LDLR variant classification through activity-normalized prime editing screening.

Area of Science:

• Genetics
• Molecular Biology
• Cardiovascular Disease Research

Background:

• Inherited variants in the LDL receptor (LDLR) gene are a primary cause of familial hypercholesterolemia (FH), increasing coronary artery disease (CAD) risk.
• Many LDLR variants lack clear clinical classifications, hindering early intervention and effective patient management.

Purpose of the Study:

• To develop and validate an activity-normalized prime editing screening pipeline to assess the functional impact of thousands of LDLR coding variants.
• To improve the classification of LDLR variants and enhance understanding of their role in FH and CAD.

Main Methods:

• An innovative prime editing screening pipeline was developed, incorporating a genotypic outcome reporter for activity normalization across 5,184 LDLR coding variants.
• A statistical approach was used to denoise variant scores by leveraging data from all missense variants at a given position.
• Phenotypic measurements of LDL-cholesterol (LDL-C) uptake were adjusted for variable editing efficiency.

Main Results:

• The prime editing assay successfully separated pathogenic from benign ClinVar LDLR variants and showed concordance with UK Biobank LDL-C data.
• A majority of previously unclassified rare LDLR variants were reclassified based on integrated evidence, including functional screening data.
• The study identified novel LDLR variants enhancing LDL-C uptake via increased interaction with apolipoprotein B, providing insights into LDLR-apolipoprotein binding.

Conclusions:

• The developed prime editing approach significantly enhances the understanding of LDLR variant function and its contribution to FH.
• This method offers a powerful tool for large-scale functional variant assessment, improving clinical variant interpretation and patient care strategies.