration could be mistaken for calcium signals. Thimerosal (TMS) is used as a calcium-mobilizing agent and we analyzed the contribution of zinc ions to the signal observed with fluorescent calcium probes after TMS stimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zinc release. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes show that TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity with probes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury-induced signal of the calcium probes Fluo-4 and FURA-2 was completely absent when the zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine] was added. In contrast, the signal caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was unaffected by TPEN. In light of these observations, zinc may also contribute to calcium signals caused by mercury-containing compounds other than TMS, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered.
GENOTOXICITY OF THIMEROSAL IN CULTURED HUMAN LYMPHOCYTES WITH AND WITHOUT METABOLIC ACTIVATION SISTER CHROMATID EXCHANGE ANALYSIS PROLIFERATION INDEX AND MITOTIC INDEX
Eke D, Celik A.
Mersin University, Faculty of Science and Letters, Department of Biology, 33343 Mersin, Turkey.
Toxicol In Vitro. 2008 Jun;22(4):927-34. Epub 2008 Feb 1.
Thimerosal is an antiseptic containing 49.5% of ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. In this study, we evaluated the genotoxic effect of thimerosal in cultured human peripheral blood lymphocytes using sister chromatid exchange analysis in culture conditions with and without S9 metabolic activation. This study is the first report investigating the genotoxic effects of thimerosal in cultured human peripheral blood lymphocyte cells using sister chromatid exchange analysis. An analysis of variance test (ANOVA) was performed to evaluate the results. Significant induction of sister chromatid exchanges was seen at concentrations between 0.2 and 0.6 microg/ml of thimerosal compared with negative control. A significant decrease (p<0.001) in mitotic index (MI) and proliferation index (PRI) as well as an increase in SCE frequency (p<0.001) was observed compared with control cultures. Our results indicate the genotoxic and cytotoxic effect of TH in cultured human peripheral blood lymphocytes at tested doses in cultures with/without S9 fraction.
CELLULAR AND MITOCHONDRIAL GLUTATHIONE REDOX IMBALANCE IN LYMPHOBLASTOID CELLS DERIVED FROM CHILDREN WITH AUTISM.
James SJ, Rose S, Melnyk S, Jernigan S, Blossom S, Pavliv O, Gaylor DW.
Department of Pediatrics; andDepartment of Biostatistics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock, Arkansas, USA.
FASEB J. 2009 Mar 23. [Epub ahead of print]
Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.
GENETIC VARIANT OF GLUTATHIONE PEROXIDASE 1 IN AUTISM.
Ming X, Johnson WG, Stenroos ES, Mars A, Lambert GH, Buyske S.Department of Neurosciences and Neurology, UMDNJ-New Jersey Medical School, 90 Bergen Street, DOC 8100, Newark, NJ 07103, USA.
Brain Dev. 2009 Feb 3. [Epub ahead of print]
Genetic factors can contribute to autistic disorder (AD). Abnormal genes of oxidative stress pathways and increased oxidative stress have been reported in autism spectrum disorders. Polymorphisms of genes involved in glutathione metabolism, e.g. GSTP1 and GSTM1 are reportedly associated with autistic disorder. We investigated a GCG repeat polymorphism of a human glutathione peroxidase (GPX1) polyalanine repeat (ALA5, ALA6 and ALA7) in 103 trios of AD (probands and parents) using the transmission disequilibrium test. Significant transmission disequilibrium (p=0.044) was found in the overall transmission of the three alleles. The ALA6 allele was under transmitted (p=0.017). These results suggest that possessing this ALA6 allele may be protective for AD. Future study of interaction of the GPX1 GCG repeat and other gene polymorphisms such as the MnSOD ALA16 or the GPX1 Pro198Leu polymorphism in this cohort of AD families may shed light in whether the combination of the ALA6 allele with another polymorphism of antioxidant allele contributes to the increased oxidative stress in autism.
