Defining the compendium of pathogenic germline alleles in cancer susceptibility genes via multiplex assays of variant effect (CG-MAVE)

▪Background: There are >100 cancer susceptibility genes (CSGs) for which testing is currently offered within the NHS Genomic Medicine Service.

Whilst next-generation sequencing has enabled dramatic expansion in the size of panels analysed as well as the number of patients we can test, this has resulted in an increase in the rate of detection of variants of uncertain significance (VUS), particularly in non-European patients.

Thus, as illustrated recently by the landmark Findlay et al multiplex assay of variant effect (MAVE) for BRCA1, pre-generated robust functional data for every possible variant that might be detected represents a hugely powerful aid to variant interpretation.

The overarching hypothesis of the studies proposed in this grant is that we can deploy at massive scale saturation genome editing (SGE) in HAP1 and other cell systems to analyse the functional impact of every nucleotide change in each gene’s coding region, thus creating a comprehensive atlas of variant function.

Aim (1): To deliver at massively high throughput the established HAP1 SGE cell survival assay for at least eight major cancer susceptibility genes.

Aim (2): To develop and validate three novel SGE assays addressing seven additional cancer susceptibility genes, which, if successful will then be moved through to the high-throughput production phase.

Aim (3): To(a) assemble extensive clinical and population genomic datasets for MAVE validation, (b) develop international consensus analytical and validation standards for MAVE data (c) develop UK NHS MAVE clinical implementation frameworks (d) digital platforms for MAVE data dissemination. ▪Methods: The SGE method comprises CRISPR-mediated gene-editing, generation of libraries containing hundreds of single nucleotide variants (SNVs) in pooled experiments and quantitation of variant abundance using DNA sequencing at multiple timepoints to track effects on cell survival.

In addition to HAP1 survival, we shall develop new methods including a 6-thioguanine (6-TG) selection assay and engineering of new monoallelic cell lines. ▪Outputs: We shall generate complete nucleotide-resolution functional maps for at least 15 of the most frequently analysed cancer susceptibility genes.

Via methodology and NHS implementation frameworks developed in Aim 3, these functional scores will be directly implemented within the NHS Genomic Medicine service to enable interpretation of clinically-detected VUSs in CSGs.

This will facilitate identification of more individuals at elevated genetic risk of cancer who can benefit from interventions for cancer prevention and early detection.

As an exemplar project, we shall assemble sets of VUS clinically-detected in non-European patients for priority post-MAVE review and variant interpretation.