【作者】 赵云静；

【导师】 麻宏伟；

【作者基本信息】 中国医科大学 ， 儿科学， 2010， 博士

【摘要】 前言功能性构音障碍(functional articulation disorder)是儿童期最常见的言语障碍,又称为发育性发音障碍(developmental articulation/ phonological disorder)。调查显示,一半以上的构音障碍儿童以后可能会出现语言、阅读及拼写方面的困难。长期随访研究表明功能性构音障碍儿童在高中时成绩明显落后于正常儿童,学历普遍低,工作机会相对差,多从事半技能或非技术性工作。迄今为止,功能性构音障碍的病因尚不清楚。家族聚集现象及双生子研究显示功能性构音障碍在亲属及同胞中的患病率明显高于普通人群,表明功能性构音障碍的发生与遗传因素密切相关。FOXP2基因(MIM605317)是人类所发现的第一个言语相关基因,位于7q31,属于“FOX”基因家族。FOXP2基因14外显子G/A的突变导致严重的语言和言语障碍,如口头及书面语言的理解和表达障碍、发育性言语失用、口面部精细运动障碍等。FOXP2基因包括一个多谷氨酸盐束、一个锌指、一个亮氨酸拉链基序和一个叉头框DNA结合区。通过FOXP2基因mRNA在人和鼠发育中的脑组织时间及空间上的表达模式研究发现FOXP2基因在基底神经节、丘脑、下橄榄体和小脑均有表达,支持FOXP2基因在纹状体皮质和橄榄小脑回路发育中的作用,而这一回路与运动控制功能有关。此外,FOXP2基因编码的蛋白作为一种转录因子可以调控其它基因在发育中的肺组织、心血管、肠道和神经组织的表达。另外,Zeesman S等报道一个7q31-q32缺失的女孩,这个女孩患有严重的交流障碍、伴特殊面容及轻度发育落后。染色体检查发现该患儿存在父源染色体包括FOXP2基因区域的7q31-q32片段的缺失。分析可能由于FOXP2基因的单倍剂量不足导致语言交流障碍和口部运动障碍。2007年,Lennon PA报道了一个语言障碍伴有发育性言语失用病例,染色体分析显示7q31.1-7q31.31缺失,这是包括FOXP2基因在内的最小范围的染色体缺失病例,进一步证明FOXP2基因在语言及言语发育中的重要作用。因此,FOXP2及其临近基因成为语言和言语障碍的重要候选基因。基于以上证据,本文在FOXP2基因内选择5个多态位点(SNPs):rs923875,rs2396722,rs1852469,rs17137124和rs1456031,对其等位基因在辽宁地区汉族人群中的分布特点进行研究并与功能性构音障碍患儿进行比较,然后根据连锁不平衡分析结果进行单倍型分析,旨在探讨FOXP2基因与功能性构音障碍的相关性,同时,对中重度功能性构音障碍患儿进行FOXP2基因14外显子G/A的突变筛查。对象与方法1、对象功能性构音障碍患儿150例及正常健康体检者140例,功能性构音障碍患儿中男109例,女41例,平均年龄6.45±2.11岁,均为汉族。所有患儿均进行智力测试、构音评价,均符合功能性构音障碍的诊断标准。2、方法留取所有受试者外周静脉血2ml,采用酚一氯仿一异戊醇法提取基因组DNA。根据NCBI的SNP数据库选择FOXP2基因内的SNPs位点,引物选自参考文献或采用Primer Premier 5.0软件进行设计。引物由上海invitrogen公司合成,限制性内切酶购自NEB或MBI公司。常规PCR方法扩增目的片段,1.5%琼脂糖凝胶电泳检测PCR产物。FOXP2基因SNPs等位基因分型采用限制性片段长度多态性(RFLP)的方法,PCR扩增产物经相应的限制性内切酶消化,酶切产物经2%-2.5%琼脂糖凝胶电泳,凝胶自动成像系统扫描,记录各样本基因型。各SNPs位点分别选择6例进行直接测序分析,以验证PCR-RFLP结果的准确性。PCR扩增中重度构音障碍患儿FOXP2基因14外显子,采用RFLP方法筛查14外显子G/A的突变。同时,选择中重度构音障碍患儿10例进行FOXP2基因14外显子直接测序分析。3、统计学分析根据Hardy-Weinberg平衡定律对FOXP2基因各多态位点的基因型和等位基因频率进行Hardy-Weinberg平衡吻合度检验；采用在线分析程序SHEsis进行连锁不平衡检验以及病例组和对照组基因型和等位基因频率的比较,并进行单倍型分析。数据处理采用SPSS11.0软件包,以P<0.05为具有显著性差异。结果FOXP2基因5个SNP位点:rs923875,rs2396722、rs1852469、rs17137124及rs1456031在辽宁地区汉族人群中均有多态性,其基因型及等位基因频率分别为rs923875:AA 0.11, AC 0.51, CC 0.38, A 0.368, C 0.632; rs2396722:CC 0.29, CT 0.52, TT 0.19; C 0.551, T 0.449; rs1852469:AA 0.17, AT 0.58, TT 0.25, A 0.464, T 0.536; rs17137124:CC 0.40, CT 0.44, TT 0.16, C 0.621, T 0.379; rs1456031:CC 0.28, CT 0.53, TT 0.19, C 0.547, T 0.453。均符合Hardy-Weinberg平衡定律；各位点的等位基因频率在不同种族人群中存在较大差异。SNP rs1852469的基因型频率在病例组与对照组之间存在显著性差异,χ2=13.772,P=0.001；等位基因频率比较亦存在显著性差异,χ2=9.129P=0.002528。根据连锁不平衡结果选择D’>0.5的SNPs位点构建单倍型,rs923875A/+rs2396722T/+rsl 852469T单倍型在功能性构音障碍组的频率高于正常对照组(χ2=6.590,P=0.0103,OR=1.752,95%CI：1.138-2.695,Global P=5.68e-006)。提示FOXP2基因可能与功能性构音障碍存在相关性。将功能性构音障碍按照严重程度分组后分别与正常对照组比较,其中轻度组42例与对照组比较FOXP2基因5个多态位点的基因型频率和等位基因频率均无显著性差异；中重度组108例与对照组比较,rs1852469位点的基因型频率存在显著性差异(χ2=12.379,P=0.0021),rs1852469位点的等位基因频率比较也存在显著性差异(χ2=8.486,P=0.0036)。同样选择位于5’非翻译区的3个多态位点进行单倍型分析,结果显示单倍型rs923875A/+rs2396722T/+rs1852469T在中重度功能性构音障碍组的频率显著高于正常对照组(χ2=5.169, P=0.023, OR=1.702,95%CI:1.073～2.701,Global P=0.003173).单倍型rs923875C/+rs2396722C/+rs1852469A在正常对照组的频率显著高于中重度功能性构音障碍组,为保护性单倍型(χ2=5.976, P=0.0145, OR=0.561,95%CI:0.352～0.895, Global P=0.003173)。FOXP2基因可能是中重度功能性构音障碍的易感基因。在中重度构音障碍患儿中进行FOXP2基因14外显子G/A的突变筛查,均未发现FOXP2基因14外显子G/A的突变。1、FOXP2基因5个SNPs rs923875、rs2396722、rs1852469、rs17137124及rs1456031在辽宁地区汉族人群中均有多态性,可以作为遗传标记进行相关疾病的关联研究或连锁分析。2、FOXP2基因5个SNPs rs923875、rs2396722、rs1852469、rs17137124及rs1456031的等位基因频率分布在不同种族人群中存在差异。3、FOXP2基因可能与功能性构音障碍存在相关,FOXP2基因单核苷酸多态位点rs1852469T等位基因可能是决定疾病易感性的重要因素,含有rs923875A/+rs2396722T/+rsl 852469T单倍型的个体发生功能性构音障碍的相对风险增高,含有rs923875C/+rs2396722C/+rsl 852469A单倍型的个体发生功能性构音障碍的相对风险显著降低。4、FOXP2基因14外显子G/A突变不是导致功能性构音障碍的直接原因。更多还原

