【作者】 何益群；

【导师】 赵靖平；

【作者基本信息】 中南大学 ， 精神卫生学， 2008， 博士

【摘要】 目的(1)研究儿童孤独症发病的高危因素及孤独症儿童的早期发育特征。(2)研究血浆谷氨酸和丫-氨基丁酸浓度变化与儿童孤独症的关系,及其在儿童孤独症易发癫痫病理机制中的作用。(3)通过对Reelin基因上四个单核苷酸多态(SNP)位点rs736707、rs2229864、rs362691、rs2073559的检测,探讨Reelin基因多态性与儿童孤独症的关系。方法(1)共入组儿童孤独症核心家系278例及正常对照儿童核心家系200例,调查一般人口学资料、母孕期情况、围产期情况、发育性标志出现时间、养育情况、既往史、家族史及体格检查情况;对于孤独症儿童,尚需调查早期症状表现、评定儿童孤独症评定量表(CARS)及孤独症行为检查量表(ABC)。(2)27例伴癫痫的孤独症患者作为观察组,30例不伴癫痫的孤独症患者为另一观察组,36例正常儿童作为对照组。采用高效液相色谱法测定三组血浆谷氨酸和γ-氨基丁酸浓度,进行组间比较。(3)在中国汉族孤独症核心家系(孤独症患者234人,父母399人)及正常对照人群(286人)中,检测Reelin基因上四个单核苷酸多态位点rs736707、rs2229864、rs362691、rs2073559,采用TaqMan—PCR的方法确定样本各位点的基因型,进行以核心家系为基础的传递不平衡检验及病例对照的关联研究。(4)采用DNA测序和序列分析的方法,验证TaqMan-PCR等位基因鉴别分析技术的准确性。(5)使用GENEHUNTER(version 2.1)软件对孤独症核心家系进行传递不平衡分析,使用SAS软件包构建单倍体模型,以x~2检验比较组间差异。统计分析采用SPSS14.0软件包,根据资料特征分别进行独立样本t检验、x~2检验、方差分析、Logistic回归分析以及计算暴露因素的优势比(OR值)。结果(1)儿童孤独症先证者中男性233人,女性45人,男女比例为5.18:1;患者起病年龄为22.36±8.29月。家长感觉到孩子异常最早是在10个月时,最晚在48个月。(2)与正常儿童相比,孤独症患者的多项围产期和母孕期高危因素差异具有显著性。Logistic回归发现:母亲受教育年限(P=0.009)、父亲生育年龄(P=0.001)、家庭负担(P=-0.001)、抚育者(P=0.001)、妊娠反应(P=0.025)、孕期服药史(P=0.014)、分娩方式(P=-0.045)、养育方式(P＜0.001)、主要教导者(P＜0.001)、阳性家族史(P=0.002)进入回归方程。(3)计算回归方程中有临床意义的6项指标的组间优势比(OR值),从强到弱依次为父亲生育年龄＞30岁(OR=6.715)、家族史阳性(OR=5.798)、孕期服用药物(OR=4.653)、非自然分娩(OR=4.147)、母亲受教育年限＞11年(OR=1.373)、妊娠反应中/重度(OR=1.044)。(4)与对照组儿童相比,孤独症儿童的各项发育性标志出现时间、孤独症早期表现总分、头围等指标,差异具有统计学意义(P＜0.001)。(5)不伴癫痫的孤独症组(A组)血浆谷氨酸浓度为653.95±545.87pmol/L,伴有癫痫的孤独症组(B组)血浆谷氨酸浓度为1608.81±1478.22pmol/L,正常对照儿童(C组)血浆谷氨酸浓度为653.70±589.86pmol/L。A组与B组相比,差异具有显著性(P＜0.05);B组与C组相比,差异具有显著性(P＜0.05);A组与C组相比,无显著性差异(P＞0.05)。A组血浆γ-氨基丁酸浓度为71.46±65.98pmol/L,B组为111.19±85.27pmol/L,C组为70.13±51.36pmol/L。A组与B组相比,差异具有显著性(P＜0.05);B组与C组相比,差异具有显著性(P＜0.05);A组与C组相比,无显著性差异(P＞0.05)。(6)孤独症核心家系单位点SNP传递不平衡分析,四个位点均无显著性差异(P＞0.05);四个位点组成的单倍体中GTCC 5条全部未传递,未传递存在显著性差异,其余各单倍体未发现不平衡传递。对照组样本在四个SNPs多态位点上全部符合H-W平衡。病例对照关联研究中,孤独症组与对照组四个SNPs多态位点基因型及等位基因频率比较均未发现显著性差异(P＞0.05)。孤独症组与对照组单倍体分型比较中,rs362691-rs2073559相邻两位点的CC、CT、GC三种单倍体,每一单倍体频率在两组之间比较,差异均无显著性(P＞0.05);而GT单倍体频率在两组之间差异具有显著性(x~2=5.805,P=0.020)。两组单倍体频率构成具有显著性差异(x~2=9.682,P=0.021)。其余相邻两位点、三位点以及四个位点组合的每一单倍体频率、以及单倍体频率构成均无显著性差异(P＞0.05)。结论(1)儿童孤独症在母孕期、围产期高危因素明显增多,前三位的高危因素分别是父亲生育年龄大于30岁、家族史阳性、孕期服用药物史。(2)孤独症儿童整体发育落后于正常对照儿童,而且在发育过程中具有某些特征性的早期表现,有望被早期识别。(3)血浆谷氨酸/γ-氨基丁酸浓度异常在孤独症儿童中并不具有普遍性,仅在伴有癫痫的孤独症儿童中有异常升高。谷氨酸/γ-氨基丁酸系统功能异常可能是孤独症易发癫痫的病理机制之一。(4)以孤独症核心家系为基础的传递不平衡检验未发现Reelin等位基因的优势传递。病例对照关联研究发现仅rs362691-rs2073559的GT单倍体频率在两组之间存在显著性差异,提示Reelin基因与孤独症无关联。更多还原

