Risk of Autistic Disorder in Affected Offspring of Mothers with a Glutathione S-Transferase P1 Haplotype

ARTICLE Risk of Autistic Disorder in Affected Offspring of Mothers With a Glutathione S-Transferase P1 Haplotype

Tanishia A. Williams, PhD; Audrey E. Mars, MD; Steven G. Buyske, PhD; Edward S. Stenroos, BS; Rong Wang, MS; Marivic F. Factura-Santiago, MD; George H. Lambert, MD; William G. Johnson, MD

Objective: To test whether polymorphisms of the glu- Main Outcome Measures: Association of haplo- tathione S-transferase P1 gene (GSTP1) act in the mother types with AD was tested by the TDT-Phase program, during pregnancy to contribute to the phenotype of au- using the expectation-maximization (EM) algorithm for tistic disorder (AD) in her fetus. uncertain haplotypes and for incomplete parental genotypes, with standard measures of statistical significance. Design: Transmission disequilibrium testing (TDT) in case mothers and maternal grandparents. Results: The GSTP1*A haplotype was overtransmitted to case mothers (P=.01 [P=.03 using permutation test- Setting: Autistic disorder may result from multiple ing]; odds ratio, 2.67 [95% confidence interval, 1.39- genes and environmental factors acting during preg- 5.13]). Results of the combined haplotype and geno- nancy and afterward. Teratogenic alleles act in mothers type analyses suggest that the GSTP1-313 genotype alone during pregnancy to contribute to neurodevelopmental determined the observed haplotype effect. disorders in their offspring; however, only a handful have been identified. GSTP1 is a candidate susceptibil- Conclusions: Overtransmission of the GSTP1*A hap- ity gene for AD because of its tissue distribution and its lotype to case mothers suggests that action in the mother role in oxidative stress, xenobiotic metabolism, and during pregnancy likely increases the likelihood of AD JNK regulation. in her fetus. If this is confirmed and is a result of a gene- Participants: We genotyped GSTP1*G313A and environment interaction occurring during pregnancy, GSTP1*C341T polymorphisms in 137 members of 49 fami- these findings could lead to the design of strategies for lies with AD. All probands received a clinical diagnosis of prevention or treatment. AD by Autism Diagnostic Interview–Revised and Autism Diagnostic Observation Schedule–Generic testing. Arch Pediatr Adolesc Med. 2007;161:356-361

N MOST CASES IN WHICH A GENE lele homozygotes.5 Demonstrating in- has been associated with a disor- creased frequency of a putative terato- der, the disease allele acts in the genic allele in mothers but not fathers of affected individual. Alterna- affected individuals is evidence for a tera- tively, a disease allele of a gene togenic allele and is the method that has Author Affiliations: Imay act in the mother during pregnancy been used in most articles. Strong evi- Departments of Neurology to contribute to the phenotype of her af- dence of a teratogenic allele (eg, by ma- (Drs Williams and Johnson, fected child.1 So far, there is evidence for ternal transmission disequilibrium test- Mr Stenroos, and Ms Wang) such maternally acting alleles, so-called ing [TDT] as in the present study) has and Pediatrics (Drs Mars, teratogenic alleles, for only a handful of rarely been achieved,1 and there is no Factura-Santiago, and Lambert) 2 and Center for Childhood genes. strong evidence for any so far in autism. Neurotoxicology and Exposure Examples of teratogenic alleles in- Children with autistic disorder (AD) Assessment (Drs Mars, clude the following: (1) in spina bifida, the show deviation from the normal develop- Lambert, and Johnson), G allele of MTR A2756G and MTRR A66G mental pattern with impaired social inter- University of Medicine and polymorphisms and the deletion allele of actions and communication, restricted in- Dentistry of New Jersey–Robert the DHFR 19–base pair (bp) deletion poly- terests, and repetitive stereotyped patterns Wood Johnson Medical School, morphism3; (2) in Down syndrome, the G of behavior that are evident before 36 Piscataway, and Departments of 6,7 Statistics and Genetics, Rutgers allele of MTRR A66G and the T allele of months of age. Findings from clinical ge- 4 University, New Brunswick, NJ MTHFR C677T polymorphisms ; and (3) netic studies and modeling studies sug- (Dr Buyske). in orofacial clefting, the GSTT1-null al- gest that AD may be caused by multiple

