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ORIGINAL ARTICLE
Year : 2012  |  Volume : 60  |  Issue : 6  |  Page : 581-584

RELN gene polymorphisms and susceptibility to autism in Chinese Han population


Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China, China

Date of Submission16-Aug-2012
Date of Decision21-Aug-2012
Date of Acceptance22-Aug-2012
Date of Web Publication29-Dec-2012

Correspondence Address:
Peichao Tian
Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.105190

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 » Abstract 

Background: Single nucleotide polymorphisms (SNPs) in the Reelin gene (RELN) are likely candidates to confer risk for autism. The objective of the present study is to investigate the association of RELN gene SNPs with autism. Materials and Methods: A total of 367 Chinese Han subjects were recruited, including 186 autism patients and 181 unrelated healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods were used to detect RELN gene polymorphisms. The association between SNPs and autism was analyzed in this study. Results: The g.333509A>C in intron12 and g.504742G>A in exon60 were detected in the RELN gene and a significant association was found between the g.504742G>A polymorphism and autism. Allele and genotype frequencies for the g.504742G>A polymorphism in autistic patients were significantly different for healthy subjects. There was no significantly difference in g.333509A>C polymorphism and autism in the studied populations. Conclusions: Our findings indicated that g.333509A>C was not significantly associated with autism. The g.504742G>A polymorphic variant in the RELN gene might affect subjects susceptibility toward autism in Chinese Han population.


Keywords: Association, autism, RELN gene, single nucleotide polymorphisms, susceptibility


How to cite this article:
Tian P. RELN gene polymorphisms and susceptibility to autism in Chinese Han population. Neurol India 2012;60:581-4

How to cite this URL:
Tian P. RELN gene polymorphisms and susceptibility to autism in Chinese Han population. Neurol India [serial online] 2012 [cited 2020 Mar 28];60:581-4. Available from: http://www.neurologyindia.com/text.asp?2012/60/6/581/105190



 » Introduction Top


Autism is a childhood and severe genetically inherited neurodevelopmental disorder characterized by significant impairments of social interactions, difficulties with communication, and displays of stereotypic behaviors, activities, and interests. Candidate gene studies have been investigated as an alternative and effective approach for identifying variants increasing susceptibility to autism. [1],[2],[3],[4] Previous studies have evaluated possible associations between several important candidate genes polymorphisms (such as Reelin (RELN), oxytocin receptor (OXTR), serotonin transporter (5HTT), engrailed 2 (EN2), metabotropic glutamate receptor 8 (GRM8), apolipoprotein E (APOE), wingless-type MMTV integration site family member (WNT2), and autism risk. [1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17] The human RELN gene is located in the region of linkage for autism on chromosome 7 [18] and has been studied as an important candidate gene for autism. [1],[2] It expresses mainly in the brain as well as in blood, spinal cord, and other organs and tissues in the body, encodes an extracellular matrix glycoprotein with serine protease activity, acts as the signaling protein in guiding neuron migration, lamination and connection during the period of embryonic brain development. [5],[6],[19] Evidence from previous studies suggests that common polymorphisms in the RELN gene are likely candidates to confer risk for autism. [1],[2],[3],[4],[6],[10],[12],[20],[21],[22] Several RELN gene SNPs have been approved to be associated with autism. [1],[2],[3],[4],[10],[12],[23],[24] However, results from different studies have shown conflicting results due to ethnic differences in the studied samples. [3],[4],[5],[6],[10],[11],[20],[23],[25],[26] The association of the RELN gene g.333509A>C and g.504742G>A variants with autism has not been investigated. The objective of the present study was to detect the g.333509A>C and g.504742G>A polymorphisms and evaluated the association of the polymorphisms with autism.


 » Materials and Methods Top


Subjects

In this study, altogether 186 autistic patients (mean age: 5.4 years, 154 males and 32 females) and 181 unrelated healthy Chinese Han subjects (mean age: 5.6 years, 150 males and 31 females) were recruited from the First Affiliated Hospital of Zhengzhou University, People's Republic of China. All the patients fulfilled the diagnostic criteria for autism of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, third revision (DSM-IV-TR) (American Psychiatric Association, 2000) criteria. [27] This study was approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University and informed consent was obtained from all subjects for the study.

Genotyping

The genomic DNA was isolated from peripheral venous blood samples by standard extraction protocol. According to the mRNA sequences (GenBank ID: NM_005045.3) and DNA sequences (GenBank ID: NG_011877.1) of human RELN gene, primers were designed using Primer Premier 5.0 software. Details of the primers sequences, region and fragment sizes, and annealing temperature were described in [Table 1]. Polymerase chain reaction (PCR) amplifications were performed in 20 μl reaction mixture containing 50 ng mixed DNA template, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgCl 2 , and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China). The PCR protocol was 95°C for 5 minutes followed by 32 cycles of 94°C for 30 seconds, annealing at the corresponding temperature [Table 1] for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. The PCR products were separated with 1.5% agarose gel including 0.5 μg/ml of ethidium bromide. The RELN gene polymorphisms were investigated by the PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods. Aliquots of 5 μl PCR products were digested using 2U restriction enzyme (MBI, Fermentas) at 37°C for 10 hours following the supplier's manual. The digested products were separated by electrophoresis in 2.5% agarose gel stained with ethidium bromide. PCR amplified products of each genotype were sequenced by ABI 3730 sequencer (Bioasia Biotechnology Co., Ltd., Shanghai, China) to verify the accuracy of genotype determination by enzyme digestion.
Table 1: Primer pairs, PCR, and PCR-RFLP analysis used for the RELN gene

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Statistical analyses

Statistical analysis using the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA) included the chi-squared test for genotype and allele frequencies comparison. A level of P value < 0.05 was considered statistically significant.


