| Title: | Functional data for LDLR variant classification: comparative insights from high-content microscopy and flow cytometry assays |
| Journal: | Atherosclerosis |
| Published: | 8 Oct 2026 |
| Pubmed: | https://pubmed.ncbi.nlm.nih.gov/42868824/ |
| DOI: | https://doi.org/10.1016/j.atherosclerosis.2026.121921 |
Publication 21577
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Abstract
BACKGROUND AND AIMS: FH VCEP specifications for familial hypercholesterolemia variant interpretation assign a higher evidence weight to functional data from flow cytometry than microscopy assays. This restricts the use of microscopy assays for LDLR variant classification. We aimed to compare functional data obtained by both assays to determine whether microscopy-based assays could support a higher evidence level.</p>
METHODS: Fifty LDLR variants with available flow cytometry and microscopy data were compared for LDL uptake activity, including 21 newly characterized variants by microscopy. Variants were grouped by FH VCEP functional thresholds and results integrated with UK Biobank data to assess associations with lipid traits.</p>
RESULTS: We validated our microscopy assay with FH VCEP-classified control variants and compared functional activities measured by microscopy and flow cytometry assays, which showed significant correlation (r = 0.66, 95% CI: 0.473-0.795, p = 1.45 × 10-7) and a close average agreement (Bland-Altman bias = -0.05, 95% LoA: -0.69 to +0.60). Functional FH VCEP variant groups for both assays resulted in fair categorical agreement (Cohen's κ = 0.26, 95% CI: 0.018-0.479). 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.</p>
CONCLUSION: Microscopy assays show concordance with flow cytometry, consistent associations with lipid phenotypes and our OddsPath calculations support re-evaluation of evidence weights for microscopy assays.</p>
6 Authors
- Mohammad Majharul Islam
- Ana Catarina Alves
- Rafael Graça
- Joana Rita Chora
- Mafalda Bourbon
- Simon G Pfisterer
1 Application
| Application ID | Title |
|---|---|
| 87446 | Improving rare-variant association studies with functional cell data |
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