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 interacting gene loci,8,9 while environmental and epige- Seven cytosolic families of GSTs are known in hu- netic factors may contribute to variable expressivity pos- mans, including at least 16 cytosolic GST subunits (most sibly through interaction with genetic susceptibility fac- of them polymorphic), with some alleles causing func- tors.9,10 Environmental factors contributing to AD could tional alteration. For alleles with diminished function, their include toxic endogenous metabolites or exogenous tox- specific substrates might accumulate and contribute to oxi- ins or teratogens. dative damage; increased enzyme activity could also lead Neuropathological studies,11,12 cytoarchitectonic stud- to oxidative damage if the product is toxic.35 The pi class ies,13 minicolumn studies,14,15 and neonatal blood stud- of GSTs, represented by a single GST (variously known as ies16 of neurotrophins and neuropeptides support the pre- GSTP1, GSTP1-1, GSTP, GSTp, and GSTpi) coded for by natal origin of certain brain abnormalities in autism. a gene on chromosome 11q13, is expressed at the highest Consequently, it is possible that maternal genes, acting levels in most extrahepatic tissues.36 during pregnancy, could contribute to the autism phe- GSTP1 has 4 polymorphic alleles resulting from 2 notype in the fetus. amino acid changes, Ile105Val (A313G) and Ala114Val Several maternal effects have been described for au- (C341T). There is evidence37,38 that these polymorphic tism, but none reported so far gives strong evidence of a variants are functional, affecting enzyme activity and sub- teratogenic allele. For some of these, no involvement of strate specificity. For example, variation at position 105 specific maternal genes has yet been demonstrated (eg, affects thermostability of the GSTP1 enzyme39 and its cata- autism associated with maternal ingestion during preg- lytic efficiency for some substrates39,40 and correlates with nancy of thalidomide17 or valproic acid18 and increased oxidative DNA damage in breast cancer tissues.41 risk of autism spectrum disorder in children of mothers GSTP1 was recently studied in autism using a family- with diabetes mellitus or epilepsy19). There is some evi- based association study design.42 That study included only dence that maternal alleles at the MAOA locus and pos- case trios (affected individual and parents) and had nega- sibly the DBH locus may modify IQ in children with au- tive results. In the present study, we genotyped 137 in- tism.20 Diminished IQ is often seen in autism but not as dividuals in 49 families with AD for the GSTP1*G313A a cardinal feature. Although mental retardation is not part and GSTP1*C341T polymorphisms43 using maternal trios of the diagnostic criteria for autism, the 2 diagnoses could (mother of individual with AD and her parents) to look be interacting through diagnostic substitution in the popu- for a teratogenic allele. We analyzed these single nucleo- lation.21 In addition, some alleles of the glutamate recep- tide polymorphisms and the resulting haplotypes using tor 6 gene (GLUR6, GRIK2) reportedly showed in- a haplotype-based statistical approach. creased maternal transmission to male children with autism; these findings were ascribed to meiotic drive or METHODS imprinting.22 There is evidence that the major histocom- patibility complex extended haplotype, HLA B44-SC30- DR4, may act as a teratogenic