Scaling Strategies to Assess Pathogenicity of Variants of Uncertain Significance

PROJECT SUMMARY/ABSTRACT
Sequencing human genomes, especially those from individuals with Mendelian disorders, has yielded
discovery of thousands of new disease genes and has led to personalized treatments, improving the lives of
patients and their families. Unfortunately, less than half of this population is able to reap these benefits
because, despite continued advances in sequencing technology and data analysis, many patients remain
without a molecular diagnosis. A major barrier to increasing the diagnostic yield is the abundance of variants of
uncertain significance (VUS), and in particular missense VUS, which are found in more than half of all patients.
As more individuals are sequenced, the number and proportion of patients with missense VUS continues to
grow, much faster than the field is currently able to interpret the functional significance of these variants.
Therefore, it is critical to both scale up existing methods and develop new approaches to determine
the pathogenicity of these VUS in as many genes as possible to help patients and families in need.
One such method that offers hope for solving this VUS problem is Deep Mutational Scanning (DMS), which
uses cell-based assays to simultaneously test thousands of missense variants by changing each residue in a
protein of interest to all 19 other possible amino acids. However, it is currently very challenging to generate a
cell-based assay that reliably reports the activity of a given gene of interest. Fortunately, combining DMS with a
multiplexed fluorescence-based approach, SortSeq, gives an accurate assessment of deleteriousness and
pathogenicity for variants in the GLI2 gene, which operates in the Sonic Hedgehog (SHH) signaling pathway.
This proposed work will use this same well-validated assay to interrogate multiple genes involved in SHH
signaling in order to determine the feasibility of a pathway-based approach for efficiently conducting DMS in
many genes with the same molecular read-out. The scope of work is expanded to include a second pathway,
the retinal development pathway, including DMS of CRX and OTX2, and leverages identical assays for
paralogous families of transcription factors to further scale up the approach. This proposal also tests and
validates an innovative computational pipeline that includes de novo biophysical predictions to understand the
effects of variants in intrinsically disordered regions (IDRs), which is particularly synergistic with the focus on
transcription factors, which typically contain large stretches of IDRs.
Successful completion of these aims will establish multiple strategies for pathway-based and paralog-based
scaling that can be quickly and easily adapted across many disease-causing genes, resulting in improved
genome-based diagnostics and future therapeutics.

Grant Number: 1R21EY038987-01
NIH Institute/Center: NIH
Principal Investigator: Dustin Baldridge