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

Area of Science:

• Genetics
• Molecular Biology
• Cardiovascular Disease

Background:

• Familial Hypercholesterolemia (FH) is an underdiagnosed genetic disorder causing high LDL cholesterol.
• Genetic testing for LDLR, APOB, and PCSK9 genes identifies variants but faces limitations in current sequencing methods.
• Pharmacogenomic variants impact statin response in FH patients, necessitating tailored treatment.

Purpose of the Study:

• To develop a cost-effective and scalable multiplex primer-based amplicon sequencing approach for FH genetic testing.
• To simultaneously analyze FH-causative genes (LDLR, APOB, PCSK9) and key pharmacogenomic variants.
• To improve the efficiency and affordability of genetic diagnostics for FH.

Main Methods:

• Designed multiplex primers for LDLR, APOB, PCSK9 exons, and pharmacogenomic variants (SLCO1B1, ABCG2).
• Employed bead-based tagmentation for library preparation and NovaSeq 6000 for sequencing.
• Conducted analytical and clinical validation using known samples and 12 FH-suspected probands.

Main Results:

• Achieved high coverage (>100×) with no amplicon dropouts.
• Successfully detected known variants and identified novel pathogenic variants in LDLR.
• Identified 3 patients potentially requiring adjusted statin doses based on pharmacogenomic findings.
• Demonstrated reduced library preparation time (~3 hours) and cost (<$50).

Conclusions:

• The developed method efficiently sequences key FH genes and pharmacogenomic variants.
• This approach enhances the efficiency and affordability of FH genetic testing.
• Findings will guide appropriate FH screening and personalized statin dosing strategies.