Biology subjects

Fayer, S.

Publications and source records attributed to Fayer, S..

9 recordsLinked to original sources

bioRxiv2026

Gene- and domain-aware calibration increases the clinical utility of variant effect predictors

The utility of clinical genetic testing is limited because around 90% of missense variants in ClinVar remain of uncertain clinical significance. Variant effect predictors (VEPs) can score any missense variant, potentially empowering variant classification. Realizing this potential requires calibration to translate predictor scores into evidence. However, genome-wide calibration ignores predictor heterogeneity across genes, causing evidence misassignment. We developed an automated, data-adaptive framework that optimizes two complementary approaches: gene-specific calibration for genes with enough variants for calibration, and domain-aggregate calibration for other disease-associated genes, which groups variants from protein domains with similar predictor score distributions for calibration. Applied to three predictors across 2,769 genes, this framework assigned evidence to 10.6% more variants on average while generally improving evidence accuracy compared to genome-wide calibration. These calibrations and the resulting calibrated computational evidence are available through the PredictMD portal. Our framework substantially increases the clinical utility of VEPs for variant classification.

genetics↗

bioRxiv2026

A scalable approach to resolving variants of uncertain significance

Over 90% of missense variants across [~]4,000 disease-associated genes are variants of uncertain significance (VUS). Experimental variant effect measurements provide critical evidence about pathogenicity and inform disease biology, but most variants lack data and clinical translation has been limited. The Impact of Genomic Variation on Function Consortium generated experimental data for 62,215 variants across ten genes using multiplexed assays and 1,407 variants across 163 genes using arrayed assays, curated 193,139 additional community-generated variant effect measurements across 30 additional genes, and developed automated calibration methods for translating experimental data and variant effect predictions into clinical evidence. To reduce current VUS, we developed a scalable workflow using only experimental and predictive evidence, enabling reclassification of 75% of the 16,115 VUS in these genes as pathogenic or benign with <1% error. To minimize future VUS, we analyzed >90,000 unobserved variants; 62% had enough evidence to be "preclassified" as pathogenic or benign. We validated our data, evidence and classifications using All of Us and created interactive resources to enable clinical use of the calibrated data. Thus, for 40 genes, representing 1% of the clinical genome, we resolve most existing VUS and future variants, illustrating how systematic use of scalable evidence can empower genomic medicine.

genomics↗

bioRxiv2025

Functional evidence for G6PD variant classification from mutational scanning

G6PD deficiency is one of the most common enzyme deficiencies worldwide, and increases the likelihood of adverse reactions to certain drugs and foods. Identifying people at risk is challenging, since most are asymptomatic until they encounter a trigger. This is further complicated since over 60% of 1,559 known genetic variants in G6PD are variants of uncertain significance and thus cannot guide drug prescribing and dosing. To resolve which variants are clinically meaningful and avoid harm from adverse drug reactions, we conducted two high-throughput functional assays: one for G6PD activity, and one for abundance. We measured the function of 9,527 missense, nonsense, and synonymous G6PD variants. The patterns of variant effect on activity and abundance confirmed the importance of structural NADP+ for G6PD activity and abundance, and G6PD dimerization for G6PD activity. Based on the ability of our functional assay scores to accurately classify G6PD variants of known clinical effect, we generated evidence that 4,870 missense variants contribute to G6PD deficiency and 2,245 are unlikely to contribute to G6PD deficiency. Our data can be used to deepen our understanding of G6PD as a protein, and to close the gap in classification for variants of uncertain significance to improve implementation of genetic medicine for G6PD deficiency.

genetics↗

bioRxiv2025

Image-based, pooled phenotyping reveals multidimensional, disease-specific variant effects

Genetic variants often produce complex phenotypic effects that confound current assays and predictive models. We developed Variant in situ sequencing (VIS-seq), a pooled, image-based method that measures variant effects on molecular and cellular phenotypes in diverse cell types. Applying VIS-seq to [~]3,000 LMNA and PTEN variants yielded high-dimensional morphological profiles that captured variant-driven changes in protein abundance, localization, activity and cell architecture. We identified gain-of-function LMNA variants that reshape the nucleus and autism-associated PTEN variants that mislocalize. Morphological profiles predicted variant pathogenicity with near-perfect accuracy and distinguished autism-linked from tumor syndrome-linked PTEN variants. Most variants impacted a multidimensional continuum of phenotypes not recapitulated by any single functional readout. By linking protein variation to cell images at scale, we illuminate how variant effects cascade from molecular to subcellular to cell morphological phenotypes, providing a framework for resolving the complexity of variant function.

genomics↗

bioRxiv2025

Gene-based calibration of high-throughput functional assays for clinical variant classification

