Association between PTGS2 polymorphism and autism spectrum disorders in Korean trios

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Neuroscience Research 62 (2008) 66–69 www.elsevier.com/locate/neures

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Association between PTGS2 polymorphism and autism spectrum disorders in Korean trios Hee Jeong Yoo a, In Hee Cho b, Mira Park c, Eunchung Cho a, Soo Churl Cho d, Bung Nyun Kim d, Jae Won Kim d, Soon Ae Kim e,* a Department of Psychiatry, Seoul National University Bundang Hospital, Seongnam, Kyeonggi, Republic of Korea Department of Psychiatry, Gil Medical Center, Gachon University of Medicine and Science, Incheon, Republic of Korea c Department of Preventive Medicine, School of Medicine, Eulji University, Daejon, Republic of Korea d Department of Child and Adolescent Psychiatry, College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea e Department of Pharmacology, School of Medicine, Eulji University, 143-5 Youngdu-dong, Jung-gu, Daejon 301-832, Republic of Korea b

Received 20 February 2008; accepted 29 May 2008 Available online 5 June 2008

Abstract Cyclooxygenase-2 (Cox-2) is an inducible enzyme involved in neuroplasticity and the neuropathology of the central nervous system. This study evaluated the relationship between autism spectrum disorders (ASDs) and polymorphisms of PTGS2 (the gene encoding Cox-2) with 151 Korean family trios including children with ASDs. We found that the A allele of rs2745557 was preferentially transmitted in ASDs ( p < 0.01) and that the GAAA haplotype was significantly associated with ASDs ( p < 0.01). We also observed statistically significant associations between each genotype and the specific symptom domain scores of ADOS and ADI-R, including communication, qualitative abnormalities in reciprocal social interaction, and overactivity/agitation. # 2008 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. Keywords: Autism spectrum disorders (ASD); Cyclooxygenase 2; Transmission disequilibrium test (TDT); Association study

1. Introduction Autism is a severe neurodevelopmental disorder that is characterized by social and communicative impairment accompanied by repetitive and stereotypical behaviors and interests. Data from several epidemiological twin and family studies provide substantial evidence for the fact that autism spectrum disorders (ASDs) are among the most heritable complex disorders in which chromosomal abnormalities and genetic polymorphisms are thought to play a role, and that several genes interact with one another to produce the clinical phenotype (Sebat et al., 2007; Weiss et al., 2008). Therefore, ASDs represent a multi-locus model with epistasis (Folsteinl and Rosen-Sheidley, 2001). During inflammation, cyclooxygenase-2 (Cox-2) is rapidly induced by growth factors, cytokines, and proinflammatory

* Corresponding author. Tel.: +82 42 259 1672; fax: +82 42 259 1679. E-mail address: [email protected] (S.A. Kim).

molecules, and it is thought to be involved in prostanoid production under acute and chronic inflammatory conditions as well as in neurodegenerative processes, seizures, ischemia, normal neuronal functioning, neurotoxicity, and synaptic plasticity (Minghetti, 2004). Cox-2 is constitutively expressed in the neuronal tissues in regions that are critically affected in psychiatric disorders, such as the forebrain, hippocampus, hypothalamus, and amygdala (Ibuki et al., 2003). The relationships between Cox-2 and neurodegenerative and psychiatric diseases have been studied. Some studies have reported an association between decreased risk of Alzheimer’s disease and the C allele of Cox-2 (prostaglandin-endoperoxide synthase 2; PTGS2)-765 promoter polymorphism, which plays a protective role in disease development (Yermakova and O’Banion, 2001). There are three lines of indirect evidence supporting the involvement of Cox-2 in ASDs. The first is the possible relationship between Cox-2 and Rett syndrome, a variant of ASDs, in which the laminar pattern of cortical Cox-2 immunoreactivity is disrupted and Cox-2-positive neurons are decreased in number and distributed randomly (Kaufmann

0168-0102/$ – see front matter # 2008 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. doi:10.1016/j.neures.2008.05.008

