Association Of MeCP2 Gene And BDNF Gene With Autism

IntruductionAutism is a pervasive developmental disorder that is characterized by severe impairments in reciprocal social interactions, communication and patterns of stereotyped interests and activities. It is now well established that genetic factors play an important role in the pathogenesis of autism. The population prevalence of autism is 10-60 in 10,000, with a male to female ratio of 4 to 6:1. The predominance of male patients might be explained by the involvement of an X-linked gene on the X chromosome.Rett syndrome (RTT) is a severe neurodevelopmental disorder and a leading cause of mental retardation with autistic features in females. RTT is also classified as a pervasive developmental disorder both in the DSM-IV and ICD-10. The RTT gene was identified as X-linked gene, MeCP2 gene on Xq28 in 1999, which encodes the methyl-CpG-binding protein 2 (MeCP2). MeCP2 selectively binds to methylated DNA and functions as a powerful transcriptional repressor. MeCP2 mutations have also been identified in individuals with a variety of clinical syndromes, including neonatal encephalopathy in males and X-linked mental retardation in both males and females. Furthermore, MeCP2 duplications have been shown to cause a progressive postnatal neurological disorder. Autism and RTT share many characteristics in common including developmental regression of language, cognitive and social skills after normal development for the first 6 to 18 months. These evidences suggest that common pathways may underlie regression in these two disorders. Therefore, MeCP2 gene is an important candidate gene for autism.Moreover, MeCP2 is a putative transcriptional repressor and is believed to be involved in the silencing of downstream genes. It is therefore supposed that dysfunctions of these downstream genes may produce similar phenotypes to those from MeCP2 mutation. This would suggest that autism might be a disorder associated with MeCP2 target genes. Discovering which genes are misregulated in the absence of functional MeCP2 and demonstrating their role in causing neuronal dysfunction and disease manifestations are challenging but important steps for understanding the pathogenesis of Rett syndrome and related disorders. The identification of the MeCP2 target genes will provides new opportunities to elucidate the etiology of autism and neuropathological processes underlying regression both in RTT and autism.By using chromatin immunoprecipitation (ChIP) as well as gene expression analyses on Mecp2^+/y and Mecp2^-/y perinatal mouse cortical cultures that are less heterogeneous, the first mammalian neuronal target gene for MeCP2 was identified as brain-derived neurotrophic factor (Bdnf) in 2003.BDNF gene is located in 11q13, encoding a neurotrophic factor involved in neuronal survival and differentiation during the development of the central nervous system, and also in the synaptic efficiency and neuronal plasticity. BDNF has also been implicated in adult neural plasticity, including learning and memory. Eelevated BDNF level has been observed in the brain, blood and serum of autistic individuals, compared to healthy controls, suggesting that BDNF expression in autism may be abnormal.Herein, we first searched for MeCP2 mutations in a well-characterized sample of autism in attempt to understand the relationship between MeCP2 gene and autism and its possible role in the regression of autism. Furthermore, We analysed the distribution of allele frequencies of BDNF gene in Liaoning Han population, and performed a transmission disequilibrium test (TDT) analysis of SNPs in 112 parent-offspring trios to investigate the association of BDNF gene with autism. Materials and Methods1,SubjectsA total of 112 Chinese trios (singleton autistic disorder patients and their parents), including 31 unrelated boys with developmental regression, aged from 2 to 12 years, were recruited from the Clinic of Developmental Pediatrics of the 2nd affiliated hospital of China Medical University between 2001 and 2005. Diagnoses of autism were established by a team of experienced developmental pediatricians. All the patients fulfilled the DSM-â…£criteria for autism. Individuals with potentially confounding factors, such as prenatal or perinatal events were excluded。2,Molecular analysis(1) Mutation analysisBlood samples were collected, and genomic DNA was isolated from peripheral leukocytes using the phenol-chloroform method. PCR primer pairs were based on those described by Amir (1999) with some modification. PCR products were purified and directly sequenced. Sequencing results were compared with the reference human MeCP2 sequence (GenBank accession number: AF030876; X99686.).(2) Genotyping of SNPsThree SNPs, rs6265, rs11030104, and rs10835210 in the BDNF gene were selected from the dbSNP for the PCR-RFLP analysis. Of the three SNPs, rs6265 is a non-synonymous coding SNP. Rs11030104 and rs10835210 are intronic SNPs. PCR was performed and the products were ovemightly digested with restriction enzymes Eco721, Afal and Fbal respectively. The samples were loaded on 1.5% agarose gels containing gene finder for electrophoresis. Genotypes were determined from Chem imager5500 (Alpha Innotech Corporation, USA).(3) Statistical AnalysisThe Hardy-Weinberg equilibrium for genotype frequencies was evaluated by the Chi-square test. Family-based association analysis was performed using the transmission/disequilibrium test (TDT). The significance lever for all statistical tests was 0.05. Data analysis were calculated with soft SPSS 13.0 package. Linkage disequilibrium were analysed with online program SHEsis online program.ResultsWe searched for sequence variants in the MeCP2 gene in a group of autistic boys with developmental regression. One sequence variant, 1461*139 A＞G substitution in 3'UTR, was observed. No coding sequence variant was found in any of the patients tested.The allele and genotype frequencies of all three SNPs of BDNF gene were analysed. There was no significant deviation from Hardy-Weinberg equilibrium at any of the three SNPs, suggesting that the population was under Hardy-Weinberg Equilibrium. The significant differences of genotype and allele frequency distributions were detected between the Chinese North population and Caucasian population, and between the Chinese North population and Japanese populationThe TDT analysis for 112 Chinese trios (singleton autistic disorder patients and their parents) showed that the polymorphism rs11030104 had preferential transmission to the affected offspring,χ²=6.722, P=0.010. rs10835210 had preferential transmission to the affected offspring with developmental regression. No correlation between the alleles of rs11030103, rs10835210 and penotypes of autism were obsered.Conclusion1. Mutations in the coding sequence of MeCP2 gene is unlikely to be a major susceptibility factor in autism.2. The variations in 3' UTR of MeCP2 gene may have a subtle effect, It is worthwhile extending the mutation screening, with a larger patient sample of strictly defined phenotype, to the 3'UTR of MeCP2 gene.3. There are polymophisms of rs6265, rs11030104 and rs10835210 among Liaoning population of China.4. Genotype and allele frequency distributions of BDNF gene in Liaoning Han population were significantly different from those in Japanese population and Caucasian population.5. rs11030104 of BDNF had preferential transmission to the affected offspring, suggesting that BDNF gene may be a susceptible gene for autism.6. rs10835210 of BDNF had preferential transmission to the affected offspring with developmental regression, suggesting that BDNF gene may be involved in the regression of autism.