Association between the g.296596G > A genetic variant of RELN gene and susceptibility to autism in a Chinese Han population

Association between the g.296596G > A genetic variant of RELN gene and susceptibility to autism in a Chinese Han population

Xiaoyan Fu; Zhu Mei; Lixin Sun

Department of Pediatrics, Tongji Hospital of Tongji University, Shanghai, People's Republic of China

^{Send correspondence to}

ABSTRACT

Autism is a childhood neuro-developmental disorder, and Reelin (RELN) is an important candidate gene for influencing autism. This study aimed at investigating the influence of genetic variants of the RELN gene on autism susceptibility. In this study, 205 autism patients and 210 healthy controls were recruited and the genetic variants of the RELN gene were genotyped by the created restriction site-polymerase chain reaction (CRS-PCR) method. The influence of genetic variants on autism susceptibility was analyzed by association analysis, and the g.296596G > A genetic variant in exon10 of the RELN gene was detected. The frequencies of allele/genotype in autistic patients were significantly different from those in healthy controls, and a statistically significant association was detected between this genetic variant and autism susceptibility. Our data lead to the inference that the g.296596G > A genetic variant in the RELN gene has a potential influence on autism susceptibility in the Chinese Han population.

Keywords: autism, susceptibility, association analysis, RELN gene, genetic variants.

Introduction

Autism is a severe childhood neuro-developmental disorder characterized by significant impairments, such as social deficits, delay and deviance in language development and communication skills, and displays of stereotypic behaviors, activities and interests (Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Holt et al., 2010; Tian, 2012). Evidence from previous studies suggests that certain genetic variants can contribute to autism susceptibility (Zhang et al., 2002; Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Ashley-Koch et al., 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; Tian, 2012). Several candidate genes, such as Reelin (RELN), Serotonin transporter (5HTT), Oxytocin receptor (OXTR), Metabotropic glutamate receptor 8 (GRM8), Engrailed 2 (EN2), Wingless-type MMTV integration site family member (WNT2), and Apolipoprotein E (APOE), have been investigated with respect to possible associations between genetic variants and autism susceptibility (Petit et al., 1995; Cook et al., 1997; Yirmiya et al., 2001; Bonora et al., 2003; Zhong et al., 2003; Gharani et al., 2004; Li et al., 2004; Bartlett et al., 2005; Skaar et al., 2005; Wu et al., 2005; Ashley-Koch et al., 2007; Jacob et al., 2007; Dutta et al., 2008; Lerer et al., 2008; Li et al., 2008; Holt et al., 2010; He et al., 2011). It has been reported that the RELN gene is one of the most important candidate genes for autism susceptibility (Turner et al., 2000; Zhang et al., 2002; Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Ashley-Koch et al., 2007; Freitag, 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; Tian, 2012). The RELN gene is located in the region of linkage for autism on chromosome 7 (Monaco et al., 2001a,b). Recently, several studies observed the potential associations of RELN genetic variants with autism risks (Turner et al., 2000; 2001; Persico et al., 2001; Zhang et al., 2002; Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Serajee et al., 2006; Ashley-Koch et al., 2007; Freitag, 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; Tian, 2012). Some of these reports confirmed that certain RELN genetic variants are statistically associated with autism risk (Persico et al., 2001; Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Serajee et al., 2006; Ashley-Koch et al., 2007; Li et al., 2008; Holt et al., 2010; Tian, 2012). However, the results from these observations were conflicting rather than conclusive (Krebs et al., 2002; Zhang et al., 2002; Bonora et al., 2003; Devlin et al., 2004; Li et al., 2004; Skaar et al., 2005; Serajee et al., 2006; Dutta et al., 2008; Li et al., 2008; He et al., 2011). Up to date, there are no similar studies on the association of g.296596G > A genetic variant in RELN gene with autism susceptibility. Therefore, the objective of this study was to detect this genetic variant and further evaluate its influence on autism susceptibility.

Subject and Methods

Subjects

In this case-control study done from between January 2009 and December 2012 at the Tongji Hospital, a total of 415 subjects were enrolled, including 205 autism patients (mean age: 4.6 years, 148 males and 57 females) and 210 healthy subjects (mean age: 4.7 years, 151 males and 59 females). All subjects were genetically unrelated Chinese of Han ethnicity. The autism patients were diagnosed by the criteria defined by the American Psychiatric Association (2000) manual DSM-IV-TR. The controls were frequency-matched to autism patients in terms of age and gender. The protocol of this study was approved by Ethics Committee of the Tongji Hospital and informed consent was obtained from all subjects.

