{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T18:37:30Z","timestamp":1793039850604,"version":"4.5.0"},"reference-count":63,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"17","content-domain":{"domain":["www.ahajournals.org"],"crossmark-restriction":true},"short-container-title":["Circulation"],"published-print":{"date-parts":[[2026,10,27]]},"abstract":"<jats:sec>\n                    <jats:title>BACKGROUND:<\/jats:title>\n                    <jats:p>\n                      Inherited variants in the LDL (low-density lipoprotein) receptor (\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      ) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>METHODS:<\/jats:title>\n                    <jats:p>\n                      We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>RESULTS:<\/jats:title>\n                    <jats:p>\n                      We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the American College of Medical Genetics and Genomics\/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>CONCLUSIONS:<\/jats:title>\n                    <jats:p>\n                      Altogether, activity-normalized prime editing provides a scalable framework for\n                      <jats:italic toggle=\"yes\">LDLR<\/jats:italic>\n                      variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.\n                    <\/jats:p>\n                  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