A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile x (FMR1) gene in newborn and high-risk populations
A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile x (FMR1) gene in newborn and high-risk populations
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DOI：
10.2353/jmoldx.2008.070073
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发表时间：
2008-01-01
影响因子：
4.1
通讯作者：
Hagerman, Paul J.
中科院分区：
文献类型：
--
作者：
Tassone, Flora;Pan, Ruiqin;Hagerman, Paul J.
脆性X综合征是智力障碍的最常见遗传原因，也是与自闭症相关的最常见单基因，通常发生在超过200个CGG重复（全突变范围）的脆性X智力低下1（FMR 1）等位基因上。目前，在一般人群中没有对全突变FMR 1等位基因数量的无偏估计;一个主要障碍是缺乏在大人群中扩增FMR 1等位基因的有效筛选工具。我们已经开发了一种快速的聚合酶链反应（PCR）为基础的筛选工具扩展FMR 1等位基因。该方法利用随机靶向扩增的CGG区域内的嵌合PCR引物，使得PCR产物的广泛分布的存在代表扩增的等位基因的阳性结果。该方法适用于筛选男性和女性，并适用于筛选整个前突变（55至200个CGG重复）和全突变范围内的等位基因大小。此外，该方法能够使用来自单个干血斑的少至1%的DNA快速检测扩增的等位基因。这项工作中提出的方法适用于筛选大规模的新生儿或高危人群（如自闭症，卵巢早衰，共济失调，痴呆）的扩展FMR 1等位基因。本文所述的测试每个样品的材料成本低于5美元;通过适当的放大和自动化，每个样品的成本应该接近1美元。
Fragile X syndrome, the most common inherited cause of intellectual impairment and the most common single gene associated with autism, generally occurs for fragile X mental retardation 1 (FMR1) alleles that exceed 200 CGG repeats (full-mutation range). Currently, there are no unbiased estimates of the number of full-mutation FMR1 alleles in the general population; a major obstacle is the lack of an effective screening tool for expanded FMR1 alleles in large populations. We have developed a rapid polymerase chain reaction (PCR)-based screening tool for expanded FMR1 alleles. The method utilizes a chimeric PCR primer that targets randomly within the expanded CGG region, such that the presence of a broad distribution of PCR products represents a positive result for an expanded allele. The method is applicable for screening both males and females and for allele sizes throughout the premutation (55 to 200 CGG repeats) and full-mutation ranges. Furthermore, the method is capable of rapid detection of expanded alleles using as little as 1% of the DNA from a single dried blood spot. The methodology presented in this work is suitable for screening large populations of newborn or those at high risk (eg, autism, premature ovarian failure, ataxia, dementia) for expanded FMR1 alleles. The test described herein costs less than $5 per sample for materials; with suitable scale-up and automation, the cost should approach $1 per sample.