Genotyping

Venous blood samples were collected from each subject, and genomic DNA was isolated by using the DNA Blood Mini kit (QIAGEN, Valencia, CA), following the manufacturer's instructions. PCR primers were designed by Primer Premier 5.0 software and their sequences, annealing temperature, amplification region and sizes were shown in Table 1. The PCR amplifications were carried out in 20 µL reaction mixtures, containing 50 ng of mixed DNA template, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgCl₂ and 0.5 U Taq DNA polymerase (Promega, Madison, WI, USA). The PCR protocol was: 95 ºC for 5 min, followed by 32 cycles of 94 ºC for 30 s, 59.5 ºC for 30 s, and 72 ºC for 30 s, and a final extension at 72 ºC for 8 min. The genetic variants of RELN gene were genotyped by the created restriction site-polymerase chain reaction (CRS-PCR) method, where one of the primers contained a nucleotide mismatch, which enabled the use of restriction enzymes for discriminating sequence variations (Haliassos et al., 1989; Zhao et al., 2003; Yuan et al., 2012; Yuan et al., 2013a; Yuan et al., 2013b). Aliquots of 5 µL PCR amplified products were digested with 2U MaeII restriction enzyme (MBI, Fermentas) at 37 ºC for 10 h, following the manufacturer's instructions. The digested products were separated in 2.0% agarose gels stained with ethidium bromide and observed under UV light. To verify the accuracy of genotype determination by the CRS-PCR method, approximately 15% of the PCR amplified products were randomly picked and sequenced on an ABI 3730 sequencer by Bioasia Biotechnology Co., Ltd (Shanghai, China).

Statistical analyses

The chi-squared (χ²) test was used to evaluate the Hardy Weinberg equilibrium (HWE) for genotype and allele frequencies. A p value < 0.05 was considered statistically significant. All statistical analyses were down using the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA).

Results

In this case-control study, we detected the g.296596G > A genetic variant of RELN gene using the CRS-PCR method. The sequence analyses indicated that this genetic variant resulted from a non-synonymous mutation caused by a G to A mutation in exon 10 of the RELN gene, this leading to the replacement of a valine (Val) by an isoleucine (Ile) (p.Val359Ile). The PCR products was digested with MaeII restriction enzyme and divided into three genotypes: GG (191 and 23 bp), GA (214, 191 and 23 bp) and AA (214 bp, Table 1). Table 2 shows the respective allele and genotype frequencies in autism patients and healthy controls. The allele-G and genotype-GA were predominant in the studied subjects. The allele frequencies in autism patients (G, 53.66%; A, 46.34%) were significantly different from those of healthy controls (G, 62.38%; A, 37.62%; χ² = 6.4827, p = 0.0109). The genotype frequencies were also statistically significant different between autism patients and healthy controls (χ² = 7.2176, p = 0.0271, Table 2). The distributions of genotype in autism patients and healthy controls were fitted with HWE (all p-values > 0.05, Table 2).

Discussion

Recent studies indicate that autism is a disease of polygenic inheritance, wherein genetic variants play key functions in the development of autism. Candidate gene studies have proven a useful approach for identifying genetic variants associated with increasing autism susceptibility (Holt et al., 2010; Tian, 2012). The human RELN gene is an important candidate gene for influencing autism (Turner et al., 2000; Zhang et al., 2002; Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Ashley-Koch et al., 2007; Freitag, 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; Tian, 2012), and analysis of genetic variants in the RELN gene allows to effectively screen for autism risk (Bonora et al., 2003; Bartlett et al., 2005; Skaar et al., 2005; Holt et al., 2010; Tian, 2012). Several previous studies have been carried out using RELN as a candidate gene for autism susceptibility in different populations (Persico et al., 2001; Krebs et al., 2002; Zhang et al., 2002; Bonora et al., 2003; Devlin et al., 2004; Li et al., 2004; Bartlett et al., 2005; Skaar et al., 2005; Serajee et al., 2006; Ashley-Koch et al., 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; He et al., 2011). In the present study, we found the g.296596G > A genetic variant in the RELN gene and evaluated the relationship of this genetic variant with respect to autism susceptibility in a Chinese Han population by an association analysis. The allele and genotype frequencies for this genetic variant in autism patients were significantly different from those in healthy subjects (Table 2). Our data indicate that the g.296596G > A genetic variant of the RELN gene has a statistically significant association with autism susceptibility and may affect the subjects susceptibility toward autism in the Chinese Han population. Several similar studies concerned the influence of other genetic variants in the RELN gene on autism susceptibility (Persico et al., 2001; Krebs et al., 2002; Zhang et al., 2002; Bonora et al., 2003; Devlin et al., 2004; Li et al., 2004; Bartlett et al., 2005; Skaar et al., 2005; Serajee et al., 2006; Ashley-Koch et al., 2007; Dutta et al., 2008; Li et al., 2008; Holt et al., 2010; He et al., 2011; Tian, 2012), which are in accordance with our results, including a significant association between the RELN genetic variant (rs2073559) and autism susceptibility (Ashley-Koch et al., 2007). Evidence for the association of the RELN genetic variant (rs362780, p = 0.00165) with autism susceptibility was found by Holt et al. (Holt et al., 2010). Tian (2012) reported that the g.504742G > A polymorphic variant in the RELN gene might affect subjects susceptibility toward autism in the Chinese Han population, but that the g.333509A > C variant was not significantly associated with autism. These observations all corroborate that RELN genetic variants are important contributors to the genetic risk in autism susceptibility.

