Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay
Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay
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DOI：
10.1016/j.jmoldx.2014.10.001
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发表时间：
2015-03-01
影响因子：
4.1
通讯作者：
Chong, Samuel S.
中科院分区：
文献类型：
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作者：
Lian, Mulias;Rajan-Babu, Indhu-Shree;Chong, Samuel S.
DMPK CTG 重复序列的不稳定和扩张会导致 1 型强直性肌营养不良 (DM1)，这是最常见的成人发病的神经肌肉疾病。 DM1 和其他强直性疾病之间的重叠临床特征需要分子确认才能明确诊断。孕前筛查可以改善生殖计划，尤其是受 DM1 影响的女性，她们的卵巢储备能力下降，体外受精-植入前基因诊断结果不佳。我们对来自 DM1 受影响/未受影响细胞系的 17 种 DNA 优化了三重引物 PCR 和熔解曲线分析。对 60 个基因型已知的临床样本进行了盲法测试。质粒构建体 pDMPK(CTG)35 和 pDMPK(CTG)48 用于建立将 DM1 受影响的样品与未受影响的样品分开的阈值温度。筛选后三联引物 PCR 扩增子大小通过短周期标记引物延伸和毛细管电泳来实现。三重引物 PCR 熔解曲线分析未受影响和 DM1 影响样品的熔解峰温度分别低于和高于对照质粒的熔解峰温度。熔解后毛细管电泳分析扩增子的曲线与筛选结果完全一致。三重引物 PCR 熔解曲线分析是一种简单且经济高效的筛选工具，可用于快速鉴定 DM1。配套的确认协议允许在需要时快速确定 CTG 重复大小。该策略避免了对所有测试样本进行毛细管电泳大小测定的需要，从而将毛细管电泳分析仅限于筛选阳性的病例子集。
Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell Lines. A blinded test was performed on 60 genotype-known clinical samples. Plasmid constructs pDMPK(CTG)35 and pDMPK(CTG)48 were used to establish threshold temperatures separating DM1-affected from unaffected samples. Post-screen triplet-primed PCR amplicon sizing was achieved by short-cycle labeled-primer extension followed by capillary electrophoresis. Triplet-primed PCR melting curve analysis meLt peak temperatures of unaffected and DM1-affected samples were lower and higher than the control plasmids' melt peak temperatures, respectively. Capillary electrophoresis of post-melting curve analysis amplicons was completely concordant with the screening results. Triplet-primed PCR melting curve analysis is a simple and cost-effective screening tool for rapid identification of DM1. The companion confirmation protocol allows quick determination of CTG repeat size when required. This strategy avoids the need to perform capillary electrophoresis sizing on all test samples, Limiting capillary electrophoresis analysis to only a subset of cases that are screen-positive.