| Autism spectrum disorder(ASD)represents a group of childhood-onset lifelong neurodevelopmental disorders.Its core characteristics are social communication disorders and stereotyped repeated behavior,with significant clinical heterogeneity and etiological heterogeneity.The prevalence of ASD has increased dramatically,with the onset of ASD usually occurring before the age of three in all ethnic and socio-economic groups,ranking first among children with mental disabilities.The etiology and pathogenesis of ASD are still unclear.Previous studies have shown that the etiology of ASD is complex,which may be caused by the combination of environmental and genetic factors.Genetic studies have shown that ASD has a high degree of genetic heterogeneity,and its heritability is up to more than 90%.Therefore,genetic factors are considered as the main cause of ASD.Single nucleotide polymorphism(SNP),as an important genetic marker,has been applied to many aspects of research,of which the association between SNPs and ASD has been the focus of research at home and abroad.Currently,the latest theoretical consensus is that neurodevelopment,neuronal plasticity and abnormal synaptic connection function may be important molecular mechanisms for the occurrence of ASD.Studies have shown that micro RNA(mi RNA)has an important functional role in the brain,especially in neuroplasticity and neurodevelopment.In recent years,some scholars have studied the pathogenesis of ASD by mining SNPs-binding mi RNAs in the 3’UTR region of ASD-related genes.Disco-interacting protein 2(DIP2)family genes(DIP2A,DIP2 B,and DIP2C)are highly expressed in the brain.DIP2 family could participate in brain cell transcription,change neuron gene expression,and regulate synaptic plasticity and cognitive behavior.However,whether SNPs in DIP2 family are associated with ASD risks or with a specific ASD clinical phenotype,whether mi RNA binding to susceptible SNP sites in the 3’UTR region of the DIP2 family have an effect on ASD have not been reported.Objective:We investigate whether these 20 SNPs in DIP2 family are associated with ASD risks or with a specific ASD clinical phenotype in Chinese Han population in a case-control study.Moreover,we aim to explore the association between mi RNAs which bound to the SNPs of DIP2 family and ASD,so as to provide scientific basis for exploring the etiology of ASD,clarifying its pathogenesis,and searching for specific ASD molecular markers.Methods:A total of 1443 subjects with Chinese Han nationality(715 cases with ASD and728 healthy controls)were enrolled in this study.Controls were matched with the cases on the basis of age and sex.A Peripheral blood sample was collected from each subject.Genomic DNA was extracted according to the manufacturer’s instructions.We selected 20 functional SNPs(rs1007893,rs34736293,rs7279002,rs2070435,rs2255397,rs1107065,rs2248636,rs79564340,rs11169524,rs3803181,rs2280503,rs1047912,rs4768915,rs3740304,rs2288681,rs7088729,rs4242757,rs10795060,rs10904083,and rs4881274)in DIP2 family.SNP genotyping was determined by MALDI-TOF-MS.We investigated whether these SNPs are associated with ASD risks or with a specific ASD clinical manifestation in Chinese Han population.The goodness of fit Chi-squared test was used to determine whether the genotype distributions were in Hardy-Weinberg equilibrium(HWE).Fisher’s exact test and Chi-squared test were used to check whether allele frequency and genotype distributions in cases were significantly different from those in controls.Associations between the 20 SNPs and ASD under five different inheritance