【Abstract】 IntroductionFunctional articulation disorder (FAD) is the most common speech disorder of children. It is also known as developmental articulation disorder or developmental phonological disorder. The estimated prevalence is 15.6% in children at age 3 and 3.8% at age 6. It is defined by developmentally inappropriate errors in speech production that reduce intelligibility. More than half of these children encounter later academic difficulties in language, reading, and spelling. The residual effects of preschool FAD may be life long.Although the etiology of FAD is unknown, there is a lot of literature suggesting that susceptibility to FAD is genetic, including familial aggregation studies and some twin studies.Forkhead-box P2 (FOXP2) gene (MIM 605317), located on 7q31, encodes a transcription factor containing a polyglutamine tract, a zinc finger, a leucine zipper motif, and a forkhead-box DNA binding domain. The G/A mutation in exon 14 of FOXP2 was found to cause a severe developmental disorder of verbal communication, involving profound articulation deficits, accompanied by linguistic and grammatical impairments in a large family known as the KE family. Lai CS et al have determined the detailed spatial and temporal expression pattern of FOXP2 mRNA in the developing brain of mouse and human. They found expression in several structures including the cortical plate, basal ganglia, thalamus, inferior olives and cerebellum. These data support a role for FOXP2 in the development of corticostriatal and olivocerebellar circuits involved in motor control. And it is likely to regulate gene expression in defined areas of developing lung, cardiovascular, intestinal, and neural tissue. The available expression data are compatible with a conserved role (or roles) for this gene in regulating development of neural circuitry underlying motor control and sensory-motor integration in mammals and birds. Structural and functional neuroimaging with gene expression studies supported a significant relationship between FOXP2 and the language neural system. All these studies suggest that the FOXP2 gene is involved in the development of the neural system that mediates the specific motor coordination necessary for speech. Moreover, Zeesman et al found a child with developmental apraxia of speech and mild cognitive delay who had a deletion of 7q31 that included FOXP2 gene. These findings indicate that FOXP2 is a candidate gene for speech and language disorder.In the present study, we attempted to investigate the association between the FOXP2 gene and FAD using the analyses of association and haplotype.Materials and methods1、SubjectsA total of 150 patients and 140 healthy unrelated controls of similar ethnic background were recruited from Shenyang, Shengjing Hospital, China Medical University. Of the 150 cases, were 109 male and 41 female. The mean age of the children at the time of testing was 6.45±1.98 years. All the patients fulfilled the criteria for functional articulation disorder. Physical examinations were performed and patients were excluded if they had any medical or genetic conditions which could be contributing.2、Methods(1)Genotyping of SNPsGenomic DNA was obtained from peripheral blood leukocytes using standard phenol-chloroform method. Five single nucleotide polymorphisms (SNPs) rs923875, rs2396722, rs1852469, rs17137124 and rs1456031 in the FOXP2 gene were selected from the dbSNP database. PCR primer pairs were based on references or designed by Primer Premier 5.0. PCR was performed and the products were digested with restriction enzymes Apall, VspⅠ, Trull, Afl II and RsaⅠrespectively. The samples were loaded on 2% or 2.5% agarose gels containing ethidium brodmide for electrophoresis at 100 V for 50 min. Gel were read blindly by two independent raters with discrepancies resolved by re-genotyping.6 random select samples for each SNP were tested again by direct DNA sequencing. PCR products were purified using a QIAQuick PCR purification kit (Qiagen, Germany). Direct sequencing of the samples was performed on an ABI 3730 DNA sequencer (Perkin Elmer, Foster city, California, USA). Sequencing results were compared with the reference human FOXP2 sequence and the results of RFLP.