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Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants (RNA-Seq)

ABSTRACT: Predicting the pathogenic effect of rare genetic variants is hampered by the limited cohort of diagnosed patients and the difficulty in evaluating the effect of existing and novel variants. To address this issue, we leveraged a protein Multiplexed Assay of Variants Effect (MAVE) to functionally assess the effect of ~2,300 missense variants of the TP63 gene, some of which cause autosomal dominant developmental disorders. The activity of each variant was measured using an optimized fibroblast-to-keratinocyte conversion protocol, and a subset of mutants was validated using a wide range of functional tests. To expand MAVEs to any disease-driving gene without a specific predefined assay, we developed SCRAMseq (Single Cell RNAseq Associated with MAVEs by sequencing), which can retrieve each variant and characterize the functional consequences at the single-cell level through full-length scRNAseq. The dataset generated and validated here reclassified hundreds of variants present in the general population and definitively classified a Variant of Unknown Significance as pathogenic in a patient with ectodermal dysplasia. This work provides a robust and easy-to-use workflow to dissect the effect of gene variants for any possible disease-driving gene.

ORGANISM(S): Homo sapiens

PROVIDER: GSE275994 | GEO | 2025/08/28

REPOSITORIES: GEO

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Project description:Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>5000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 99% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of HAP1 haploid cells endogenously targeted using a CRISPR/cas9 knockin approach. A total of 6959 SNVs were characterized for effects on cell survival. The assay was validated relative to nonsense and synonymous variants, ClinVar pathogenic/likely pathogenic and benign/likely benign variants and homology directed repair cell based DNA repair assay results, all of which showed >94% sensitivity and specificity. Variants were assigned posterior probabilities of pathogenicity using a VarCall two component Bayesian mixture model and were further grouped according to ACMG strength of evidence under the PS3/BS3 rule resulting in Benign Strong (n=5430), Benign Moderate (n=190), Benign Supporting (n=61), VUS (122), Pathogenic Strong (n=1021), Pathogenic Moderate (n=88), and Pathogenic Supporting (n=47). Breast cancer case-control association studies showed that pooled SNVs encoding functionally pathogenic missense variants were associated with increased risks of breast (odds ratio (OR)3.81, 95%CI: 2.88-5.07) and ovarian cancer (OR 5.93, 95%CI: 4.12-8.52). The functional data were also combined with other sources of information in the ClinGen BRCA1/2 VCEP ACMG/AMP-classification model. A total of 785 SNVs, including 261 missense SNVs, were classified as pathogenic or likely pathogenic, while 5566 SNVs, including 3786 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers.

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Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants (RNA-Seq)

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