A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening

Abstract

Abstract 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.