In conclusion, the present study revealed that the g.296596G > A genetic variant in RELN gene is statistically associated with autism susceptibility in a Chinese Han population. Gene function studies and association studies on larger population are still necessary to confirm these findings and to investigate the biological mechanism underlying RELN-mediated autism susceptibility.

Received: May 9, 2013

Accepted: June 20, 2013.

Associate Editor: Mara H. Hutz

License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

• Ashley-Koch AE, Jaworski J, Ma de Q, Mei H, Ritchie MD, Skaar DA, Robert Delong G, Worley G, Abramson RK, Wright HH, et al. (2007) Investigation of potential gene-gene interactions between APOE and RELN contributing to autism risk. Psychiatr Genet 17:221-226.
• Bartlett CW, Gharani N, Millonig JH and Brzustowicz LM (2005) Three autism candidate genes: A synthesis of human genetic analysis with other disciplines. Int J Dev Neurosci 23:221-234.
• Bonora E, Beyer KS, Lamb JA, Parr JR, Klauck SM, Benner A, Paolucci M, Abbott A, Ragoussis I, Poustka A, et al. (2003) Analysis of reelin as a candidate gene for autism. Mol Psychiatry 8:885-892.
• Cook Jr EH, Courchesne R, Lord C, Cox NJ, Yan S, Lincoln A, Haas R, Courchesne E and Leventhal BL (1997) Evidence of linkage between the serotonin transporter and autistic disorder. Mol Psychiatry 2:247-250.
• Devlin B, Bennett P, Dawson G, Figlewicz DA, Grigorenko EL, McMahon W, Minshew N, Pauls D, Smith M, Spence MA, et al. (2004) Alleles of a reelin CGG repeat do not convey liability to autism in a sample from the CPEA network. Am J Med Genet B Neuropsychiatr Genet 126B:46-50.
• Dutta S, Sinha S, Ghosh S, Chatterjee A, Ahmed S and Usha R (2008) Genetic analysis of reelin gene (RELN) SNPs: No association with autism spectrum disorder in the Indian population. Neurosci Lett 441:56-60.
• Freitag CM (2007) The genetics of autistic disorders and its clinical relevance: A review of the literature. Mol Psychiatry 12:2-22.
• Gharani N, Benayed R, Mancuso V, Brzustowicz LM and Millonig JH (2004) Association of the homeobox transcription factor, ENGRAILED 2, 3, with autism spectrum disorder. Mol Psychiatry 9:474-484.
• Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J, Kaplan JC and Kitzis A (1989) Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 17:3606.
• He Y, Xun G, Xia K, Hu Z, Lv L, Deng Z and Zhao J (2011) No significant association between RELN polymorphism and autism in case-control and family-based association study in Chinese Han population. Psychiatry Res 187:462-464.
• Holt R, Barnby G, Maestrini E, Bacchelli E, Brocklebank D, Sousa I, Mulder EJ, Kantojarvi K, Jarvela I, Klauck SM, et al. (2010) Linkage and candidate gene studies of autism spectrum disorders in European populations. Eur J Hum Genet 18:1013-1019.
• Jacob S, Brune CW, Carter CS, Leventhal BL, Lord C and Cook Jr EH (2007) Association of the oxytocin receptor gene (OXTR) in Caucasian children and adolescents with autism. Neurosci Lett 417:6-9.
• Krebs MO, Betancur C, Leroy S, Bourdel MC, Gillberg C and Leboyer M (2002) Absence of association between a polymorphic GGC repeat in the 5' untranslated region of the reelin gene and autism. Mol Psychiatry 7:801-804.