Earlier pre-2009 studies relevant to the previous new study on glutathione, autism and mercury:
GLUTATHIONE-S-TRANSFERASE POLYMORPHISM, METALLOTHIONEIN EXPRESSION, AND MERCURY LEVELS AMONG STUDENTS IN AUSTRIA
Gundacker C, Komarnicki G, Jagiello P, Gencikova A, Dahmen N, Wittmann KJ, Gencik M.
Sci Total Environ. 2007 Oct 15;385(1-3):37-47.
BACKGROUND: Detoxification is an essential process in all living organisms. Humans accumulate heavy metals primarily as a result of lifestyle and environmental contamination. However, not all humans experience the estimated individual exposure. This suggests the presence of genetic regulatory mechanisms.
OBJECTIVE: In order to identify genetic factors underlying the inter-individual variance in detoxification capacity for the heavy metal mercury, 192 students were investigated. We focused on the relationship between polymorphisms in glutathione-S-transferase (GST) genes and mercury concentrations in blood, urine, and hair. The correlation between blood mercury levels, GSTT1 and GSTM1 polymorphism, and gene expression of certain metallothionein subgroups (MT1, MT3) was evaluated in a further group of students (N=30).
METHODS: Mercury levels in acid digested samples were measured by cold vapor AAS. Genotyping of the GSTT1 and GSTM1-gene deletion polymorphism was performed by means of PCR. Gene expression of several MT genes was analyzed in lymphocytes from fresh peripheral blood by semiquantitative RT-PCR.
RESULTS: The following was noted: a) hair mercury concentrations are significantly increased in persons with the double deleted genotype (GSTT1-/- and GSTM1-/-) as compared to persons with the intact genotype, and b) MT1X expression is higher in persons with the intact genotype (GSTT1+/+ and GSTM1+/+).
CONCLUSIONS: We conclude that the epistatic effect of the GSTT1 and the GSTM1 deletion polymorphism is a risk factor for increased susceptibility to mercury exposure. The relationship between MT gene expression and GST gene polymorphisms needs further investigation. If MT expression depends on GST polymorphisms it would have important implications on the overall metal detoxification capability of the human organism.
RISK OF AUTISTIC DISORDER IN AFFECTED OFFSPRING OF MOTHERS WITH A GLUTATHIONE S-TRANSFERASE P1 HAPLOTYPE.
Williams TA, Mars AE, Buyske SG, Stenroos ES, Wang R, Factura-Santiago MF, Lambert GH, Johnson WG.
Department of Neurology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Arch Pediatr Adolesc Med. 2007 Apr;161(4):356-61.
OBJECTIVE: To test whether polymorphisms of the glutathione S-transferase P1 gene (GSTP1) act in the mother during pregnancy to contribute to the phenotype of autistic disorder (AD) in her fetus.
DESIGN: Transmission disequilibrium testing (TDT) in case mothers and maternal grandparents. SETTING: Autistic disorder may result from multiple genes and environmental factors acting during pregnancy and afterward. Teratogenic alleles act in mothers during pregnancy to contribute to neurodevelopmental disorders in their offspring; however, only a handful have been identified. GSTP1 is a candidate susceptibility gene for AD because of its tissue distribution and its role in oxidative stress, xenobiotic metabolism, and JNK regulation. PARTICIPANTS: We genotyped GSTP1*G313A and GSTP1*C341T polymorphisms in 137 members of 49 families with AD. All probands received a clinical diagnosis of AD by Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule-Generic testing. MAIN OUTCOME
MEASURES: Association of haplotypes with AD was tested by the TDT-Phase program, using the expectation-maximization (EM) algorithm for uncertain haplotypes and for incomplete parental genotypes, with standard measures of statistical significance. RESULTS: The GSTP1*A haplotype was overtransmitted to case mothers (P = .01 [P = .03 using permutation testing]; odds ratio, 2.67 [95% confidence interval, 1.39-5.13]). Results of the combined haplotype and genotype analyses suggest that the GSTP1-313 genotype alone determined the observed haplotype effect.
CONCLUSIONS: Overtransmission of the GSTP1*A haplotype to case mothers suggests that action in the mother during pregnancy likely increases the likelihood of AD in her fetus. If this is confirmed and is a result of a gene-environment interaction occurring during pregnancy, these findings could lead to the design of strategies for prevention or treatment.
