Project description:Multiplexed assays of variant effect are powerful methods to profile the consequences of rare variants on gene expression and organismal fitness. Yet, few studies have integrated several multiplexed assays to map variant effects on gene expression in coding sequences. Here, we pioneered a multiplexed assay based on polysome profiling to measure variant effects on translation at scale, uncovering single-nucleotide variants that increase and decrease ribosome load. By combining high-throughput ribosome load data with multiplexed mRNA and protein abundance readouts, we mapped the cis-regulatory landscape of thousands of catechol-O-methyltransferase (COMT) variants from RNA to protein and found numerous coding variants that alter COMT expression. Finally, we trained machine learning models to map signatures of variant effects on COMT gene expression and uncovered both directional and divergent impacts across expression layers. Our analyses reveal expression phenotypes for thousands of variants in COMT and highlight variant effects on both single and multiple layers of expression. Our findings prompt future studies that integrate several multiplexed assays for the readout of gene expression
Project description:Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type - amino acid (AA) substitutions - despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate the first comprehensive atlas of diverse variant effects for a disease protein, amyloid beta (Aß) that aggregates in Alzheimer’s disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD variants and many mutations beyond substitutions that accelerate Aß aggregation. Truncations, substitutions, insertions, single- and multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are strongly enriched in the polar N-terminus of Aß. This first comparative atlas for any disease gene highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.
Project description:Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.
Project description: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.
Project description:Identifying molecular effects between herring and beef diet in Ldlr-/- mice The transcriptome of the three tissues, which are liver, muscle and adipose tissue, were used for indentify the influence of diet on metabolism. Ldlr-/- mice were fed with either a beef diet or a herring diet.
Project description:In this study, we used multiplexed DNA repair assays of variants in the BRCA1 carboxy-terminus to functionally characterize 2271 variants for homology-directed repair function (HDR) and 1427 variants for cisplatin resistance (CR). We found a high level of consistent results in the two multiplexed functional assays with non-functional variants located within regions of the BRCA1 protein necessary for its tumor suppression activity.
