Lian Jiabian, Liang Yaoji, Wang Yunling, Chen Ying, Li Xun, Xia Lu
Center for Precision Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Department of Laboratory Medicine, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China; Xiamen Cell Therapy Research Center, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, 361003, China.
Biochee Biotech Co.,Ltd., Xiamen, 361102, China; Amogene Biotech Co.,Ltd., Xiamen, 361102, China.
Clin Chim Acta. 2024 Jul 15;561:119761. doi: 10.1016/j.cca.2024.119761. Epub 2024 Jun 5.
Determination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes.
Following protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/28, 4 with UGT1A1 28/28, 22 with UGT1A16 G/G, 6 with UGT1A16 G/A, 4 with UGT1A16 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination.
The new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/μL of input genomic DNA.
Our developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.
在5-氟尿嘧啶和伊立替康治疗前确定DPYD和UGT1A1基因多态性对于避免严重的药物不良反应至关重要。因此，迫切需要针对最常见的DPYD和UGT1A1基因多态性的准确可靠的基因分型方法。在本研究中，我们介绍了一种用于区分DPYD基因c.1236G>A、c.1679T>G、c.2846A>T、IVS14+1G>A以及UGT1A1*1、*28、*6（G71R）基因型的新型聚合酶链反应（PCR）熔解曲线分析方法。
在优化方案后，采用该技术对厦门大学附属第一医院2023年3月至2023年10月招募的28例患者进行基因分型。这些患者包括20例UGT1A11/1、8例UGT1A11/28、4例UGT1A128/28、22例UGTIA16 G/G、6例UGTIA16 G/A、4例UGTIA1*6 A/A、27例DPYD（c.1236）G/G、3例DPYD（c.1236）G/A、2例DPYD（c.1236）A/A、27例DPYD（c.1679）T/T、2例DPYD（c.1679）T/G、3例DPYD（c.1679）G/G、28例DPYD（c.2846A/T）A/A、2例DPYD（c.2846A/T）A/T、2例DPYD（c.2846A/T）T/T、28例DPYD（c.IVS14+1）G/G、2例DPYD（c.IVS14+1）G/A以及2例DPYD（c.IVS14+1）A/A，以及3种质粒标准品。通过将结果与桑格测序或多重定量PCR（qPCR）的结果进行比较来评估方法的准确性。计算熔解温度（Tm）的批内和批间精密度以评估可靠性，并通过检测限检查评估灵敏度。
新方法准确鉴定了所有基因型，并且比多重qPCR具有更高的准确性。Tm的批内和批间变异系数均≤1.97%，标准差≤0.95°C。检测限为输入基因组DNA 0.09 ng/μL。
我们开发的PCR熔解曲线分析能够准确、可靠、快速、简单且经济高效地检测DPYD和UGT1A1基因多态性。其应用可轻松扩展至配备荧光PCR平台的临床实验室。
