【摘要】 【目的】采用优化的PCR体系对脆性X智力障碍基因-1(FMR-1)片段扩增,分析汉族孤独症谱系障碍(autism spectrum disorder,ASD)患儿FMR-1基因异常突变发生情况。【方法】以466例汉族ASD患儿为研究对象,其中典型孤独症433名,未分类广泛性发育障碍(PDD-NOS)33名。取患儿外周血3～5mL提取DNA,采用优化的PCR体系对466例患儿DNA进行FMR-1基因片段扩增;对于扩增成功的PCR产物进一步采用短串联重复序列(short tand-erm repeat,STR)荧光检测技术进行检测。【结果】466个ASD患儿中,464个ASD患者(99.57%)DNA样品PCR扩增成功;2个典型孤独症患者(0.43%)DNA样品PCR扩增失败(排除DNA质量和操作问题),推断FMR-1基因发生异常突变。对于扩增成功的PCR产物进一步采用STR荧光检测技术进行检测,结果显示PCR产物均小于500bp,其(CGG)n重复个数属正常突变范围。【结论】研究采用优化的PCR体系对FMR-1基因片段扩增,进一步采用STR荧光检测技术准确推断出基因片段中三核苷酸(CGG)重复个数,优化后的PCR体系可适用于大样本孤独症谱系障碍儿童的FMR-1基因突变检测。检测结果显示汉族孤独症谱系障碍儿童FMR-1基因异常突变率为0.43%。更多还原

【Abstract】 【Objective】 To analyze the mutation condition of fragile X mental retardation 1(FMR-1) in Chinese Han autism spectrum disorder(ASD) children using optimized PCR system and STR fluorescence technology.【Methods】 From January 2007 to October 2010,466 Chinese Han ASD children were recruited for this study by the Children Development Behavior Research Center of Harbin Medical University.Among 466 Chinese Han ASD children,433 children were diagnosed as typical autism;33 children were diagnosed as pervasive developmental disorder-not otherwise specified(PDD-NOS).Total genomic DNA was extracted from peripheral blood leukocytes(3 to 5mL whole blood),and PCR amplifications were performed in a total volume of 25 μL containing 50 ng of genomic DNA.The expanded range of allele sizes were detected by gel visualization and STR fluorescence technology.【Results】 464(99.57%)PCR products of ASD children were amplified successfully.Those PCR implications which fragment size was less than 500 bp,were further verified by STR fluorescence technologies.At the same time,the DNA samples of two autism patients(0.43%) were not amplified successfully by PCR,excluding the possibility of DNA quality and operational problems,and deduced them as abnormal mutation of fragile X mental retardation 1(FMR-1).【Conclusions】 Using optimized PCR system and STR fluorescence technology can significantly provide the CGG repeat numbers of alleles.The incidence of the mutation of FMR-1 gene in the Chinese Han ASD children is 0.43%.更多还原