Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, People's Republic of China.
Department of Mathematics, Utah Valley University, Orem, UT, USA.
BMC Genomics. 2018 Jun 20;19(1):485. doi: 10.1186/s12864-018-4833-4.
Copy number variation (CNV) has been implicated in the genetics of multiple human diseases. Spinal muscular atrophy (SMA) and 22q11.2 deletion syndrome (22q11.2DS) are two of the most common diseases which are caused by DNA copy number variations. Genetic diagnostics for these conditions would be enhanced by more accurate and efficient methods to detect the relevant CNVs.
Competitive PCR with limited deoxynucleotide triphosphates (dNTPs) and high-resolution melting (HRM) analysis was used to detect 22q11.2DS, SMA and SMA carrier status. For SMA, we focused on the copy number of SMN1 gene. For 22q11.2DS, we analyzed CNV for 3 genes (CLTCL1, KLHL22, and PI4KA) which are located between different region-specific low copy repeats. CFTR was used as internal reference gene for all targets. Short PCR products with separated Tms were designed by uMelt software.
One hundred three clinical patient samples were pretested for possible SMN1 CNV, including carrier status, using multiplex ligation-dependent probe amplification (MLPA) commercial kit as gold standard. Ninety-nine samples consisting of 56 wild-type and 43 22q11.2DS samples were analyzed for CLTCL1, KLHL22, and PI4KA CNV also using MLPA. These samples were blinded and re-analyzed for the same CNVs using the limited dNTPs PCR with HRM analysis and the results were completely consistent with MLPA.
Limited dNTPs PCR with HRM analysis is an accurate method for detecting SMN1 and 22q11.2 CNVs. This method can be used quickly, reliably, and economically in large population screening for these diseases.
拷贝数变异（CNV）与多种人类疾病的遗传有关。脊髓性肌萎缩症（SMA）和 22q11.2 缺失综合征（22q11.2DS）是由 DNA 拷贝数变异引起的两种最常见的疾病。通过更准确和有效的方法来检测相关的 CNV，可以增强对这些病症的遗传诊断。
采用竞争性聚合酶链反应（PCR）结合有限脱氧核苷酸三磷酸（dNTP）和高分辨率熔解（HRM）分析，用于检测 22q11.2DS、SMA 和 SMA 携带者状态。对于 SMA，我们主要关注 SMN1 基因的拷贝数。对于 22q11.2DS，我们分析了位于不同区域特异性低拷贝重复之间的 3 个基因（CLTCL1、KLHL22 和 PI4KA）的 CNV。CFTR 被用作所有靶标内部参考基因。通过 uMelt 软件设计了具有分离 Tm 的短 PCR 产物。
使用多重连接依赖性探针扩增（MLPA）商业试剂盒作为金标准，对 103 例临床患者样本进行了可能的 SMN1 CNV 检测，包括携带者状态。使用 MLPA 对 99 例样本（包括 56 例野生型和 43 例 22q11.2DS 样本）进行了 CLTCL1、KLHL22 和 PI4KA CNV 分析。这些样本是盲样，并用有限 dNTPs PCR 结合 HRM 分析重新分析了相同的 CNV，结果与 MLPA 完全一致。
有限 dNTPs PCR 结合 HRM 分析是一种准确的检测 SMN1 和 22q11.2 CNV 的方法。该方法可用于快速、可靠和经济地对这些疾病进行大规模人群筛查。
