High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

BACKGROUND: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. METHODS: In this study, we leveraged the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein-coding KCNE1 variants. RESULTS: We comprehensively assayed KCNE1 variant cell surface expression (2554/2709 possible single-amino-acid variants) and function (2534 variants). Our study identified 470 loss- or partial loss-of-surface expression and 574 loss- or partial loss-of-function variants. Of the 574 loss- or partial loss-of-function variants, 152 (26.5%) had reduced cell surface expression, indicating that most functionally deleterious variants affect channel gating. Nonsense variants at residues 56-104 generally had WT-like trafficking scores but decreased functional scores, indicating that the latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation (with > 70% loss-of-function variants) were in predicted close contact with binding partners KCNQ1 or calmodulin. Our functional assay data were consistent with gold standard electrophysiological data (ρ = - 0.64), population and patient cohorts (32/38 presumed benign or pathogenic variants with consistent scores), and computational predictors (ρ = - 0.62). Our data provide moderate-strength evidence for the American College of Medical Genetics/Association of Molecular Pathology functional criteria for benign and pathogenic variants. CONCLUSIONS: Comprehensive variant effect maps of KCNE1 can both provide insight into I Ks channel biology and help reclassify variants of uncertain significance.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

Defining and Reducing Variant Classification Disparities.

Background: Multiplexed Assays of Variant Effects (MAVEs) can test all possible single variants in a gene of interest. The resulting saturation-style data may help resolve variant classification disparities between populations, especially for variants of uncertain significance (VUS). Methods: We analyzed clinical significance classifications in 213,663 individuals of European-like genetic ancestry versus 206,975 individuals of non-European-like genetic ancestry from All of Us and the Genome Aggregation Database. Then, we incorporated clinically calibrated MAVE data into the Clinical Genome Resource's Variant Curation Expert Panel rules to automate VUS reclassification for BRCA1, TP53, and PTEN . Results: Using two orthogonal statistical approaches, we show a higher prevalence ( p ≤5.95e-06) of VUS in individuals of non-European-like genetic ancestry across all medical specialties assessed in all three databases. Further, in the non-European-like genetic ancestry group, higher rates of Benign or Likely Benign and variants with no clinical designation ( p ≤2.5e-05) were found across many medical specialties, whereas Pathogenic or Likely Pathogenic assignments were higher in individuals of European-like genetic ancestry ( p ≤2.5e-05). Using MAVE data, we reclassified VUS in individuals of non-European-like genetic ancestry at a significantly higher rate in comparison to reclassified VUS from European-like genetic ancestry ( p =9.1e-03) effectively compensating for the VUS disparity. Further, essential code analysis showed equitable impact of MAVE evidence codes but inequitable impact of allele frequency ( p =7.47e-06) and computational predictor ( p =6.92e-05) evidence codes for individuals of non-European-like genetic ancestry. Conclusions: Generation of saturation-style MAVE data should be a priority to reduce VUS disparities and produce equitable training data for future computational predictors.

Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq.

Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of ~20,000 variant effects for ~1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRaf's signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.

Pacybara: accurate long-read sequencing for barcoded mutagenized allelic libraries.

MOTIVATION: Long-read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some applications, e.g. sequencing mutagenized libraries where multiple distinct clones differ by one or few variants, require the use of barcodes or unique molecular identifiers. Unfortunately, sequencing errors can interfere with correct barcode identification, and a given barcode sequence may be linked to multiple independent clones within a given library. RESULTS: Here we focus on the target application of sequencing mutagenized libraries in the context of multiplexed assays of variant effects (MAVEs). MAVEs are increasingly used to create comprehensive genotype-phenotype maps that can aid clinical variant interpretation. Many MAVE methods use long-read sequencing of barcoded mutant libraries for accurate association of barcode with genotype. Existing long-read sequencing pipelines do not account for inaccurate sequencing or nonunique barcodes. Here, we describe Pacybara, which handles these issues by clustering long reads based on the similarities of (error-prone) barcodes while also detecting barcodes that have been associated with multiple genotypes. Pacybara also detects recombinant (chimeric) clones and reduces false positive indel calls. In three example applications, we show that Pacybara identifies and correctly resolves these issues. AVAILABILITY AND IMPLEMENTATION: Pacybara, freely available at https://github.com/rothlab/pacybara, is implemented using R, Python, and bash for Linux. It runs on GNU/Linux HPC clusters via Slurm, PBS, or GridEngine schedulers. A single-machine simplex version is also available.

