The Association Analysis Of RELN And GRM8 Genes With Autism And The Autistic Social Impairment Study

Background and objective:Autism is a severe neuropsychiatric disorder characterized by three core symptoms:deficits in communication,social interactions and repetitive and stereotyped behaviors and interests.Autism is generally diagnosed before 3 years old and the prevalence is not less than 1.3/1000 with a male to female sex ratio of around 4.3:1. Twin and family studies from last fourty years reveal that genetic factors are very likely to play a major role in the aetiology of autism,although the susceptibility genes are still unknown to date.Many independent genome-wide scans for autism susceptibility loci have been carried out.The region on chromosome 7q stands out as the region of suggestive linkage to aetiology of autism with the greatest concordance in many independent genome-wide scans.RELN and GRM8,the two genes selected in this study,are located within this region.In this study,we selected 12 SNPs located in these two genes as the gene markers.By using the case-control association analysis,we will investigate the relationship between autism and the single SNP and the haplotypes which consist of these SNPs.As a result,we will try to find out if these two genes are the susceptibility genes of the autism.The association study between autism and these two genes may make it more clear for the genetic bases of the aetiology of autism.In addition,it is the very first and important step for the molecular mechanism research of the autism in the future.The phenotypic heterogeneity of autism was thought to play a important role for making the results of the human genetic analysis appear to be conflicting.In order to resolve this problem,many researchers start to use endophenotype,measures of a disease component or associated traits that can take on meaningful values apart from disease status,to define more homogenous groups of subjects.Throughout the three domains of autism,the deficits in social interaction is the most common and essential manifestation.Though there is no mental retardation in most high functioning autistic disorders or Asperger syndrome,the social reciprocal behavior(RSB)is also obviously destroyed.The impairment of this domain was thought to be distributed continuously across the whole autistic spectrum and to share common genetic determinants and neuromechanism.Social Responsiveness Scale(SRS)is recognized as the only quantitative measure that could assess the degree of impairment of RSB in autistic spectrum disorders up to now.In this study,we use SRS to assess the deficits of RSB in autism patients,intellectual matched Down syndrome patients and the general population control.Our purpose is to replicate the result that in Chinese Han population of autistic disorders,the single index score of SRS could also reflect the general impairment in RSB and there is no culture difference for this inherent autistic social impairment.As a result,the autistic social impairment could also be used as endophenotype for genetic analysis in the future in Chinese Han population. PartⅠThe association analysis of RELN gene with autistic spectrum disorder in Chinese Han populationSubjects and methods:1.Blood sample collection and DNA extraction: Blood samples were obtained from unrelated 213 cases(mean age:5.3 years,181 males and 32 females)recruited from the out-patients of our hospital and from several special autism-training schools.All patients were diagnosed by the diagnostic and statistical manual of mental disorders(DSM)-Ⅳcriteria or International Classification of Diseases-10(ICD-10).160 unrelated controls(mean age:6.7 years,135 males and 25 females)with no psychiatric disorders were recruited from the outpatients of our hospital.DNA samples were extracted from leukocytes by standard methods.2.SNP selection and genotyping:We selected eight SNPs(SNP1～SNP8)in the RELN through the SNP database(http://www.ncbi.nlm.nih.gov/snp/).Genotying was done by SNPstream technology(Beckman Coulter SNPstream)which is the combination of the fidelity polymerase mediate reaction,single base primer extension and microarray methods.3.Statistical analysis:The deviation from Hardy-Weinberg equilibrium (HWE)was examined in controls by theχ~2 test;Based on the logistic regression method,the case-control association of genotypes in five inheritance models (codominant,dominant,recessive,overdominant,log-additive)was tested for all the 12 single SNPs;Pairwise Linkage Disequilibrium(LD)was calculated for the cases and controls in Chinese Han population;Constructed the haplotype blocks based on the results of LD analyses;The association analysis of the haplotypes with autism was similar to that of genotypes of single SNP by logistic regression.The significance of all these tests was 0.05. Results:1.A significant genetic association between SNP2(rs736707,located in intron 59 of RELN)and autism was observed,and the log-additive model was accepted as the best inheritance model fitting this data(OR:0.72,95%CI:0.54-0.97, P=0.03).2.We found strong LD(D'＞0.75)between some pairs of the markers in RELN gene.Therefore,we constructed two different haplotype blocks consisting of some nearby SNPs which are in strong LD,these two haplotypes are SNP1/SNP2/SNP3/SNP4 and SNP6/SNP7.3.Haplotype-specific association analysis revealed that the combination of SNP1/SNP2/SNP3/SNP4 which are in strong linkage