Korean Journal of Biological Psychiatry

교신저자：김순애, 301-746 대전광역시 중구 용두동 143-5
책임저자：전화) (042) 259-1672, 전송) (042) 259-1668, E-mail) sakim@eulji.ac.kr

Introduction

   Autism spectrum disorders(ASDs) are neuropsychiatric developmental disorders characterized by impairment in social communication and a preference for repetitive and solitary interests and behaviors. Several family and twin studies have reported the role of genetics in the etiology of autism.¹⁾ In association genetic studies for ASDs, several neurotransmitter genes such as serotonin-related genes are considered as candidate genes because of the possible involvement of neurochemical effects in pathogenesis of ASDs.
   Serotonin(5-hydroxytryptamine or 5-HT) has been considered to play an important role in ASD development. A relatively high platelet serotonin level was observed in a subset of ASD subjects and their first-degree relatives²⁾；the blood serotonin level was also found to correlate with the verbal intelligence quotient(IQ) and the severity of the disorder.³⁾⁴⁾⁵⁾ The use of selective serotonin reuptake inhibitor drugs(SSRIs) was found to improve some symptoms such as repetitive behavior, aggression, and language use in individuals with autistic disorder.⁶⁾⁷⁾⁸⁾ Numerous genes, for example, the serotonin transporter gene, the tryptophan hydroxylase gene, and the monoamine oxidase A gene, are involved in regulating serotonin levels, and some of them have been screened in ASD individuals.^9)10)11)12)
   The serotonin transporter is a major component of the serotonergic system and is thought to play a role in autism pathogenesis. The serotonin transporter gene (SLC6A4) is one of the most widely studied candidate genes for ASD；it is located in chromosome 17q11.2 and has 14 exons. Several linkage studies and genome-wide screening studies have found evidence of a linkage near the serotonin transporter gene.^13)14)15) Some studies have also reported an association between autism and polymorphic markers in the SLC6A4 gene, such as 5-HTTLPR insertion-deletion polymorphisms in the promoter region. The association between the 5-HTTLPR polymorphism of SLC6A4 and ASD has produced controversial results that touch upon ethnic diversity, methods of genetic analysis, and symptom profiles of ASD.^{9)14)16)17)18)19)20)} It is suggested that hyperserotonemia in ASD may be associated with the effects of regulation and expression of SLC6A4 via the 5-HTTLPR promoter polymorphism.²¹⁾²²⁾ The results of the association analysis using single nucleotide polymorphisms(SNPs) of SLC6A4(i.e., rs2020 942, rs2066713, and rs2020936) in several studies were controversial. Kim et al.¹⁷⁾ reported an association between autism and these SNPs in 115 trios examined, but their results were not consistent with those of McCauley et al.¹⁴⁾ or Wu et al.²³⁾ In their extensive study with large cohort samples, Ramoz et al.²⁴⁾ reported the absence of a linkage or association with 9 SNPs and 5-HTTLPR covering SLC6A4 or any haplotypes.
   By using the transmission/disequilibrium test(TDT), we found a significant preferential transmission of the long allele of 5-HTTLPR in Korean ASD.²⁵⁾ To further investigate the role of SLC6A4 in susceptibility of Korean population to ASD, we evaluated the association between ASD and common SNPs of SLC 6A4 by using 151 ASD trios in the Korean population.

Methods

1. Subjects
   The subjects were recruited from a family-based genetic association study of ASD conducted by the same research group.²⁵⁾²⁶⁾ Subject ascertainment and diagnostic methods have been described previously. Briefly, ASD was diagnosed using the Autism Diagnostic Interview-Revised(ADI-R)²⁷⁾ and the Korean version of the Autism Diagnostic Observation Schedule(ADOS)²⁸⁾ with the best estimates of 2 board-certified child psychiatrists. Subjects diagnosed to have or strongly suspected of having neurofibromatosis, tuberous sclerosis, metabolic encephalopathies, Down's syndrome, Fragile X syndrome, or other known chromosomal abnormalities were excluded. We obtained written informed consent from all participants, and the study was approved by the Institutional Review Boards(IRB) of the participating institutions. This study included 151 complete trios, consisting of patients with ASD(79.9±35.6 months, 86.1% males, 87.4% autism, 13.5% Pervasive Developmental Disorder-Not Otherwise Specified(PDD-NOS), and 1.6% Asperger's disorder) and their biological parents. Their psychological characteristics were almost similar, as described previously.²⁶⁾
   For a case-control association study, we used Korean HapMap project data of 7 SNPs(rs3813034, rs 1042173, rs6352, rs140701, rs2020942, rs6354, and rs2020936) in the SLC6A4 gene from 90 Korean adult samples with a 1：1 male to female ratio(http: //sysbio.kribb.re.kr:8080/khapmap/index.jsp).

