Multiplexed Assays of Variant Effects (MAVE) of PALB2 single nucleotide variants in exon 10.

To validate a high-throughput screening data in human cells using Multiplexed Assays for Variant Effects (MAVE), we performed a high-throughput deep mutational scanning of single nucleotide changes in exon 10 encoding p.G1000 to p.I1037 of the WD40 domain of PALB2 using a cell survival assay in haploid human HAP1 cells. We obtained MAVE scores for 276 single-nucleotide variants, leading to 9 nonsense and 68 synonymous changes, as well as 199 amino acid substitutions. Both variant groups showed an asymmetric distribution that is skewed towards low MAVE scores of nonsense and damaging variants, respectively. These MAVE data included scores for 218 unique single-nucleotide variants, leading to 9 nonsense changes and 209 amino acid substitutions. We observed a good and significant correlation between the outcomes from the MAVE and high-throughput screens (n=179, r=-0,6439, p<0.0001), indicating concordance between the outcomes of high-throughput analysis of PALB2 variants in human and mouse cells. Saturation genome editing was used to introduce variants and the variant count on day 5 and day 20 were measured. The log2 ratio between D5 and D20 was used to measure the variant effects