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bioRxiv · 10.64898/2026.08.19.745862

Multiplexed Quantification of Variant Abundance in the Globin Gene Family: Integrating Saturation Mutagenesis with Cross-Paralog Prediction

Cai, X.·Wang, D.·Hu, J.·Huang, Y.·Guo, W.·Shi, Y.·Zhou, Y.·Xiao, C.·Ye, Y.·Wang, C.·Zhou, W.·Xu, X.·Jia, X.
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Abstract

Widespread genetic testing has expanded variant identification, yet functional characterization remains a bottleneck in genome guided medicine. Here, we present a modified Variant Abundance by Massively Parallel Sequencing (VAMP-seq) platform integrating experimental and computational approaches for high-resolution abundance profiling of protein variants. Utilizing a lentiviral integration system, we systematically assessed the stability effects of 2,696 amino acid substitutions in {zeta}-globin (HBZ) via saturation mutagenesis in human cells, achieving complete variant coverage with high reproducibility. Representative variants showed strong concordance with orthogonal low-throughput validation assays. We further developed a deep learning framework leveraging VAMP-seq derived HBZ data to predict variant abundance across thalassemia-associated globin paralogs (HBA, HBB, and HBG1) not experimentally tractable. Our hybrid framework demonstrates how targeted experimental profiling combined with AI-driven extrapolation can accelerate variant interpretation across protein family members.

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Cai, X., Wang, D., Hu, J., Huang, Y., Guo, W., Shi, Y., Zhou, Y., Xiao, C., Ye, Y., Wang, C., Zhou, W., Xu, X., Jia, X.. 2026-08-24. Multiplexed Quantification of Variant Abundance in the Globin Gene Family: Integrating Saturation Mutagenesis with Cross-Paralog Prediction. https://doi.org/10.64898/2026.08.19.745862

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