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for cSNP for determining the importance of biological meaning for this study, the cSNPs of PTGS2 did not meet our criteria. Therefore, we selected four SNPs in the intronic regions (rs2745557), 30 -untranslated region (rs5275), 50 -proximal gene region (rs689466), and 30 -near gene region (rs4648308) of the PTGS2. In addition, we genotyped these SNPs by using the GoldenGateTM assay (Illumina).

et al., 1997). Second, Cox-2 could play a role in long-term potentiation, which is a major model of synaptic plasticity and subsequent learning and memory. This notion emerged from a study in which rats subjected to selective destruction of basal forebrain cholinergic neurons during the first post-natal week showed decreased levels of hippocampal Cox-2 in adulthood, accompanied by impaired social memory, which is a critical deficit in human ASDs (Ricceri et al., 2004). Third, it has been hypothesized that a dysregulated or abnormal immune response is involved in some forms of ASDs. Since cytokines and other products of immune activation have widespread effects on neuronal pathways and can alter behaviors such as mood and sleep, it is possible that Cox-2 is involved in these abnormal immune processes in ASDs (Ashwood et al., 2006). Based on previous reports, we hypothesized that PTGS2 polymorphisms are related to ASD pathogenesis, and we conducted a family-based association study of PTGS2 in Korean children with ASDs by using the transmission disequilibrium test (TDT) and haplotype analysis. To the best of our knowledge, this is the first study to investigate the relationship between PTGS2 and ASDs.

2.3. Genotypes and symptom severity As a measure of symptom severity, we used the sum of the three behavioral domain scores of the ADI-R diagnostic algorithms, i.e., qualitative abnormalities in reciprocal social interaction; qualitative abnormalities in communication; and restricted, repetitive, and stereotypical patterns of behavior. On the ADOS, the mean domain scores for each genotypic group were compared, since the composition of items differed among the modules. The ADOS and ADI-R item scores ranged from 0 (no abnormality) to 3 (most abnormal). A score of 3 was converted to 2 and 8 (not applicable) or 9 (unknown or not asked) to 0, as specified in the algorithm.

2.4. Statistical analyses To check the data quality and presence of genotypic errors, we evaluated the Hardy–Weinberg equilibrium and Mendelian inheritance of genotypes within the trios. We tested the family-based association for each individual polymorphism and haplotype by using the standard TDT method in Haploview (http://www.broad.mit.edu/mpg/haploview/). An analysis of variance (ANOVA) was used to compare the ADOS and ADIR domain scores according to the specific genotype of each SNP. When the results of the ANOVA were significant, post hoc Bonferroni tests were performed to assess the differences between the genotype groups. SPSS ver. 15.0 software (SPSS, Chicago, IL, USA) was used for the analysis. Statistical significance was defined as p < 0.05.

2. Materials and methods 2.1. Subjects The subjects were recruited from a family-based genetic association study of ASD conducted by the same research group (Cho et al., 2007; Kim et al., 2006, 2007), and we recruited 25 trios in addition to the 126 trios described in Kim et al. (2007). Subject ascertainment and diagnostic methods have been previously described. Briefly, ASD was diagnosed using the Autism Diagnostic Interview-Revised (ADI-R) (Yoo, 2007) and the Korean version of the Autism Diagnostic Observation Schedule (ADOS) (Yoo and Kwak, in press) with the best estimate of two board-certified child psychiatrists. Subjects who were diagnosed with or strongly suspected of having neurofibromatosis, tuberous sclerosis, any kind of metabolic encephalopathy, Down’s syndrome, Fragile X syndrome, or other known chromosomal abnormalities were excluded. Written informed consent was obtained from the parents/primary caregivers, and this study was approved by the review boards of the institutions at which it was conducted.