 » Results Top


In the present study, the g.333509A>C and g.504742G>A SNPs were identified by PCR-RFLP and DNA sequencing methods, including A→C mutation at position 333509 and G→A mutation (resulting in Glycine (Gly) to Serine (Ser) amino acid replacement, p.Gly3244Ser) at position 504742 of the human RELN gene, respectively in intron12 and exon60 (reference sequence NG_011877.1, NM_005045.3 and NP_005036.2). The PCR product of g.333509A>C was digested with BspH I enzyme and divided into three genotypes, AA (180 bp and 43 bp), AC (223 bp, 180 bp and 43 bp), and CC (223bp) [Table 1]. The allele and genotype frequencies of the g.333509A>C polymorphisms were presented in [Table 2]. Allele frequencies in autistic patients and control group were 71.24% and 69.34% for A allele, and 28.76% and 30.66% for C allele, respectively. Frequencies of the AA, AC, and CC genotypes were 52.69%, 37.10%, and 10.21% in autistic patients, while the frequencies of these genotypes in healthy subjects were determined to be 48.62%, 41.44%, and 9.94%, respectively. Allele A and genotype AA frequencies were predominant in the studied subjects. The results of the chi-squared test between autistic individuals and controls for the g.333509A>C SNP indicated that the polymorphism sites were moderate polymorphism and fitted with Hardy-Weinberg equilibrium (P>0.05) [Table 2]. As for the g.504742G>A SNP, the PCR product of g.504742G>A was digested with the Hpa II enzyme; the three possible genotypes were defined by three distinct banding patterns: GG (173 bp and 67 bp), GA (240 bp, 173 bp and 67 bp), and AA (240 bp) [Table 1]. Overall genotype and allele frequencies for this polymorphism in autistic patients and healthy controls were determined in [Table 3]. Allele frequencies in patients and healthy controls were 53.49% and 62.71% for G allele, and 46.51% and 37.29% for A allele, respectively. Frequencies of the GG, GA, and AA genotypes were 30.11%, 46.77%, and 23.12% in autistic patients, while the frequencies of these genotypes in healthy controls were determined to be 37.02%, 51.38%, and 11.60%, respectively. Allele G and genotype GA frequencies were predominant in the studied subjects. The test for Hardy--Weinberg equilibrium in both groups of subjects did not show a significant deviation from Hardy--Weinberg equilibrium (P > 0.05) [Table 3]. However, the case--control analysis showed significant differences in the distributions of alleles and genotypes between autistic individuals and controls for g.504742G>A (χ2 = 6.3936, P = 0.0115 and χ2 = 0.7467, P = 0.6884) [Table 3].
Table 2: Genotype and allele frequencies of the RELN gene intron12 g.333509A>C polymorphism in the studied populations

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Table 3: Genotype and allele frequencies of the RELN gene exon60 g.504742G>A polymorphism in the studied populations

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 » Discussion Top


The current investigation confirms the findings of the association between the g.333509A>C and g.504742G>A variants of RELN gene and autism in 186 autism patients and 181 unrelated healthy Chinese Han subjects. Analysis of variants in candidate genes allows effective screening of the individuals at risk of autism. [1],[2],[3],[4] It is well known that autism is a disease of polygenic inheritance, and the RELN gene is an important candidate gene for autism. [1],[2] In this study, we observed a significant association between the g.504742G>A variant allele and autism. Sequence analysis suggested that g.504742G>A variant was located in the exon60 of human RELN gene, and corresponding Glycine (Gly) to Serine (Ser) amino acid replacement (p.Gly3244Ser). Allele and genotype frequencies for g.504742G>A polymorphism in autistic patients were significantly different for healthy subjects [Table 3]. The g.504742G>A polymorphic variant in the RELN gene might affect subjects' susceptibility toward autism in the Chinese Han population. Results from the present study indicated that g.333509A>C in intron12 was not significantly associated with autism. Although introns were not coding sequences, the evidence was provided constantly to prove that introns played an important role on regulating mRNA splicing, transcription, and gene expression and regulation. [28],[29] Whether the g.333509A>C locus in RELN gene intron12 affected the gene expression and regulation needs to be further verified. Several studies have been carried out for autism using RELN as a candidate gene in different populations. [1],[2],[3],[4],[5],[6],[10],[11],[12],[20],[23],[24],[25],[26] Furthermore, there were similar studies concerning associations of other SNPs within RELN gene to autism, [1],[2],[3],[4],[5],[6],[10],[11],[12],[20],[23],[24],[25],[26] but not contained the g.333509A>C and g.504742G>A SNPs. Our present results provided the strongest evidence for significant association between the g.504742G>A polymorphism and autism, while not showing significant differences in the g.333509A>C polymorphism. These analyses suggested the potential of RELN as an important contributor to genetic risk in autism.

In conclusion, the current preliminary study suggests that the g.504742G>A SNP of RELN gene is likely to be associated with autism in the Chinese Han population. Our results support the possible involvement of the RELN gene as a risk factor for autism susceptibility. It is worthy to take further studies with larger samples to validate these findings and to investigate the biological mechanism of the RELN gene with autism susceptibility.

 
 » References Top

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    Tables

  [Table 1], [Table 2], [Table 3]

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