Abstract Variants of uncertain significance constitute the vast majority of missense variants, hampering the utility of genetic testing, despite its remarkable impacts on clinical diagnosis and decision-making. Although the sequencing and interpretation of genetic variants have been drastically improved in recent years, only about 2% of novel missense variants are currently clinically actionable. Therefore, a robust and scalable pipeline is desperately needed to provide functional evidence critical for variant interpretation. In this work, we demonstrate Paracell, a deep learning-based phenotypic profiling pipeline that leverages subcellular segmentation, high-dimensional single-cell phenotyping, and machine learning to classify the functional impact of variants. Paracell captures heterogeneity in cellular phenotypes and protein colocalization between variants and their signaling partners, as well as subcellular markers, and enables analysis at a single-cell resolution. Using a classification model trained on single-cell features extracted by Paracell, we were able to accurately classify cells from loss-of-function (LoF) variants based on their impact. After aggregating cellular profiles on a variant level, our pipeline was able to distinguish LoF variants from functional ones with high sensitivity and specificity. Our work demonstrates that Paracell is a robust and scalable method that can sensitively detect differences in single-cell phenotypic profiles. The systematic application of this pipeline will provide valuable functional evidence for variant interpretation, enhancing their clinical utility and accelerating personalized cancer care. Citation Format: David L. Nguyen, Jesse T. Chao. Paracell: A high throughput, deep learning-based pipeline for single-cell phenotypic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3536.
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