disequilibrium(D'＞0.75)showed significant association with autism(P=0.027).Conclusion:1.This is the first time to study the association between RELN gene with autism in Chinese Han population,a significant genetic association between SNP2(rs736707,located in intron 59 of RELN)and autism was observed,and the log-additive model was accepted as the best inheritance model.2.We constructed two different haplotype blocks consisting of some nearby SNPs which are in strong LD (D'＞0.75),and haplotype-specific association analysis revealed that the combination of SNP1/SNP2/SNP3/SNP4 showed significant association with autism,the log-additive model was accepted as the best inheritance model.3.Our study suggest the possible involvement of RELN gene in the susceptibility to autism.Future replications are warranted before definitive conclusion can be drawn.PartⅡThe association analysis of GRM8 gene with autistic spectrum disorder in Chinese Han populationSubjects and methods:(1)Blood sample collection and DNA extraction: Blood samples were obtained from unrelated 213 cases(mean age:5.3 years,181 males and 32 females)recruited from the out-patients of our hospital and from several special autism-training schools.All patients were diagnosed by the diagnostic and statistical manual of mental disorders(DSM)-Ⅳcriteria or International Classification of Diseases-10(ICD-10).160 unrelated controls(mean age:6.7 years,135 males and 25 females)who were diagnosed of no psychiatric disorders before were recruited from the outpatients of our hospital.DNA samples were extracted from leukocytes by standard methods.(2)SNP selection and genotyping:We selected four SNPs (SNP9～SNP12)in the GRM8 genes through the SNP database(http:// www.ncbi.nlm.nih.gov/snp/).Genotying was done by SNPstream technology (Beckman Coulter SNPstream)which is the combination of the fidelity polymerase mediate reaction,single base primer extension and microarray methods.(3)Statistical analysis:The deviation from Hardy-Weinberg equilibrium(HWE)was examined in controls by theχ~2 test;Based on the logistic regression method,the case-control association of genotypes in five inheritance models(codominant,dominant,recessive, overdominant,log-additive)was tested for all the four single SNPs;Pairwise Linkage Disequilibrium(LD)was calculated for the cases and controls in Chinese Han population;Constructed the haplotype blocks based on the results of LD analyses;The association analysis of the haplotypes with autism was similar to that of genotypes of single SNP by logistic regression.The significance of all these tests was 0.05.Results:(1).None of the single SNPs showed significant association between autism and the markers of GRM8 gene(P＞0.05).(2)We constructed one haplotype block consisting of some nearby SNPs(SNP9/SNP10/SNP11)which are in strong LD(D'＞0.75).(3)Haplotype-specific association analysis showed no significant association between autism and the haplotype of GRM8 gene(P＞0.05).Conclusion:1.This is the first time to study the association between GRM8 gene with autism in Chinese Han population,none of the single SNPs showed significant association between autism and the markers of GRM8 gene.2. Haplotype-specific association analysis showed no significant association between autism and the haplotype of GRM8 gene.3.Our study suggest that GRM8 might not be the susceptibility gene of autism in Chinese Han population,future replications are warranted before definitive conclusion can be drawn.PartⅢAssessment of autistic social impairment in Chinese Han populationSubjects and methods:(1)RSB assessment using SRS:After assessing ability of the RSB with SRS in different groups including autism patients,intellectual matched Down syndrome patients and the general population control,a single scale score was generated.Higher SRS scores indicate higher degrees of social impairment. (2)One-way ANOVA Analysis:With SPSS 11.5 version,a comparison of mean SRS scores between various groups of subjects was conducted using one-way analysis of variance followed by Post-Hoc t tests.The significance of all these tests was 0.05.Results:Assessment of SRS and statistical analysis:Comparison of the SRS scores showed significance among these three groups(SRS raw score:F=149.57,P＜0.01;SRS standard score:F=106.11,P＜0.01).Post-Hoc test showed that the mean SRS raw scores for children with autism(96.77±15.27)were significantly higher than those for children with Down syndrom(60.17±18.85,P＜0.01),and the general population of control(29.68±10.63 P＜0.01).In addition,the mean SRS raw scores for children with Down syndrom were significantly higher than those for the general population of control(P＜0.01).Post-Hoc test showed that the mean SRS standard scores for children with autism(80.92±8.15)were significantly higher than those for children with Down syndrom(65.67±11.52,P＜0.01),and the general population of control(49.32±5.70 P＜0.01).In addition,the mean SRS standard scores for children with Down syndrom were significantly higher than those for the general population of control(P＜0.01).Conclusion:1.This is the first time to assess the social impairment in Chiese Han population of autistic disorders by using the social responsiveness scale,our study suggest that in Chinese Han population of autistic disorders,the single index score of SRS can also reflect the general impairment in RSB.2.There is no culture difference for the inherent autistic social impairment reflected by SRS.