• Lerer E, Levi S, Salomon S, Darvasi A, Yirmiya N and Ebstein RP (2008) Association between the oxytocin receptor (OXTR) gene and autism: Relationship to Vineland Adaptive Behavior Scales and cognition. Mol Psychiatry 13:980-988.
• Li H, Li Y, Shao J, Li R, Qin Y, Xie C and Zhao Z (2008) The association analysis of RELN and GRM8 genes with autistic spectrum disorder in Chinese Han population. Am J Med Genet B Neuropsychiatr Genet 147B:194-200.
• Li J, Nguyen L, Gleason C, Lotspeich L, Spiker D, Risch N and Myers RM (2004) Lack of evidence for an association between WNT2 and RELN polymorphisms and autism. Am J Med Genet B Neuropsychiatr Genet 126B:51-57.
• Monaco AP and Bailey AJ (2001a) Autism. The search for susceptibility genes. Lancet 358:S3.
• Monaco PA and Bailey AJ (2001b) A genomewide screen for autism: Strong evidence for linkage to chromosomes 2q, 7q, and 16p. Am J Hum Genet 69:570-581.
• Persico AM, D'Agruma L, Maiorano N, Totaro A, Militerni R, Bravaccio C, Wassink TH, Schneider C, Melmed R, Trillo S, et al. (2001) Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder. Mol Psychiatry 6:150-159.
• Petit E, Herault J, Martineau J, Perrot A, Barthelemy C, Hameury L, Sauvage D, Lelord G and Muh JP (1995) Association study with two markers of a human homeogene in infantile autism. J Med Genet 32:269-274.
• Serajee FJ, Zhong H and Mahbubul Huq AH (2006) Association of Reelin gene polymorphisms with autism. Genomics 87:75-83.
• Skaar DA, Shao Y, Haines JL, Stenger JE, Jaworski J, Martin ER, DeLong GR, Moore JH, McCauley JL, Sutcliffe JS, et al. (2005) Analysis of the RELN gene as a genetic risk factor for autism. Mol Psychiatry 10:563-571.
• Tian P (2012) RELN gene polymorphisms and susceptibility to autism in Chinese Han population. Neurol India 60:581-584.
• Turner M, Barnby G and Bailey A (2000) Genetic clues to the biological basis of autism. Mol Med Today 6:238-244.
• Wu S, Jia M, Ruan Y, Liu J, Guo Y, Shuang M, Gong X, Zhang Y, Yang X and Zhang D (2005) Positive association of the oxytocin receptor gene (OXTR) with autism in the Chinese Han population. Biol Psychiatry 58:74-77.
• Yirmiya N, Pilowsky T, Nemanov L, Arbelle S, Feinsilver T, Fried I and Ebstein RP (2001) Evidence for an association with the serotonin transporter promoter region polymorphism and autism. Am J Med Genet 105:381-386.
• Yuan ZR, Li J, Li JY, Gao X and Xu SZ (2013) SNPs identification and its correlation analysis with milk somatic cell score in bovine MBL1 gene. Mol Biol Rep 40:7-12.
• Yuan ZR, Li JY, Li J, Gao X and Xu SZ (2013) Effects of DGAT1 gene on meat and carcass fatness quality in Chinese commercial cattle. Mol Biol Rep 40:1947-1954.
• Yuan ZR, Li JY, Li J, Zhang LP, Gao X, Gao HJ and Xu SZ (2012) Investigation on BRCA1 SNPs and its effects on mastitis in Chinese commercial cattle. Gene 505:190-194.
• Zhang H, Liu X, Zhang C, Mundo E, Macciardi F, Grayson DR, Guidotti AR and Holden JJ (2002) Reelin gene alleles and susceptibility to autism spectrum disorders. Mol Psychiatry 7:1012-1017.
• Zhao CJ, Li N and Deng XM (2003) The establishment of method for identifying SNP genotype by CRS-PCR. Yi Chuan 25:327-329.
• Zhong H, Serajee FJ, Nabi R and Huq AH (2003) No association between the EN2 gene and autistic disorder. J Med Genet 40:e4.

Publication Dates

• Publication in this collection
  03 Sept 2013
• Date of issue
  2013

History

• Received
  09 May 2013
• Accepted
  20 June 2013