Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Clin Chem. 2012 Mar;58(3):568-79. doi: 10.1373/clinchem.2011.174615. Epub 2012 Jan 5.
CGG repeat expansions in the FMR1 (fragile X mental retardation 1) gene are associated with fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. We evaluated the use of melting curve analysis (MCA) of triplet-primed PCR (TP-PCR) assays as a rapid screening tool for the positive identification of expanded FMR1 alleles in men and women.
Both 5'- and 3'-weighted direct TP-PCRs (dTP-PCRs) were evaluated on 29 cell line-derived DNA samples and 44 blinded clinical samples. The presence of expansions was identified by the melting curve profiles generated automatically through MCA on the LightCycler 480 Real-Time PCR System. All samples were also analyzed by capillary electrophoresis to confirm the identities of the PCR fragments that gave rise to the observed melt peak profiles.
The presence of expanded alleles in samples from both males and females produced melt peak profiles that were distinct from those of individuals with the normal allelic form. In the blinded test, positive and negative calls for the presence of an expanded allele corroborated with previously determined genotype classifications for all samples.
The approach of dTP-PCR plus MCA offers a single-step strategy with high diagnostic sensitivity and specificity for rapid screening detection of FMR1 CGG repeat expansions, regardless of sex. The combined use of 5'- and 3'-weighted dTP-PCR assays minimizes the incidence of false-negative results arising from repeat-flanking deletions.
FMR1（脆性 X 智力低下 1 号基因）基因中的 CGG 重复扩展与脆性 X 综合征、脆性 X 相关震颤/共济失调综合征和脆性 X 相关原发性卵巢功能不全有关。我们评估了三核苷酸引物 PCR（TP-PCR）分析的熔解曲线分析（MCA）作为一种快速筛选工具，用于阳性鉴定男性和女性中扩展的 FMR1 等位基因。
对 29 个细胞系衍生的 DNA 样本和 44 个盲样临床样本进行了 5'-和 3'-加权直接 TP-PCR（dTP-PCR）评估。通过 MCA 在 LightCycler 480 实时 PCR 系统上自动生成的熔解曲线图谱来识别扩增的存在。所有样本均通过毛细管电泳分析，以确认产生观察到的熔解峰图谱的 PCR 片段的身份。
来自男性和女性的样本中扩增等位基因的存在产生的熔解峰图谱与具有正常等位基因形式的个体明显不同。在盲测中，存在扩增等位基因的阳性和阴性检测结果与所有样本的先前确定的基因型分类相符。
dTP-PCR 加 MCA 的方法提供了一种单一步骤的策略，具有高诊断灵敏度和特异性，可快速筛选检测 FMR1 CGG 重复扩展，无论性别如何。5'-和 3'-加权 dTP-PCR 分析的联合使用最大限度地减少了由于重复侧翼缺失而导致的假阴性结果的发生。