2. Genotyping
   Blood samples from all subjects were collected in tubes containing EDTA and stored at -70℃. Genomic DNA was extracted using the G-spin Genomic DNA Extraction Kit(Intron, Daejeon, Korea).
   We evaluated the genetic structure of SLC6A4 by using the Entrez SNP Database(http://www.ncbi.nlm. nih.gov/) and determined SNP in the coding region (cSNP) and a common genetic variation of SLC6A4 (i.e., SNPs in the gene region with minor allele frequencies >5% in 2 Asian populations). We selected 5 cSNPs(rs6352, rs28914834, rs28914832, rs2228673, and rs28914828), 4 SNPs in the intronic regions(rs 3794808, rs140701, rs2020942, and rs2020936), 1 SNP in the 5'-untranslated region(rs6354), 1 SNP in the 3'-untranslated region(rs1042173), and 1 SNP in the 3'-near gene region(rs3813034) of SLC6A4. Genotyping was performed using the GoldenGateTM assay(Illumina, San Diego, USA).

　

3. Statistical analysis
   To evaluate the data quality and the presence of genotypic errors, we evaluated the Hardy-Weinberg equilibrium and Mendelian inheritance of the 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/). Statistical significance was defined as p<0.05.
   To evaluate the power of the samples to detect an excessive transmission of alleles, we used the program PBAT.²⁹⁾ We assume that autism prevalence to be K=0.006, with a targeted significance level of 0.05. In terms of the frequency of the disease allele, we used observed frequency of the highly transmitted allele and we employed the additive model.
   Tests to compare the alleles, genotypes and haplotype frequencies of cases with those of Korean Hap Map controls were conducted using SNPAlyze 5.0.4 (Dynacom, Chiba, Japan).

Results

   The genotypic distribution for all the SNPs did not deviate from that expected based on the Hardy-Weinberg equilibrium. Four cSNPs(rs28914834, rs2891 4832, rs2228673, and rs28914828) were monomorphic. Information on these SNPs is given in Table 1. In the TDT analysis, we did not find any statistically significant overtransmitted SNP alleles in the ASDs (Table 2). Estimation of the pairwise linkage disequilibrium(LD) to determine the extent of LD for the SNPs showed that all the SNPs were in strong disequilibrium(D'>0.9) with each other(Table 3). Because of strong LDs among the SNP markers, in the haplotype analysis, we found only 3 haplotypes consisting of 7 SNPs in SLC6A4, and we did not observe any significant association between the haplotypes and ASD(Table 2).
   We used an odds ratio(OR) of 1.7 for carrying one disease allele. Considering the prevalence of ASD in 0.6% of general population, the power of TDT for rs 1042173, rs6352, rs3794808, rs6354, and rs2020936 was 0.373, 0.131, 0.378, 0.218, and 0.218, respectively. A type I error rate of 0.05 was used in all calculations.
   In the case-control association analysis, we did not observe any statistically significant differences in allele, genotype, and haplotype frequencies between our ASD cases and the Korean HapMap controls(Table 4).

Discussion

   Several previous reports have indicated the possibility of a linkage and/or association between SLC6A4 and ASD. Kim et al.¹⁷⁾ revealed an association between autism and SNPs of SLC6A4 in 115 trios. However, studies involving TDTs have failed to reveal any consistent evidence of this association. In the present study, we did not observe any statistically significant transmission from biological parents to Korean ASD children with more SNPs(i.e., 12 SNPs in the SLC 6A4 gene, of which 8 SNPs were analyzed with statistical tools) in the TDT and haplotype analysis. Moreover, the power values of our study were very small, and our study has potential limitations with regard to detection of causative variants or variants with more modest effects. Furthermore, the control subjects were not age and sex matched with the study subjects, and psychiatric disorders in the control subjects were not evaluated. Nonetheless, the results of the case-control analysis did not reveal any statistically significant results.
   The reason for these differences in association results might be as follows. First, ethnic differences might contribute to different genetic associations. This study included only Asian Korean population, similar to the study of Wu et al.(175 Han Chinese trios)²³⁾；thus, the sample population differed from that used in the study by Kim et al.(94 Caucasians, 7 Americans, 8 Asian Americans, and 6 Hispanics).¹⁷⁾ Second, the sample size of the study by Kim et al. was relatively small compared to that of our study. Therefore, the results of the study by Kim et al need to be considered. Finally, the genetic heterogeneity of ASDs and the possibility of involvement of other genes or environmental factors such as gene-gene interaction or gene-environmental interaction must be considered.
   Although our study had certain limitations such as relatively small sample size and a lack of information on phenotypes and environmental risk factors, our findings suggest that common SNPs(minor allele frequency of more than 0.01) and haplotypes for SLC6A4 do not appear to significantly contribute to ASD in the Korean population. Nonetheless, we believe that the association study of the 5-HTTLPR(not SNPs) marker for SLC6A4 must be replicated in a larger Korean population, together with a phenotypic analysis, because of its functional effects and a previous report on positive association.²⁵⁾