A mutational atlas for Parkin proteostasis.

Proteostasis can be disturbed by mutations affecting folding and stability of the encoded protein. An example is the ubiquitin ligase Parkin, where gene variants result in autosomal recessive Parkinsonism. To uncover the pathological mechanism and provide comprehensive genotype-phenotype information, variant abundance by massively parallel sequencing (VAMP-seq) is leveraged to quantify the abundance of Parkin variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, shows that most low abundance variants are proteasome targets and are located within the structured domains of the protein. Half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) reveals an exposed degron region proximal to the so-called "activation element". This work provides examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation.

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes.

BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encodes the sarcomeric protein (ß-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that ß-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of ß-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of ß-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

Will variants of uncertain significance still exist in 2030?

In 2020, the National Human Genome Research Institute (NHGRI) made ten "bold predictions," including that "the clinical relevance of all encountered genomic variants will be readily predictable, rendering the diagnostic designation 'variant of uncertain significance (VUS)' obsolete." We discuss the prospects for this prediction, arguing that many, if not most, VUS in coding regions will be resolved by 2030. We outline a confluence of recent changes making this possible, especially advances in the standards for variant classification that better leverage diverse types of evidence, improvements in computational variant effect predictor performance, scalable multiplexed assays of variant effect capable of saturating the genome, and data-sharing efforts that will maximize the information gained from each new individual sequenced and variant interpreted. We suggest that clinicians and researchers can realize a future where VUSs have largely been eliminated, in line with the NHGRI's bold prediction. The length of time taken to reach this future, and thus whether we are able to achieve the goal of largely eliminating VUSs by 2030, is largely a consequence of the choices made now and in the next few years. We believe that investing in eliminating VUSs is worthwhile, since their predominance remains one of the biggest challenges to precision genomic medicine.

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts.

Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.

Genetically encoded fluorescent indicators (GEFIs) are protein-based optogenetic tools that change their fluorescence intensity when binding specific ligands in cells and tissues. GEFI encoding DNA can be expressed in cell subtypes while monitoring cellular physiological responses. However, engineering GEFIs with physiological sensitivity and pharmacological specificity often requires iterative optimization through trial-and-error mutagenesis while assessing their biophysical function in vitro one by one. Here, the vast mutational landscape of proteins constitutes a significant obstacle that slows GEFI development, particularly for sensors that rely on mammalian host systems for testing. To overcome these obstacles, we developed a multiplexed high-throughput engineering platform called the optogenetic microwell array screening system (Opto-MASS) that functionally tests thousands of GEFI variants in parallel in mammalian cells. Opto-MASS represents the next step for engineering optogenetic tools as it can screen large variant libraries orders of magnitude faster than current methods. We showcase this system by testing over 13,000 dopamine and 21,000 opioid sensor variants. We generated a new dopamine sensor, dMASS1, with a >6-fold signal increase to 100 nM dopamine exposure compared to its parent construct. Our new opioid sensor, µMASS1, has a ∼4.6-fold signal increase over its parent scaffold's response to 500 nM DAMGO. Thus, Opto-MASS can rapidly engineer new sensors while significantly shortening the optimization time for new sensors with distinct biophysical properties.

An Atlas of Variant Effects to understand the genome at nucleotide resolution.

Sequencing has revealed hundreds of millions of human genetic variants, and continued efforts will only add to this variant avalanche. Insufficient information exists to interpret the effects of most variants, limiting opportunities for precision medicine and comprehension of genome function. A solution lies in experimental assessment of the functional effect of variants, which can reveal their biological and clinical impact. However, variant effect assays have generally been undertaken reactively for individual variants only after and, in most cases long after, their first observation. Now, multiplexed assays of variant effect can characterise massive numbers of variants simultaneously, yielding variant effect maps that reveal the function of every possible single nucleotide change in a gene or regulatory element. Generating maps for every protein encoding gene and regulatory element in the human genome would create an 'Atlas' of variant effect maps and transform our understanding of genetics and usher in a new era of nucleotide-resolution functional knowledge of the genome. An Atlas would reveal the fundamental biology of the human genome, inform human evolution, empower the development and use of therapeutics and maximize the utility of genomics for diagnosing and treating disease. The Atlas of Variant Effects Alliance is an international collaborative group comprising hundreds of researchers, technologists and clinicians dedicated to realising an Atlas of Variant Effects to help deliver on the promise of genomics.