3. Results The present study included 151 complete trios, consisting of patients with ASD (79.9  35.6 months, 86.1% male, 87.4% autism) and their biological parents. The psychological properties are fairly similar, as described previously (Kim et al., 2007). We checked the Hardy–Weinberg equilibrium and Mendelian inheritance of genotypes within the trios. For all the SNPs, the genotypic distribution did not deviate from that expected based on the Hardy–Weinberg equilibrium (0.509 < p < 0.937). One child genotyping data for four SNPs was missed and three Mendelian inheritance errors were found. These four trios were excluded from the data set in this TDT and haplotype analysis. The TDT showed that the A allele was overtransmitted in ASDs (x2 = 7.143, p = 0.008) for 1 SNP (rs2745557, intronic) (Table 1). After applying the Bonferroni correction, this result was found to be statistically significant. When we estimated the pairwise linkage disequilibrium (LD) to

2.2. Genetic analysis Blood samples from all the subjects were collected in EDTA-containing tubes and stored at 70 8C. Genomic DNA was extracted using the G-spin Genomic DNA Extraction Kit (Intron, Daejeon, Korea). We evaluated the genetic structure of PTGS2 by using Entrez SNP database (http:// www.ncbi.nlm.nih.gov/) and publicly available genotype data for Asian populations from the International HapMap project (http://www.hapmap.org). We assessed the common genetic variation of PTGS2 (SNPs in the gene region with minor allele frequencies >5% in two Asian populations). Although we looked Table 1 Transmission disequilibrium test of the PTGS2 gene in autism spectrum disorders SNP

Minor allele

Minor allele frequency

Overtransmitted allele

T

NT

x2

p

rs4648308 rs5275 rs2745557 rs689466

A G A G

0.044 0.171 0.024 0.481

– A A G

12 47 12 76

12 45 2 73

0 0.043 7.143 0.06

1 0.835 0.008* 0.806

T, transmitted; NT, non-transmitted; *p < 0.01.

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Table 2 Haplotype analysis for the genetic association between PTGS2 gene and autism spectrum disorders Block

Haplotype

Frequency

T

NT

x2 (d.f. = 1)

p

rs4648308 + rs5275 + rs2745557 + rs689466

AGGA GGGA GAGG GAGA GAAA

0.045 0.125 0.484 0.321 0.024

12.0 33.2 76.8 53.6 12.0

12 34.2 72.2 66.2 2.0

0 0.014 0.143 1.327 7.143

1 0.905 0.706 0.249 0.008*

T, transmitted; NT, non-transmitted; *p < 0.01.

Table 3 Association between the genotypes of rs2745557 and the clinical symptoms of patients with ASD SNP

Genotype of rs2745557 GG

AG

ADI-R Qualitative abnormalities n reciprocal social interaction

22.59(6.84)

21.11(5.56)

0.63

Qualitative abnormalities in communication Verbal subjects Nonverbal subjects Restricted, repetitive & stereotyped patterns of behavior

18.78(4.07) 12.36(3.05) 6.38(3.22)

16.17(3.06) 14.00(0.00) 5.44(2.07)

1.53 6.51 0.86

0.13 0.00a 0.39

1.07(0.39) 1.40(0.39) 1.35(0.65) 0.54(0.40) 0.80(0.76) 0.34(0.61) 0.33(0.56)

1.05(0.32) 1.49(0.36) 1.30(0.75) 0.50(0.29) 0.20(0.42) 0.70(0.82) 0.60(0.70)

0.14 0.70 0.27 0.34 4.00 1.73 1.44

0.89 0.49 0.78 0.73 0.00a 0.08 0.15

26.21(12.60) 29.20(19.13) 73.34(140.13)

23.33(10.26) 32.13(19.65) 46.00(7.77)

0.67 0.42 0.78

0.51 0.68 0.64

ADOS Language and communication Reciprocal social interaction Imagination/creativity Stereotyped behaviors and restricted interests Overactivity/agitation Tantrums, aggression, negative or disruptive behavior Anxiety Onset criteria First recognition of symptoms (months) Onset of first-words (months) Onset of first-phrase (months) a

t

p 0.53

p < 0.001.