models were analyzed using online SNPStats analysis program.The linkage disequilibrium(LD)degree between the SNPs was calculated using Haploview 4.2 software.Haplotype analysis was performed using SNPStats.Interactions among susceptible SNPs of DIP2 family were investigated using GMDR software.Fisher’s exact test and Chi-squared test were used to check the association between allele frequency and genotype distributions and ASD severity.F test or Rank-sum test was used to analyze the associations between the genotypes and ASD phenotypes defined using ABC and CARS.A total of 60 subjects with Chinese Han nationality(30 cases and 30 healthy controls)were selected from the 1443 subjects.The controls were matched cases by age and sex.A Peripheral blood sample was collected from each subject.Mi RNA was extracted from serum according to the manufacturer’s instructions.The effect of mi RNAs,which bound to the SNPs sites of DIP2 family were investigated in Polymi RTS Database 3.0.According to the articles and Target Scan7.2 database,we selected mi RNAs with significant differential expression in ASD serum.The expression of mi RNAs in the serum of ASD cases and controls was detected using RT-q PCR.Statistical analyses were performed using SPSS24.0 software.The statistical tests were 2-tailed,and P<0.05 was defined as statistical significance.Results:1.A total of 715 ASD cases(541 males and 174 females)with an average age of4.80±2.447,and 728 health controls(548 males and 180 females)with an average age of 4.77±2.443 were selected.A total of 609 mild-to-moderate cases(448 males and161 females)and 106 severe cases(93 males and 13 females)were defined using CARS.No significant sex or age differences were found between ASD cases and controls(P>0.05).2.For controls,the genotype distributions of the 20 SNPs in DIP2 family were in HWE(P>0.05).3.Associations between ASD risk and allele frequency and associations between ASD risk and genotype distribution.Regarding DIP2 A,the allele frequencies and genotype distributions of the seven SNPs in the cases were not different from those in the controls(P>0.05).Regarding DIP2 B,the allele frequencies of SNPs(rs11169524,rs2280503,and rs4768915)in the cases were statistically different from those in the controls(P<0.05).Regarding DIP2 C,the allele and genotype frequencies of SNPs(rs3740304,rs2288681,rs7088729,and rs4242757),and the genotype frequencies of SNPs(rs10795060 and rs10904083)in the cases were significantly different from those in the controls(P<0.05).4.Influences of SNPs in DIP2 family identified by genetic models on ASD risk.For DIP2 A,we did not find the seven SNPs of DIP2 A were associated with ASD risk under any genetic models(P>0.05).For DIP2 B,the log-additive genetic model of rs11169524 and rs2280503 were positively associated with ASD(rs11169524:OR=1.19,95% CI=0.91-1.57,P=0.037;rs2280503: OR=1.18,95% CI=1.00-1.38,P=0.045).The CA/CC genotype of rs2280503 and TA/AA genotype of rs11169524 in the dominant model were associated with ASD(rs2280503: ORCA/CC vs AA=1.25,95%CI=1.01-1.54,P=0.037,rs11169524: ORTA/AA vs TT=1.23,95% CI=0.85-1.77,P=0.039).For DIP2 C,the log-additive model of rs3740304 and rs7088729 were positively associated with ASD(rs2288681: OR=1.21,95% CI=1.03-1.42,P=0.02,rs7088729:OR=1.23,95% CI=1.03-1.46,P=0.021).The CT genotype of rs4242757,GT genotype of rs10795060 in the codominant model,and GA genotype of rs10904083 in the overdominant model were negatively associated with ASD(ORCT vs CC=0.58,95%CI=0.38-0.89,P=0.009;ORGTvs GG=0.69,95% CI=0.54-0.89,P=0.014,rs10904083).5.We observed that LD existed