allele for autism because The study was approved by the Institutional Review Board of the frequency of this haplotype was increased in moth- University of Medicine and Dentistry of New Jersey–Robert ers of children with autism compared with controls.23 So Wood Johnson Medical School, and informed consent was ob- far, this has not been confirmed using a stronger study tained from the participants. Families with a child diagnosed as having autism were invited to participate through advertise- design such as maternal TDT. This haplotype frequency ments in the newsletter of the New Jersey Center for Outreach was also increased in autism cases compared with con- and Services for the Autism Community. A few families were 23 trols, suggesting action in the cases as well. recruited through the Department of Pediatrics. Selection cri- Some recent investigations in humans have linked oxi- teria for families were as follows: (1) participation of a pro- dative stress to autism.24 For example, significantly de- band with the clinical diagnosis of autism by his or her neu- creased levels of glutathione, a significantly lower ratio rodevelopmental pediatrician as assessed by telephone interview of reduced glutathione to oxidized glutathione, and other with the primary caregiver, (2) clinical diagnosis of AD con- metabolic abnormalities in individuals with autism were firmed for each proband by Autism Diagnostic Interview– 25,26 Revised and Autism Diagnostic Observation Schedule– interpreted as evidence of oxidative stress. Glutathi- 44,45 one is the most important endogenous antioxidant27 and Generic testing, and (3) blood sampling from the mother and at least 1 maternal grandparent. All probands were tested is the most abundant nonprotein thiol.28 Recently, in- ␣ using the Autism Diagnostic Interview–Revised and the Au- creased urinary excretion of 8-isoprostane-F2 ,abio- tism Diagnostic Observation Schedule–Generic by one of us marker of lipid peroxidation and oxidative stress, was (A.E.M.), a trained and certified examiner, and all received the found in autism,29 a finding that has been confirmed.30 clinical diagnosis of AD by results of these tests. Accumulating data support the importance of the glu- Venous blood was collected in specimen tubes (Vacutain- tathione S-transferase (GST) supergene family as a protec- ers; Becton Dickinson, Mountainview, Calif) containing EDTA. tive factor against reactive oxygen species and the prod- The samples were frozen immediately in cryovials at –70°C or ucts of oxidative stress.31,32 Glutathione S-transferases, a were frozen briefly (2-3 days) at –20°C for transport to the labo- category of phase 2 enzymes,33 catalyze the conjugation of ratory and prepared there. Genomic DNA was obtained from glutathione to different toxic electrophiles that have been whole blood or white blood cells isolated by centrifugation. DNA extraction was performed using commercially available blood activated by phase 1 enzymes. Glutathione S-transferases kits (QIAamp DNA; Qiagen, Valencia, Calif). Genomic DNA conjugate and detoxify products of oxidative stress. They was stored at 4°C. 34 also conjugate toxins that produce oxidative stress. Con- Genotyping of the GSTP1*A313G (Ile105Val) and jugation of glutathione to a compound by GST can some- GSTP1*C341T (Ala114Val) polymorphisms was carried out by times increase its toxicity or even create toxicity.35 polymerase chain reaction [PCR]–restriction fragment length