(2)Mutation analysisFor the moderate to severe patients, the G/A mutation of exon 14 was screened. PCR primer pairs were based on reference. PCR was performed and the products were digested with restriction enzyme Tail. The samples were loaded on 2% agarose gels containing ethidium brodmide for electrophoresis at 100 V for 50 min. Direct DNA sequencing was used in 10 samples to test the results of PCR-RFLP and other mutations.(3)Statistical analysisSignificance level was previously established at 0.05. The Hardy-Weinberg equilibrium for genotype frequencies was evaluated by the Chi-square test. The comparisons of allelic frequencies and genotype analyses between patients and controls were performed using SHEsis program online. The pairwise linkage disequilibrium (LD) and haplotype analysis were also estimated by the SHEsis program. The Bonferroni test was applied to correct for multiple comparisons.ResultsThe allele and genotype frequencies of all five SNPs of FOXP2 gene were analyzed. The genotype and allele frequencies are rs923875:AA 0.11, AC 0.51, CC 0.38, A 0.368, C 0.632; rs2396722:CC 0.29, CT 0.52, TT 0.19; C 0.551, T 0.449; rs1852469:AA 0.17, AT 0.58, TT 0.25, A 0.464, T 0.536; rs17137124:CC 0.40, CT 0.44, TT 0.16, C 0.621, T 0.379; rs1456031:CC 0.28, CT 0.53, TT 0.19, C 0.547, T 0.453, respectively. All of them were found to be in Hardy-Weinberg equilibrium in both patient and control samples. The significant differences of genotype and allele frequency distributions were detected between different populations.Statistical analyses of the SNPs showed that neither genotype nor allele frequency distributions were different between FAD patients and control subjects with the exception of the SNP rs1852469. In this case, there were significant differences in the genotype (P=0.001) and allele (P=0.002528) frequencies. These P values remained significant after Bonferroni correction (P=0.005; P=0.0126 respectively).To test for LD between the FOXP2 SNPs, D’values were calculated for all pairs of SNPs on patients and controls. We considered the SNPs with D’>0.5 to perform the haplotype analysis. Significant difference in the haplotype frequency was observed between patients and controls for the haplotype rs923875A/+rs2396722T/+ rs1852469T(P=0.0103).After the division in accordance with the degree of severity, neither genotype nor allele frequency distributions were different between mild FAD patients and control subjects; There were significant differences in the genotype (P=0.0021) and allele (P=0.0036) frequencies of SNP rs1852469 between moderate to severe FAD and controls. These P values remained significant after Bonferroni correction (P=0.0105; P=0.018 respectively).We considered 3 SNPs with D’>0.5 to perform the haplotype analysis. A risk haplotype was detected, rs923875A/+ rs2396722T/+rs1852469T (P=0.023) which was significantly associated to moderate to severe FAD.Alternatively, a protective haplotype rs923875C/+ rs2396722C/+rs1852469A (P=0.0145) was also identified. But the G/A change found in the KE family were not detected in any of the moderate to severe patients.Conclusion1. There are polymorphisms of SNPs rs923875, rs2396722、rs1852469、rs17137124 and rs1456031 among Liaoning population. They can be used as genetic markers for association or linkage analysis.2. The distributions of the five SNPs exhibit ethnic heterogeneity.3. There is an association between SNP rs1852469 and FAD. The individuals with haplotype rs923875A/+rs2396722T/+rsl852469T are more susceptible to FAD. The individuals with haplotype rs923875C/+rs2396722C/+rs1852469A are in relatively lower risk.4. The G/A mutation are not the direct factor of functional articulation disorder.更多还原