High-throughput assays measure a broad range of variant effects on gene function and hold promise for supporting genomic medicine. Current clinical guidelines for rare Mendelian diseases rely on establishing gene-specific score thresholds for each assay that separate pathogenic from benign variants. This introduces inconsistencies and subjectivity, ultimately lacking the rigor of calibration; i.e., mapping a variant score to a probability of pathogenicity. To address this problem, we introduce a semi-supervised framework for calibrating experimental assay data and propose Experimental score CALIBRator (ExCALIBR), a method that jointly models pathogenic, benign, synonymous, and population variants using skew normal mixtures to produce variant-specific probabilities of pathogenicity. Evaluated across 80 datasets from 39 genes, all meeting fit quality criteria, ExCALIBR substantially outperformed existing field standards and was further validated on the All of Us biobank data. Our results demonstrate that calibrated experimental assays generate indispensable evidence that will dramatically reduce variants of uncertain significance.

bioinformatics↗

bioRxiv2024

Multiplex, multimodal mapping of variant effects in secreted proteins

Despite widespread advances in DNA sequencing, the functional consequences of most genetic variants remain poorly understood. Multiplexed Assays of Variant Effect (MAVEs) can measure the function of variants at scale, and are beginning to address this problem. However, MAVEs cannot readily be applied to the [~]10% of human genes encoding secreted proteins. We developed a flexible, scalable human cell surface display method, Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), to measure secreted protein variant effects. We used MultiSTEP to study the consequences of missense variation in coagulation factor IX (FIX), a serine protease where genetic variation can cause hemophilia B. We combined MultiSTEP with a panel of antibodies to detect FIX secretion and post-translational modification, measuring a total of 44,816 effects for 436 synonymous variants and 8,528 of the 8,759 possible missense variants. 49.6% of possible F9 missense variants impacted secretion, post-translational modification, or both. We also identified functional constraints on secretion within the signal peptide and for nearly all variants that caused gain or loss of cysteine. Secretion scores correlated strongly with FIX levels in hemophilia B and revealed that loss of secretion variants are particularly likely to cause severe disease. Integration of the secretion and post-translational modification scores enabled reclassification of 63.1% of F9 variants of uncertain significance in the My Life, Our Future hemophilia genotyping project. Lastly, we showed that MultiSTEP can be applied to a wide variety of secreted proteins. Thus, MultiSTEP is a multiplexed, multimodal, and generalizable method for systematically assessing variant effects in secreted proteins at scale.

genomics↗

bioRxiv2023

Assigning credit where it's due: An information content score to capture the clinical value of Multiplexed Assays of Variant Effect

BackgroundA variant can be pathogenic or benign with relation to a human disease. Current classification categories from benign to pathogenic reflect a probabilistic summary of current understanding. A primary metric of clinical utility for multiplexed assays of variant effect (MAVE) is the number of variants that can be reclassified from uncertain significance (VUS). However, we hypothesized that this measure of utility underrepresents the information gained from MAVEs and that an information theory approach which includes data that does not reclassify variants will better reflect true information gain. We used this information theory approach to evaluate the information gain, in bits, for MAVEs of BRCA1, PTEN, and TP53. Here, one bit represents the amount of information required to completely classify a single variant starting from no information. ResultsBRCA1 MAVEs produced a total of 831.2 bits of information, 6.58% of the total missense information in BRCA1 and a 22-fold increase over the information that only contributed to VUS reclassification. PTEN MAVEs produced 2059.6 bits of information which represents 32.8% of the total missense information in PTEN and an 85-fold increase over the information that contributed to VUS reclassification. TP53 MAVEs produced 277.8 bits of information which represents 6.22% of the total missense information in TP53 and a 3.5-fold increase over the information that contributed to VUS reclassification. ConclusionsAn information content approach will more accurately portray information gained through MAVE mapping efforts than counting the number of variants reclassified. This information content approach may also help define the impact of modifying information definitions used to classify many variants, such as guideline rule changes.

genetics↗

bioRxiv2023

Multiplexed functional assessments of MYH7 variants in human cardiomyocytes at scale

BackgroundSingle, autosomal-dominant missense mutations in MYH7, which encodes a sarcomeric protein (MHC-{beta}) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, [~]75% of MYH7 variants are of unknown significance (VUS), causing diagnostic challenges for clinicians and emotional distress for patients. Deep mutational scans (DMS) can determine variant effect at scale, but have only been utilized in easily-editable cell lines. While human induced pluripotent stem cells (hiPSCs) can be differentiated to numerous cell types that enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates. However, CRaTER enrichment has recently enabled the pooled generation of a saturated five position MYH7 variant hiPSC library suitable for DMS for the first time. ResultsAs a proof-of-concept, we differentiated this MYH7 variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-{beta} variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-{beta} protein loss occurs in a failing human heart with a pathogenic MYH7 mutation. We find the multiplexed assessment of MHC-{beta} abundance and hiPSC-CM survival both accurately segregate all pathogenic variants from synonymous controls. Overall, functional scores of 68 amino acid substitutions across these independent assays are [~]50% consistent. ConclusionsThis study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants at scale for the first time. This proof-of-concept demonstrates the ability to DMS previously restricted, clinically-actionable genes to reduce the burden of VUS on patients and clinicians.

genetics↗

bioRxiv2023

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 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.

genetics↗