用于家族性高胆固醇血症基因筛查的基于多重 PCR 的下一代扩增片段测序快速可扩展方法。

IF 1.8 Q3 MEDICAL LABORATORY TECHNOLOGY
Mohamed Imran, V R Arvinden, Pabithadevi Balaiah Mehanathan, Raskin Erusan Rajagopal, Suriya Prabha Muthu, Arul Subbiah Arunachalam, Rahul C Bhoyar, Harie Vignesh, Samya Mitra, Ganga Nath Jha, Aayush Gupta, Manoj Kumar, Rohit Bhowmick, Niladri Sekhar Bhunia, Atanu Kumar Dutta, Vinod Scaria, Sridhar Sivasubbu
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引用次数: 0

摘要

背景:家族性高胆固醇血症(FH家族性高胆固醇血症(FH)是一种经常被漏诊的遗传性疾病,其特点是低密度脂蛋白(LDL)水平升高。对 LDLR、APOB 和 PCSK9 基因的基因检测可鉴定出高达 80% 临床诊断患者的变异基因。然而,时间、可扩展性和成本方面的限制阻碍了对这些基因进行有效的新一代测序。此外,药物基因组变异与 FH 患者他汀类药物引起的不良反应有关。为了应对这些挑战,我们开发了一种基于多重引物的扩增子测序方法,用于 FH 基因检测:方法:针对 LDLR、APOB 和 PCSK9 基因的外显子以及药物基因组变异 rs4149056(SLCO1B1:c.521T > A)、rs2306283(SLCO1B1:c.388A > G)和 rs2231142(ABCG2:c.421C > A)设计了多重引物。利用已知致病变异样本进行了分析验证,并对 12 名疑似 FH 患者进行了临床验证。文库制备采用基于珠标记的方法,测序在 NovaSeq 6000 平台上进行:结果:我们的方法确保了无扩增片段丢失,每个扩增片段的覆盖率超过 100 倍。成功检测到 2 个样本中的已知变异。此外,我们还在患者中发现了一个杂合型 LDLR(p.Glu228Ter)变异体和两个同源型 LDLR(p.Lys294Ter)和 LDLR(p.Ser177Leu)变异体。药物基因组学分析显示,共有 3 名患者可能需要减少他汀类药物的剂量。我们的方法缩短了文库制备时间(约3小时),提高了可扩展性,降低了FH基因检测的成本(低于50美元):我们的方法能有效地对 LDLR、APOB 和 PCSK9 基因(包括药物基因组变异)进行测序,为适当的筛查和他汀类药物剂量提供指导,从而提高了效率和经济性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening.

Background: Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing.

Methods: Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform.

Results: Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing.

Conclusions: Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.

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来源期刊
Journal of Applied Laboratory Medicine
Journal of Applied Laboratory Medicine MEDICAL LABORATORY TECHNOLOGY-
CiteScore
3.70
自引率
5.00%
发文量
137
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