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 GSTP1*C haplotypes were undertransmitted at almost Table 1. Estimated Haplotype Frequencies* the same rates. When those 2 rates are constrained to be equal, the odds ratio for the GSTP1*A haplotype is 1 di- Haplotype Components Frequency vided by 0.375 (odds ratio, 2.67 [95% confidence inter- GSTP1*A 313A/341C 0.71 val, 1.39-5.13]). GSTP1*B 313G/341C 0.20 To better understand the haplotype effect, we exam- GSTP1*C 313G/341T 0.09 ined the individual genotypes. The individual geno- GSTP1*D 313A/341T Not observed in sample; frequency set to 0 types (Table 3 and Table 4) were not significantly overtransmitted using TDT (P=.06 for GSTP1-313 and P=.36 *Maximum likelihood estimation based on founders only. for GSTP1-341), although the A allele at GSTP1-313 was close to being significantly overtransmitted. Testing by means of maximum likelihood estimation using the EM polymorphism methods as previously described.43 Polymerase algorithm for missing data gave a highly significant over- chain reaction was carried out in a thermal cycler (GeneAmp 9600; transmission for GSTP1-313*A (P=.005 [P=.004 using Perkin-Elmer Cetus, Norwalk, Conn). For GSTP1*A313G permutation testing]), while GSTP1-341 was not signifi- (Ile105Val), the PCR product was restricted using Alw26I; prod- cantly overtransmitted (P=.10). Both loci were in Hardy- ucts were separated on 8% polyacrylamide gels and were visual- Weinberg equilibrium among maternal grandparents ized using ethidium bromide. Digestion produced 110- and 90-bp fragments for the G allele and a 200-bp band consistent with the (P=.31 for GSTP1-313 and P=.60 for GSTP1-341) and PCR amplimer for the A allele, which had no Alw26I restriction among mothers of individuals with AD (P=.90 for GSTP1- site. For GSTP1*C341T (Ala114Val), the PCR product was re- 313 and P=.71 for GSTP1-341). stricted using AciI; products were separated on 8% polyacryl- Findings from the combined haplotype and geno- amide gels using 1ϫ Tris-borate-EDTA buffer and were visual- type analyses suggest that the GSTP1-313 genotype alone ized using ethidium bromide. Digestion produced approximate determined the observed haplotype effect. The haplo- 120- and 90-bp fragments for the C allele and an approximate type data enabled some matings that were uninforma- 210-bp band consistent with the PCR amplimer for the T allele, tive for classic TDT to be resolved and enabled greater which had no AciI restriction site. The GSTP1*A haplotype com- power for testing. prises 313A/341C, the GSTP1*B haplotype comprises 313G/ 341C, and the GSTP1*C haplotype comprises 313G/341T. The GSTP1*D haplotype (313A/341T) is rare and was not found in COMMENT the present data set. The 2-locus haplotypes were analyzed, with the individual genotypes examined in a secondary analysis. Haplotype fre- The GSTP1*A haplotype was significantly more frequencies were determined by means of maximum likelihood quently transmitted to mothers of individuals with AD estimation using the expectation-maximization (EM) algo- in maternal trios (Table 2), suggesting that it may be act- rithm.46 Association of haplotypes with AD was tested by means ing in mothers during pregnancy to contribute to the phe- of the TDT-Phase program,47 using the EM algorithm for un- notype of autism in the fetus. Findings from the com- certain haplotypes and for incomplete parental genotypes. The bined haplotype and genotype analyses suggest that the program fits an unconditional logistic regression analysis using GSTP1-313 genotype alone determined the observed hap- transmitted haplotypes as pseudocases and untransmitted hap- 48 lotype effect. Therefore, the GSTP1-313*A allele may be lotypes as pseudocontrols, modeling the haplotype relative risk. 2,3,50 Incorporating uncertain haplotypes subjects the algorithm to acting as a teratogenic allele. population stratification effects. Permutation testing, whereby The action of a GSTP1 polymorphic variant in the the transmitted and untransmitted labels are reassigned, gives mother (possibly during pregnancy) to affect her off- a result more robust against population stratification. spring may not be unprecedented. Suggesting action dur- Individual genotypes were examined using classic TDT,49 ing pregnancy, a recent study found evidence that the same as implemented in TDT-Phase (by specifying 1 genotype at a GSTP1 polymorphisms in mothers were significantly cor- time and without using the EM algorithm). They were also ex- related with lung function in their children with asthma, amined by means of maximum likelihood estimation using the an effect that was independent of transmission of alleles EM algorithm to loop over uncertain parental genotypes. to the child.51 Asthma is a disorder in which oxidative stress plays a role51 and in which isoprostanes have been RESULTS implicated,52 as in autism.29,30 Although we did not have information about maternal asthma in our study, a re- We genotyped the GSTP1*G313A and GSTP1*C341T cent study53 reported a greater than 2-fold increased risk single nucleotide polymorphisms in 137 members of 49 of autism spectrum disorder in offspring if maternal di- families with an AD proband. There were 49 mothers, agnoses of asthma and allergies were present during the 49 maternal grandmothers, and 39 maternal grandfa- second trimester of pregnancy. thers in 39 complete maternal trios and 10 incomplete At least 15 other examples of the action of teratogenic maternal trios. alleles have been reported involving at least 12 different The distribution of GSTP1 haplotype frequencies alleles.2,3,50 One of these was a GST, the GSTT1*0 allele, (Table 1) was comparable with published frequen- implicated as a possible teratogenic allele for orofacial cleft- cies.43 The haplotype analysis (Table 2) demonstrated ing.2 The interaction of a maternal deletion of GSTT1 significant overtransmission (P=.01 [P=.03 using per- (which detoxifies products of cigarette smoke) with a ma- mutation testing]). The GSTP1*A haplotype was over- ternal environmental effect (ie, smoking) was associated transmitted to case mothers, while the GSTP1*B and with increased risk of having a child with oral clefting.5

