Chaudhary Adeel G, Hussein Ibtessam R, Abuzenadah Adel, Gari Mamdouh, Bassiouni Randa, Sogaty Samira, Lary Sahira, Al-Quaiti Maha, Al Balwi Mohammed, Al Qahtani Mohammed
Faculty of Medical Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
Pediatr Neurol. 2014 Apr;50(4):368-76. doi: 10.1016/j.pediatrneurol.2013.11.020. Epub 2013 Dec 4.
Fragile X syndrome, the most common form of inherited intellectual disability, is caused by expansion of CGG trinucleotide repeat at the 5' untranslated region of the FMR1 gene at Xq27. In affected individuals, the CGG repeat expansion leads to hypermethylation and the gene is transcriptionally inactive. Our aim was to identify fragile X syndrome among children with intellectual disability in Saudi Arabia.
The study included 63 patients (53 males, 10 females) presented with intellectual disability, 29 normal subjects, and 23 other family members. DNA samples from six patients previously diagnosed with fragile X syndrome by Southern blot technique were used as positive controls. The method was based on bisulfite treatment of DNA followed by two different techniques. The first technique applied polymerase chain reaction amplification using one set of primers specific for amplifying methylated CpG dinucleotide region; another set designed to amplify the unmethylated CGG repeats. The second technique used the methylation-specific melting curve analysis for detection of methylation status of the FMR1 promoter region.
Molecular testing using methylation sensitive polymerase chain reaction had shown amplified products in all normal subjects using unmethylated but not methylated primers indicating normal alleles, whereas amplified products were obtained using methylated polymerase chain reaction primers in fragile X syndrome-positive samples and in 9 of 53 males, indicating affected individuals. Molecular testing using melting curve analysis has shown a single low melting peak in all normal males and in (44/53) patients indicating unmethylated FMR1 gene, whereas high melting peak indicating methylated gene was observed in the fragile X syndrome-positive samples and in 9 of 53 patients. We found 100% concordance between results of both techniques and the results of Southern blot analysis. Three samples have shown both methylated and unmethylated alleles, indicating possible mosaicism. No female patients or carriers could be detected by both techniques.
The technique can be applied for the rapid screening for fragile X syndrome among patients with intellectual disability. The impact of mosaicism on clinical severity needs further investigation.
脆性X综合征是遗传性智力障碍最常见的形式，由位于Xq27的FMR1基因5'非翻译区的CGG三核苷酸重复序列扩增所致。在受影响个体中，CGG重复序列扩增导致高甲基化，该基因转录失活。我们的目的是在沙特阿拉伯智力障碍儿童中识别脆性X综合征。
该研究纳入了63例智力障碍患者（53例男性，10例女性）、29例正常受试者以及23名其他家庭成员。6例先前通过Southern印迹技术诊断为脆性X综合征的患者的DNA样本用作阳性对照。该方法基于对DNA进行亚硫酸氢盐处理，随后采用两种不同技术。第一种技术应用聚合酶链反应扩增，使用一组特异性扩增甲基化CpG二核苷酸区域的引物；另一组引物设计用于扩增未甲基化的CGG重复序列。第二种技术使用甲基化特异性熔解曲线分析来检测FMR1启动子区域的甲基化状态。
使用甲基化敏感聚合酶链反应进行的分子检测显示，在所有正常受试者中，使用未甲基化而非甲基化引物可扩增出产物，表明为正常等位基因；而在脆性X综合征阳性样本以及53例男性中的9例中，使用甲基化聚合酶链反应引物可获得扩增产物，表明为受影响个体。使用熔解曲线分析进行的分子检测显示，所有正常男性以及（53例中的）44例患者中出现单一低熔解峰，表明FMR1基因未甲基化；而在脆性X综合征阳性样本以及53例患者中的9例中观察到高熔解峰，表明基因为甲基化。我们发现两种技术的结果与Southern印迹分析的结果完全一致。3个样本同时显示出甲基化和未甲基化等位基因，表明可能存在嵌合体现象。两种技术均未检测到女性患者或携带者。
该技术可用于对智力障碍患者进行脆性X综合征的快速筛查。嵌合体对临床严重程度的影响需要进一步研究。