determine the extent of LD for the four SNPs, the results showed that all the SNPs were in strong disequilibrium (D0 > 0.987) with each other. The five haplotypes consisting of all the PTGS2 SNPs were analyzed (Table 2). The GAAA haplotype was found to be significantly associated with ASD (x2 = 7.143, p = 0.008). We explored the variation in the quantitative measures of social interaction, communication, repetitive behavior, and restricted interest assessed using the ADOS and ADI-R among the genotypes of rs2745557, which showed a significantly preferential transmission of the A allele in ASD. As a result, overactivity/agitation on the ADOS is significantly more severe for subjects with the GG genotype than for those with the AG genotype, and qualitative abnormalities in communication in nonverbal subjects on the ADI-R were significantly higher for subjects with the AG genotype than for those with the GG genotype (t = 4.00, p < 0.001 and t = 6.51, p < 0.001, respectively) (Table 3). No significant difference was observed among the genotypes in rs2745557 with regard to the onset of the first words and phrases ( p > 0.05).

4. Discussion The result of this first family-based study that explored the association between the PTGS2 and ASDs showed that one intronic SNP (rs2745557) and the GAAA haplotype were significantly associated with ASDs. The level of Cox-2 in the hippocampus is reported to correlate with neuronal activity and may contribute to the process of memory consolidation (Wei and Hemmings, 2004; Yamagata et al., 1993). Further, Cox-2 contributes to alterations in the synaptic connectivity associated with epileptogenesis by up-regulating mRNA and protein expression in the hippocampus during development (Claycomb et al., 2005). Since Cox-2 is an inducible enzyme, we suggest that the genetic variation of PTGS2 affects the biosynthesis of inflammatory prostaglandins, which could subsequently influence the susceptibility to ASDs; we also suggest that the aberrantly functioning genetic variants of Cox-2 may play roles in dysfunctional learning and memory and may be related to the high prevalence of seizures in autism. With regard to disease susceptibility and the functional relationships associated with the SNPs of PTGS2, some reports

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have addressed a few medical illnesses other than ASDs (Zhang et al., 2005). The rs2745557 SNP, which showed preferential transmission in the present study, was the first genetic locus to be investigated in relation to a neuropsychiatric illness. This SNP was reported to be associated with advanced prostate cancer in the European population (Cheng et al., 2007). Its exact functional impact is unknown; however, it is possible that either this polymorphism itself or another linked marker influences Cox-2 activity. The LD analysis in this study revealed that the four SNPs of PTGS2 were in strong disequilibrium with each other and that the GAAA haplotype was significantly associated with ASDs. This implies that the specific haplotype of these four SNPs is transmitted from parents to their offspring with ASDs with stronger linkage disequilibrium than expected. This in turn suggests that these four SNPs interact in the development of ASDs, such that the functional SNP is indirectly associated with the other SNPs due to LD. Defining phenotypic subsets may increase the likelihood of detecting genetic effects in complex disorders, such as autism (Buxbaum et al., 2004). We identified genotype groups and compared the ADOS and ADI-R diagnostic algorithm scores of these groups in order to explore genotype-specific phenotypes. Although neither ADI-R nor ADOS was developed as a rating scale, both have been used in behavioral and genetic studies to rank behavioral severity within autism (Brune et al., 2006). Despite the potential problems associated with multiple testing and the rarity of the allele, in addition to the limited size of the individuals included in the study, the results of the phenotype analysis conducted in this study indicate that the genotype of rs2745557 may be associated with the symptom phenotypes of ASDs including overactivity/agitation and qualitative abnormality in communication in nonverbal subjects. The study must be replicated with several larger populations and more genetic markers, and the relationship between PTGS2 gene and the specific ASD phenotypes needs to be examined more extensively. Acknowledgments This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-E00266). Mira Park was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (R14-2003002-01001-0). In Hee Cho was supported by a Grant for Young Scientists funded by the Gachon University of Medicine and Science. References Ashwood, P., Wills, S., Van de Water, J., 2006. The immune response in autism: a new frontier for autism research. J. Leukoc. Biol. 80, 1–15. Brune, C.W., Kim, S.J., Salt, J., Leventhal, B.L., Lord, C., Cook, E.H., 2006. 5HTTLPR genotype-specific phenotype in children and adolescents with autism. Am. J. Psychiatry 163, 2148–2156.

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