Biology subjects

Fayer, S.

Publications and source records attributed to Fayer, S..

3 recordsLinked to original sources

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↗