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and ß1-ß2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and ß1-ß2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.

Machine vision reveals micronucleus rupture as a potential driver of the transcriptomic response to aneuploidy.

Micronuclei are aberrant nuclear compartments that trap a portion of a cell's chromatin in a distinct organelle separate from the nucleus and are drivers of inflammation, DNA damage, chromosome instability, and chromothripsis. Many of the consequences of micronucleus formation stem from micronucleus rupture: the sudden loss of micronucleus compartmentalization, resulting in mislocalization of nuclear factors and the exposure of chromatin to the cytosol for the remainder of interphase. Micronuclei form primarily from segregation errors during mitosis, errors that also give rise to other, non-exclusive phenotypes, including aneuploidy and chromatin bridges. The stochastic formation of micronuclei and phenotypic overlap confounds the use of population-level assays or hypothesis discovery, requiring labor-intensive techniques to visually identify and follow micronucleated cells individually. In this study, we present a novel technique for automatically identifying and isolating micronucleated cells generally and cells with ruptured micronuclei specifically using a de novo neural net combined with Visual Cell Sorting. As a proof of concept, we compare the early transcriptomic responses to micronucleation and micronucleus rupture with previously published responses to aneuploidy, revealing micronucleus rupture to be a potential driver of the aneuploidy response.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

Background: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. Results: Here, we demonstrate the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein coding KCNE1 variants. We comprehensively assayed KCNE1 variant cell surface expression (2,554/2,709 possible single amino acid variants) and function (2,539 variants). We identified 470 loss-of-surface expression and 588 loss-of-function variants. Out of the 588 loss-of-function variants, only 155 had low cell surface expression. The latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were highly concordant with gold standard electrophysiological data (ρ = -0.65), population and patient cohorts (32/38 concordant variants), and computational metrics (ρ = -0.55). Our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. Conclusions: Comprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs.

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different variants. We differentiated these hiPSCs to cardiomyocytes and show MHC-ß fusion proteins can localize as expected. Additionally, single-cell contractility analyses revealed cardiomyocytes with a pathogenic, hypertrophic cardiomyopathy-associated MYH7 variant exhibit salient HCM physiology relative to isogenic controls. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of functional transgenic cell lines at unprecedented scale.

Pacybara: Accurate long-read sequencing for barcoded mutagenized allelic libraries.

Long read sequencing technologies, an attractive solution for many applications, often suffer from higher error rates. Alignment of multiple reads can improve base-calling accuracy, but some applications, e.g. sequencing mutagenized libraries where multiple distinct clones differ by one or few variants, require the use of barcodes or unique molecular identifiers. Unfortunately, sequencing errors can interfere with correct barcode identification, and a given barcode sequence may be linked to multiple independent clones within a given library. Here we focus on the target application of sequencing mutagenized libraries in the context of multiplexed assays of variant effects (MAVEs). MAVEs are increasingly used to create comprehensive genotype-phenotype maps that can aid clinical variant interpretation. Many MAVE methods use long-read sequencing of barcoded mutant libraries for accurate association of barcode with genotype. Existing long-read sequencing pipelines do not account for inaccurate sequencing or non-unique barcodes. Here, we describe Pacybara, which handles these issues by clustering long reads based on the similarities of (error-prone) barcodes while also detecting barcodes that have been associated with multiple genotypes. Pacybara also detects recombinant (chimeric) clones and reduces false positive indel calls. In three example applications, we show that Pacybara identifies and correctly resolves these issues.

A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.

CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs.

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CR ISPR a On- T arget E diting R etrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) in MYH7 , a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different MYH7 SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.

Measuring Pharmacogene Variant Function at Scale Using Multiplexed Assays.

As costs of next-generation sequencing decrease, identification of genetic variants has far outpaced our ability to understand their functional consequences. This lack of understanding is a central challenge to a key promise of pharmacogenomics: using genetic information to guide drug selection and dosing. Recently developed multiplexed assays of variant effect enable experimental measurement of the function of thousands of variants simultaneously. Here, we describe multiplexed assays that have been performed on nearly 25,000 variants in eight key pharmacogenes (ADRB2, CYP2C9, CYP2C19, NUDT15, SLCO1B1, TMPT, VKORC1, and the LDLR promoter), discuss advances in experimental design, and explore key challenges that must be overcome to maximize the utility of multiplexed functional data.