ANALYSIS OF CASE-PARENT TRIOS AT A LOCUS WITH A DELETION ALLELE: ASSOCIATION OF GSTM1 WITH AUTISM.
Buyske S, Williams TA, Mars AE, Stenroos ES, Ming SX, Wang R, Sreenath M, Factura MF, Reddy C, Lambert GH, Johnson WG.
Departments of Statistics and Genetics, 110 Frelinghuysen Rd, Rutgers University, Piscataway, NJ 08854, USA. buyske@stat.rutgers.edu
BMC Genet. 2006 Feb 10;7:8.
BACKGROUND: Certain loci on the human genome, such as glutathione S-transferase M1 (GSTM1), do not permit heterozygotes to be reliably determined by commonly used methods. Association of such a locus with a disease is therefore generally tested with a case-control design. When subjects have already been ascertained in a case-parent design however, the question arises as to whether the data can still be used to test disease association at such a locus. RESULTS: A likelihood ratio test was constructed that can be used with a case-parents design but has somewhat less power than a Pearson's chi-squared test that uses a case-control design. The test is illustrated on a novel dataset showing a genotype relative risk near 2 for the homozygous GSTM1 deletion genotype and autism.
CONCLUSION: Although the case-control design will remain the mainstay for a locus with a deletion, the likelihood ratio test will be useful for such a locus analyzed as part of a larger case-parent study design. The likelihood ratio test has the advantage that it can incorporate complete and incomplete case-parent trios as well as independent cases and controls. Both analyses support (p = 0.046 for the proposed test, p = 0.028 for the case-control analysis) an association of the homozygous GSTM1 deletion genotype with autism.
ABERRATIONS IN FOLATE METABOLIC PATHWAY AND ALTERED SUSCEPTIBILITY TO AUTISM.
Mohammad NS, Jain JM, Chintakindi KP, Singh RP, Naik U, Akella RR.
Center for DNA Fingerprinting and Diagnostics bInstitute of Child Health, Niloufer Hospital, Hyderabad, India.
Psychiatr Genet. 2009 May 13. [Epub ahead of print]
OBJECTIVE: To investigate whether genetic polymorphisms are the underlying causes for aberrations in folate pathway that was reported in autistic children.
BASIC METHODS: A total of 138 children diagnosed as autistic based on Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria and Autism Behavior Checklist scoring and 138 age and sex matched children who are nonautistic were tested for five genetic polymorphisms, that is, cytosolic serine hydroxyl methyl transferase (SHMT1 C1420T), methylene tetrahydrofolate reductase (MTHFR C677T and MTHFR A1298C), methionine synthase reductase (MTRR A66G), methionine synthase (MS A2756G) using PCR-restriction fragment length polymorphism methods. Fisher's exact test and logistic regression analysis were used for statistical analyses.
RESULTS: MTHFR 677T-allele frequency was found to be higher in autistic children compared with nonautistic children (16.3 vs. 6.5%) with 2.79-fold increased risk for autism [95% confidence interval (CI): 1.58-4.93]. The frequencies of MTRR 66A allele (12.7 vs. 21.0%) and SHMT 1420T allele (27.9 vs. 45.3%) were lower in autistic group compared with nonautistic group with odds ratios 0.55 (95% CI: 0.35-0.86) and 0.44 (95% CI: 0.31-0.62), respectively, indicating reduced risk. MTHFR 1298C-allele frequency was similar in both the groups (53.3 vs. 53.6%) and hence individually not associated with any risk. However, this allele was found to act additively in the presence of MTHFR 677T allele as evidenced by 8.11-fold (95% CI: 2.84-22.92) risk associated with MTHFR 677CT+TT/1298AC+CC genotypes cumulatively.
CONCLUSION: MTHFR C677T is a risk factor, whereas MTRR A66G and SHMT C1420T polymorphisms reduce risk for autism. MTHFR A1298C acts additively in increasing the risk for autism.
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