Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain
- James L. Shepherdson1,2,
- David M. Granas1,2,
- Jie Li1,2,
- Zara Shariff1,2,
- Stephen P. Plassmeyer3,4,
- Alex S. Holehouse3,4,
- Michael A. White1,2 and
- Barak A. Cohen1,2
- 1Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA;
- 2Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA;
- 3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA;
- 4Center for Biomolecular Condensates, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA
Abstract
The transcription factor (TF) cone-rod homeobox (CRX) is essential for the differentiation and maintenance of photoreceptor cell identity. Several human CRX variants cause degenerative retinopathies, but most are variants of uncertain significance. We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitutions in CRX using a cell-based transcriptional reporter assay, curating a high-confidence list of nearly 2000 variants with altered transcriptional activity. In the structured homeodomain, activity scores closely aligned to a predicted structure and demonstrated position-specific constraints on amino acid substitution. In contrast, the intrinsically disordered transcriptional effector domain displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. These compositional constraints were consistent with the acidic exposure model of transcriptional activation. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, identifying pathogenic variants with high specificity and moderate sensitivity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate utility for integration into the clinical variant classification pipeline.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279415.124.
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Freely available online through the Genome Research Open Access option.
- Received March 29, 2024.
- Accepted September 11, 2024.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