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Transmitted Not Transmitted Odds Ratio Haplotype No. Frequency No. Frequency (95% Confidence Interval) GSTP1*A 80 0.82 61.25 0.63 1 GSTP1*B 13 0.13 25.61 0.26 0.389 (0.184-0.819) GSTP1*C 5 0.05 11.14 0.11 0.344 (0.114-1.039)

*The expectation-maximization (EM) algorithm was used for uncertain phase and missing genotype information.

Table 3. Transmission Disequilibrium Testing (TDT-Phase) of Individual Genotypes*

Transmitted Not Transmitted Odds Ratio Genotype No. Frequency No. Frequency (95% Confidence Interval) GSTP1-313 *A 23 0.66 12 0.34 1 GSTP1-313 *G 12 0.34 23 0.66 0.522 (0.260-1.049) GSTP1-341*C 7 0.64 4 0.36 1 GSTP1-341*T 4 0.36 7 0.64 0.571 (0.167-1.952)

*Using 1 marker at a time and without the expectation-maximization (EM) algorithm, yielding classic TDT.

Table 4. Transmission Disequilibrium Testing (TDT-Phase) Results of Individual Genotypes*

Transmitted Not Transmitted Odds Ratio Genotype No. Frequency No. Frequency (95% Confidence Interval) GSTP1-313 *A 80 0.82 61.95 0.63 1 GSTP1-313 *G 18 0.18 36.05 0.37 0.387 (0.200-0.745) GSTP1-341*C 93 0.95 86.60 0.88 1 GSTP1-341*T 5 0.05 11.40 0.12 0.409 (0.137-1.216)

*Using maximum likelihood estimation and the expectation-maximization (EM) algorithm for missing genotype information.

To our knowledge, the present study is only the sec- MAPKs are well situated to affect brain development and ond to document action of a teratogenic allele or haplo- differentiation, and some genes in this pathway could type using a stringent case-parent study design1 and is be candidates for autism susceptibility genes. The stress the first for autism. Although TDT is robust against popu- kinases are transiently activated in response to various lation stratification, the unconditional logistic regres- environmental or metabolic stimuli, including UV or sion analysis used in this study can be affected by strati- x-irradiation, heat shock, osmotic shock, or inflamma- fication. The permutation testing included herein is tory cytokines. JNK is regulated by factors besides GSTP1, considered to give results more robust to population including reactive oxygen species and changes in the re- stratification. dox potential. The selective and potent regulation by GSTP1 has a role in preventing and controlling oxi- GSTP1 is independent of other functions of GSTP1 such dative stress, and oxidative stress has been linked to AD, as detoxification and excretion of toxins and xenobiot- as discussed earlier. Thalidomide12,17 and valproic acid54 ics by conjugating them with glutathione. are teratogens associated with AD, and both induce oxi- GSTP1 not only inhibits JNK but also physically binds dative stress.55,56 Both have been linked to depletion of to it.58 Four domains of GSTP were implicated in its regu- glutathione, with thalidomide associated with glutathi- lation of JNK activation through binding or inhibi- one depletion in a sensitive species (rabbit) but not a re- tion.59 Both alleles of the GSTP1*A haplotype that were sistant species (rat)56 and with valproic acid associated associated herein with autism occur within the region con- with glutathione depletion in humans.57 tributing to binding of GST to the Jun-JNK complex; GSTP1 has an additional function, regulation of Jun GSTP1 residue 105, which contributed most or all of the N-terminal kinase (JNK), a mitogen-activated protein ki- haplotype effect observed, lies within the H site, the re- nase (MAPK) and stress kinase. Activation of MAPKs in gion where electrophilic toxins, xenobiotics, or metabo- response to extracellular stimuli leads to regulatory lites bind to GST for conjugation with glutathione.39 changes in different cellular functions such as mitosis, JNK activation and a clinical therapeutic response using differentiation, apoptosis, and cell survival. Therefore, CNI-1493, an inhibitor of JNK and p38 MAPK activa-

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 tion, were reported60 in Crohn disease. A panenteric in- Financial Disclosure: None reported. flammatory bowel–like disease has been reported in re- Funding/Support: This study was supported by grants gressive AD.61 Therefore, a therapeutic target for the R21-44244 and K25-AA015346 from the National Insti- inflammatory bowel–like disease in AD might be rel- tutes of Health, by the New Jersey Governor’s Council evant if the present results linking GSTP1 alleles and au- on Autism, by the National Alliance for Autism Re- tism are confirmed. search, by the New Jersey Center for Outreach and Ser- Several genes reported to be associated with autism vices for the Autism Community, and by Pediatric En- are related to MAPKs or to MAPK pathways, as is GSTP1. vironmental Health Center grant P50-ES09598 and Center For example, the reelin gene62 and the apolipoprotein E for Childhood Neurotoxicology and Exposure Assess- (APOE)63 gene have been associated with autism, and both ment grant P01-ES11256 from the National Institutes of proteins competitively bind the same receptor, Health and United States Environmental Protection ApoER2.64,65 A direct molecular link between ApoER2 and Agency Centers of Excellence. the JNK signaling pathway has been demonstrated.64,65 Acknowledgment: We are grateful for the participation of The finding is strengthened by a previous study66 in which the individuals with autism and their family members. homozygosity for the GSTM1 deletion allele was associated with AD. 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