between the 20 SNPs.The degree of LD in DIP2 family was relatively high.Regarding DIP2 A,D’ is range from 0.783 to 1.0,and r2 is range from 0.134 to 0.976.Regarding DIP2 B,D’ is range from 0.917 to 1.0,and r2 is range from 0.064 to 0.994.Regarding DIP2 C,D’ is range from 0.378 to 1.0,and r2 is range from 0.004 to 0.954.6.Contributions of haplotypes in DIP2 family to ASD risk.Regarding DIP2 A,we did not identify risk haplotypes,and the frequency of any haplotype was not different between the cases and the controls(P>0.05).Regarding DIP2 B,our results showed that,compared with the corresponding wild-type haplotypes,ASD risk was associated with the following mutant haplotypes,including two-SNP-formed haplotypes(for rs11169524-rs3803181,AT haplotype: OR=1.29,95% CI=1.08-1.54,P=0.006),three-SNP-formed haplotypes(for rs11169524-rs3803181-rs2280503,ATC haplotype: OR=1.28,95% CI=1.07-1.54,P=0.006;for rs380318-rs2280503-rs1047912,TCC and TCT haplotypes: OR=1.28,95% CI=1.02-1.59,P=0.031;OR=1.27,95% CI=1.01-1.59,P=0.041),four-SNP-formed haplotypes(for rs11169524-rs3803181-rs2280503-rs1047912,ATCC and ATCT haplotypes: OR=1.29,95%CI=1.03-1.60,P=0.025;OR=1.28,95% CI=1.02-1.61,P=0.032),five-SNP-formed haplotypes(for rs11169524-rs3803181-rs2280503-rs1047912-rs4768915,ATCCG,ATCTG,and GCTCG haplotypes: OR=1.28,P=0.03;OR=1.27,P=0.043;OR=2.21,P=0.002),six-SNP-formed haplotypes(for rs79564340-rs11169524-rs3803181-rs2280503-rs1047912-rs4768915,CACCCG haplotype: OR=2.01,95% CI=1.20-3.36,P=0.008).Regarding DIP2 C,our results showed that,compared with the corresponding wild-type haplotypes,ASD risk was associated with the following mutant haplotypes,including two-SNP-formed haplotypes(for rs3740304-rs2288681,CG haplotype: OR=1.27,95% CI=1.06-1.51,P=0.008;rs2288681-rs7088729,CT haplotype: OR=1.25,95% CI=1.05-1.50,P=0.012;rs7088729-rs4242757,TC haplotype: OR=1.23,95% CI=1.03-1.47,P=0.02),three-SNP-formed haplotypes(forrs3740304-rs2288681-rs7088729,GCT,haplotype: OR=1.25,95% CI=1.04-1.49,P=0.015;rs2288681-rs7088729-rs4242757,CTC haplotype: OR=1.26,95% CI=1.06-1.51,P=0.011;rs7088729-rs4242757-rs10795060,T-C-G haplotype: OR=1.24,95% CI=1.02-1.49,P=0.028),four-SNP-formed haplotypes(for rs3740304-rs2288681-rs7088729-rs4242757,GCTC haplotype: OR=1.26,95% CI=1.05-1.51,P=0.012;rs2288681-rs7088729-rs4242757-rs10795060,CTCG haplotype: OR=1.28,95% CI=1.06-1.55,P=0.011;rs7088729-rs4242757-rs10795060-rs10904083,TCGG haplotype: OR=1.23,95%CI=1.02-1.49,P=0.031;rs4242757-rs10795060-rs10904083-rs4881274,CGGA haplotype:OR=1.24,95%CI=1.04-1.52,P=0.02),five-SNP-formed haplotypes(for rs3740304-rs2288681-rs7088729-rs4242757-rs10795060,GCTCG haplotype: OR=1.26,95%CI=1.04-1.52,P=0.002;rs2288681-rs7088729-rs4242757-rs10795060-rs10904083,CTCGG haplotype: OR=1.28,95% CI=1.05-1.55,P=0.012;rs7088729-rs4242757-rs10795060-rs10904083-rs4881274,TCGGA haplotype:OR=1.24,95% CI=1.02-1.50,P=0.028),six-SNP-formed haplotypes(for rs3740304-rs2288681-rs708872-rs4242757-rs10795060-rs10904083,GCTCGG haplotype:OR=1.26,95% CI=1.04-1.52,P=0.019;rs2288681-rs7088729-rs4242757-rs10795060-rs10904083-rs4881274,CTCGGA haplotype: OR=1.29,95% CI=1.06-1.56,P=0.01),seven-SNP-formed haplotypes(for rs3740304-rs2288681-rs7088729-rs4242757-rs10795060-rs10904083-rs4881274,GCTCGGA haplotype: OR=1.27,P=0.01).7.The best model of gene-gene interaction consists of 20 SNPs of DIP2 family:rs1007893-rs1107065-rs2070435-rs2248636-rs2255397-rs34736293-rs7279002-rs1047912-rs11169524-rs2280503-rs3803181-rs4768915-rs79564340-rs10795060-rs10904083-rs2288681-rs3740304-rs4242757-rs4881274-rs7088729(CVC=10/10,P=0.001).The combination of SNPs(rs4768915 