【Abstract】 Objective(1) To study on risk factors and early manifestation in autism.(2) To explore whether plasma level of glutamate and gamma-aminobutyric acid were related with autism, as well as the high prevalence of epilepsy in autism.(3) To explore the relationship between Reelin gene polymorphisms and autism by detecting four single-nucleotide polymorphisms (SNPs) including rs736707、rs2229864、rs362691、rs2073559 located on RELN.Method(1) 278 autism trioes and 200 control trioes were recruited. At the screening, all the children would fulfill clinical questionnaires, including demography, conditions of pregnancy and perinatal period, postnatal development, raising patterns, previous history and family history, physical examination. Autism children were assessed with CARS and ABC and their early manifestation were recorded.(2) The plasma level of glumatic acid and gamma-aminobutyric acid was measured with high efficiency liquid chromatography in 27 autism children with epilepsy, compared with that of 30 autism children without epilepsy and 36 control children.(3) The four SNPs, rs736707、rs2229864、rs362691、rs2073559, on Reelin gene were genotyped by Taqman-PCR in 234 autistic children, 399 parents, and 286 controls in Chinese Han. And the family-based transmission disequilibrium test (TDT) and case-control association anlysis were performed at the same time.(4) To check the accuracy of the allele identified technique inTaqMan-PCR by DNA sequencing.(5) GENEHUNTER (version 2.1) was used in transmission disequilibrium test. The haplotype model was constructed with SAS and all analyses were conducted with SPSS14.0 for Windows. We used t-test, chi-square test, analysis of variance, logistic regression analysis, and odds ratio according to the type of the data.Results(1) Childhood autism probands consisted of 233 males and 45 females (male/female = 5.18/1) . The mean onset age of autism was 22.36±8.29 months. The emergence time of abnormality being detectedby their caregivers was different, ranging from 10 to 48 months.(2) There were significant difference in many pregnancy and perinatal period risk factors between autistic children and control. Logistic regression found that many items entered regression equation as the following: mother’s school life (P=0.009) , father’s reproductive age(P=0.001), family burden (P=0.001), dry nurse (P=0.001), pregnancy reaction (P=0.025) ,drug history during mother’s pregnancy period (P=0.014), delivery modality (P=0.045 ), nurture modality (P<0.001), main instructor (P<0.001) , positive family history (P=0.002) .(3) We calculated odds ratios of six items with clinical signific -ance in regression equation: father’s reproductive age more than 30years(OR=6.715), positive family history (OR=5.798 ), drug history during mother’s pregnancy period (OR=4.653 ) ,un-spontaneous delivery (O R = 4.147) , mother’s school life more than 11 years (OR=1.373) ,moderate /severe pregnancy reaction (OR= 1.044) .(4) There were significant differences between autism patients and control in the following items: the emergence time of early developmental sign, the total score of early manifestation, and head circumference (P<0.001) .(5) Plasma level of glutamic acid was 653.95 pmol/L (SD =545.87) in autism children (group A), 1608.81 pmol/L (SD=1478.22) in autism children with epilepsy (group B ) and 653.70 pmol/L(SD=589.86) in control group (group C). Compared with Group B, the average plasma level of glutamic acid in group A and group C was significantly lower (p<0.05) , but there was no significant difference between group A and group C (p>0.05) . In terms of plasma level of gamma-aminobutyric acid (GABA) , group A was 71.46 pmol/L(SD=65.98 ) , group B was 111.19 pmol/L ( SD=85.27 ) and group C was 70.13 pmol/L (SD=51.36) . The plasma level of GABA in group B was significantly higher than group A and group C (p<0.05 ) . But there was no significant difference between group A and Group C (p>0.05 ) .(6) No significant differences were found on family-based TDT for single-locus marker in four SNPs in autism. There was no significant transmission disequilibrium but haplotype GTCC, in which 5 haplotypes were non-transmission.All these 4 SNPs (rs736707, rs2229864, rs362691, rs2073559) among control samples met Hardy-Weinberg equilibrium. In case-control association analysis, no significant difference were found in allelic and genotypic distribution of the 4 SNPs between autism group and control group (P>0.05) .There were no significant difference in frequency of CC,CT,GC of rs362691-rs2073559 between autism and control (P>0.05 ) , but that of GT showed significant difference between the two groups ( x~2=5.805, P =0.020) . The frequency composition of haplotypes between the two groups was significantly different (x~2=9.682, P=0.021) . No significant differences were found in other multiple-locus haplotypes frequencies and frequency compositions between autism and control (P>0.05 ) .Conclusion( 1 ) Autistic children have more risk factors in duration of pregnancy and peripartum. The first three risk factors were father’s reproductive age exceeding 30, positive family history, taking medicine in duration of pregnancy.(2) The global development in autism children is behind control. There are some characteristic early manifestation in the development of autistic children, which can be identified earlier.(3) The increasing of plasma level of glutamate and gamma-aminobutyric acid is not general in all autistic children but only in those with epilepsy. It suggests that disfunction of glutamatergic and GABAergic system make a role in high prevalence of epilepsy in autism.(4) Transmission disequilibrium test based on autism trios found no preferential transmission in alleles of Reelin. Case-control association study showed that only frequency of GT in rs362691-rs2073559 was significantly different between the two groups, which suggested that Reelin gene may have no association with autism.更多还原