Functional data for LDLR variant classification: comparative insights from high-content microscopy and flow cytometry assays

Background and aimsCurrent FH VCEP specifications of ACMG/AMP guidelines for familial hypercholesterolemia (FH) variant interpretation assign a higher evidence weight to functional data obtained with flow cytometry than microscopy assays, due to lack of existing evidence. This restricts the use of microscopy-derived functional data for variant classification. We aimed to systematically compare functional data of LDLR variants obtained by high-content microscopy and flow cytometry to determine their concordance and assess whether microscopy-based assays could support a higher evidence level. MethodsFifty LDLR variants with available flow cytometry and high-content microscopy data were compared for LDL uptake activity, including 21 newly characterized variants by microscopy in this study. Variants were grouped by FH VCEP functional thresholds (<70% activity, abnormal function; >90% activity, normal function) and results were integrated with UK Biobank data to assess associations with lipid traits. ResultsFirst, we validated our scalable microscopy assay with FH VCEP-classified control variants. Then we compared functional activity measured by microscopy and flow cytometry assays for 50 variants, which showed significant correlation (r = 0.66, p<0.0001) and a close average agreement (Bland-Altman bias = -0.05). Applying FH VCEP functional classification thresholds yielded broadly consistent classification in both methods, with minor shifts among categories. Integration with UK Biobank data showed that carriers of variants with reduced LDLR activity (<70% and <50%) had higher LDL-C, total cholesterol and ApoB levels compared to those with normal activity (>90%) for both microscopy and flow cytometry assays, with more pronounced differences observed at the <50% LDLR activity threshold. ConclusionHigh-content microscopy provides reliable and scalable measurements of LDLR function, showing high concordance with flow cytometry and consistent associations with lipid phenotypes. These findings support reconsideration of the evidence weight assigned to validated microscopy assays within FH VCEP variant classification frameworks, namely to Strong (Level 1).