and rs79564340),SNPs(rs2070435 and rs4881274),and SNPs(rs1047912 and rs3740304)showed a strong synergistic effect,which synergistic increased the risk of ASD.The combination of SNPs(rs2248636and rs2280503),SNPs(rs2255397 and rs3803181)showed non-additive interaction between two SNPs,which may increase the risk of ASD.The combination of SNPs(rs10795060 and rs2288681)and SNPs(rs11169524 and rs4242757)did not increase the risk of ASD.8.The allele frequency of rs34736293 to DIP2 A in the mild-to-moderate group was statistically different from that in the severe group(P<0.05).rs1007893,rs7279002,rs2070435,and rs2248636 had statistically significant differences in visual response score among different genotypes;rs1107065 had statistically significant differences in intelligence score among different genotypes;rs79564340had statistically significant differences in visual response score among different genotypes;rs1107065 had statistically significant differences in activity level score among different genotypes;rs1047912 had statistically significant differences in relating to nonliving organisms among different genotypes;rs4768915 had statistically significant differences in mental level score among different genotypes;rs10795060 and rs10904083 had statistically significant differences in visual response score among different genotypes;rs10795060 had statistically significant differences in interpersonal relationship score among different genotypes;rs1047912 had statistically significant differences in sensory score among different genotypes(all P<0.05).9.According to the results,rs4768915 located in the 3’UTR region was selected.The mi RNA binding to the mutant allele(G)of rs4768915 which was retrieved from the Polymi RTS database is mi R-4655-3p which was finally selected as candidate mi RNAs in this study.10.The results of RT-q PCR showed that the expressions of mi R-4655-3p was up-regulated in the serum of ASD,but the differences were not statistically significant in ASD cases and in the controls(P>0.05).Conclusions:1.DIP2 B and DIP2 C may be genetic susceptibility genes for ASD.2.The SNPs(rs11169524,rs2280503,and rs4768915)of DIP2 B and SNPs(rs3740304,rs2288681,rs7088729,rs4242757,rs10795060 and rs10904083)of DIP2 C may be the genetic SNPs of ASD.3.Under the log-additive genetic mode,SNPs(rs11169524 and rs2280503)of DIP2 B and SNPs(rs3740304,rs2288681,and rs7088729)of DIP2 C may increase ASD risks.Under the dominant genetic model,the TA/AA genotype of rs11169524 in DIP2 B,and the CA/CC genotype of rs2280503 in DIP2 C may increase ASD risks.4.DIP2 B haplotype contains any two SNPs at rs11169524(A),rs2280503(C)and rs4768915(G)with SNPs ≥ 4,which may increase the risk of ASD.DIP2 C haplotype contains any two SNPs of rs3740304(G),rs2288681(C),rs7088729(T),rs4242757(C),rs10795060(G),and rs10904083(G),which may increase ASD risks.5.Interaction between rs4768915 and rs79564340,rs2070435 and rs4881274,rs1047912 and rs3740304,rs2248636 and rs2280503,and rs2255397 and rs3803181 may synergiously increase the risk of ASD.6.rs34736293 of DIP2 A may be a susceptible SNP affecting the severity of ASD.7.rs1007893,rs7279002,rs2070435,rs10904083,rs2248636,and rs10795060 are associated with abnormal visual response in ASD.rs1107065 and rs4768915 are associated with mental retardation in ASD.rs1047912 is associated with ASD relating to nonliving organisms and sensory impairment.rs10795060 is associated with ASD interpersonal relationship disorder.rs79564340 was associated with abnormal auditory response and impaired activity level in ASD. |
PDF Full Text Request