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Cited by 0Genomics and Rare DiseasesGenetic Associations and EpidemiologyCRISPR and Genetic Engineering

Massively parallel functional genomic assays in endocrinology: from promise to delivery

Open access
Xu, Jingxiang, Aslanzadeh, Vahid, Kudla, Grzegorz, Semple, Robert K·2026·Journal of Molecular Endocrinology
DOI 10.1530/jme-25-0134PMC PMC13250644
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Abstract

The late 20th century saw major advances in mapping genes and mutations in endocrine disorders, driven by biochemical studies that identified defects endocrinologically and by family-based studies that mapped them genetically. In the 21st century, however, the world of genetics has been transformed by the mapping of the human genome and the development of next-generation sequencing technologies that allow genomes to be sequenced at population scale. This has led us to be swamped by a surfeit of information about genetic variation, with the so-called genetic 'variants of unknown significance' (VUS) greatly outnumbering pathogenic mutations in genes of clinical relevance. A revolution in functional genomics has since gathered pace, driven by the need to evaluate the function of rare genetic variants at scale. Recent advances in saturation mutagenesis enable the synthesis of libraries of thousands of gene variants. When coupled to increasingly creative use of a growing repertoire of downstream assays, this permits assessment of disease-relevant function of thousands of variants simultaneously. Such assays, widely known as multiplexed assays of variant effect (MAVEs), have major potential in endocrinology when applied to hormone receptors and their downstream signalling pathways. They help determine functional significance or otherwise of VUS, enable patient stratification for rare disease treatments, and yield new insights into structure-function relationships for target genes.…

Verified 2026-11-05 · 1 reference not checked yet

AI snapshot

Written from the paper’s full text.

TL;DR

This review argues that multiplexed assays of variant effect (MAVEs) can help solve the endocrine genetics “VUS problem” by functionally characterizing thousands of gene variants in parallel, especially for hormone receptors. It surveys MAVE technologies, assay designs, and endocrine applications, concluding that clinical delivery still requires standardization, replication, and better benign variant truthsets.

Key findings

  • VUS have rapidly outstripped pathogenic variants in ClinVar, and every sequenced person is estimated to harbour 20,000 rare coding variants; MAVEs can provide strong PS3/BS3 functional evidence and complement variant effect predictors, though MAVE–VEP correlation was only around Spearman ρ 0.5–0.6 in one recent study.
  • Endocrine receptor MAVEs have been reported for PPARγ, β2AR, MC4R, INSR, and MET; discriminatory performance was high, with areas under ROC curves for identifying pathogenic mutations generally in excess of 0.90.
  • Independent Parkin MAVEs using different cellular systems and assay designs showed strong agreement (R = 0.77) and high diagnostic performance (ROC AUC 0.92 and 0.94), whereas abundance-based VAMP-seq assays showed lower classification performance (ROC AUC ∼0.70–0.78).
  • Non-receptor endocrine examples include GCK, with nearly all possible missense and nonsense variants studied in yeast complementation assays, and HNF1A, with deep mutational scanning quantifying 11,970 protein-coding variants in human hepatocytes.

Methods

  • Narrative review of MAVEs in endocrinology, synthesizing published studies, community resources, and prior literature rather than generating new experimental data.
  • Covers mutational library generation, choice of cell/organism, functional assay types (survival, FACS, imaging, single-cell transcriptomics), data analysis standards, and applications to endocrine disease genes.
  • Reports performance metrics and examples from cited MAVE studies; no systematic search strategy or meta-analysis is described.

Limitations

  • Stated in the paper: MAVE reproducibility has lagged behind novel assay development; few genes have been studied across different model systems or laboratories, and none for hormone receptors; benign variant truthsets are limiting; standardization, reporting, and clinical use remain under deliberation; iPSC-based MAVEs face cost, editing, promoter silencing, and differentiation bottlenecks; no canonical nuclear hormone receptors and no cytokine-type or TGF-family receptors have yet been subjected to MAVEs.
  • Evident from the text: as a narrative review, it provides no primary data, no systematic search, and no quantitative synthesis; the examples and performance metrics are drawn from heterogeneous studies and may not be exhaustive.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1530/jme-25-0134

Cites

121 of 122 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

71 of 122 cited sources checked · 38 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Cas9 introduces a targeted double-strand break, which is repaired by homology-directed repair using donor templates, resulting in the incorporation of a single variant at the endogenous genomic locus.

    “Cas9 introduces a targeted double-strand break, which is repaired by homology-directed repair using the donor templates, resulting in the incorporation of a single variant at the endogenous genomic locus (54, 55).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [54] Canver, 2015, BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis, Nature source

      In “Tiled pooled enhancer editing in situ” (page unavailable), the paper describes Cas9-induced double-strand breaks followed by nonhomologous end joining that produces a spectrum of indels—not donor-template homology-directed repair incorporating a single variant. The Discussion mentions homology-directed repair only as an alternative approach, not the method used here. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [55] Findlay, 2014, Saturation editing of genomic regions by multiplex homology-directed repair, Nature source

      Checked before — its earlier verdict is held back until a new check runs.

  • Premisereasoning

    Endogenous genome-editing approaches enable the detection of splice-disrupting synonymous and intronic variants, which cannot be readily assessed using cDNA-based library generation methods in MAVE studies.

    “Consequently, these approaches enable the detection of splice-disrupting synonymous and intronic variants, which cannot be readily assessed using cDNA-based library generation methods in MAVE studies (58).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [58] Olvera-León, 2024, High-resolution functional mapping of RAD51C by saturation genome editing, Cell source
  • Premisecitation

    PIK3R1 mutations produce different phenotypes depending on the variant and domain of the protein affected.

    “One striking example is PIK3R1, which encodes a regulatory subunit of phosphoinositide 3-kinase (PI3K), a crucial player in signalling from many types of hormone receptor: early nonsense PIK3R1 mutations produce immunodeficiency and enhance insulin sensitivity, while nonsense mutations late in the gene cause severe insulin resistance and developmental defects. Furthermore, missense mutations pro”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] Tomlinson, 2025, Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant, Elife source
  • Premisecitation

    MAVEs generate large-scale, experimental datasets with low circularity for VEP validation.

    “Importantly, MAVEs generate large-scale, experimental datasets with ‘low circularity’ (i.e. with minimal or no dependence on the data sources commonly used by VEPs) benchmark for VEP validation (29).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] Livesey, 2025, Variant effect predictor correlation with functional assays is reflective of clinical classification performance, Genome Biol source
  • Premisecitation

    Problems with VEPs include overfitting towards existing clinical annotations.

    “Problems with VEPs include overfitting towards existing clinical annotations (29), biased performance in populations underrepresented in sequencing data (30), and variable predictive performance across genes due to inadequate calibration of scores (31).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Livesey, 2025, Variant effect predictor correlation with functional assays is reflective of clinical classification performance, Genome Biol source
  • Classified as probably not a substantial claim. 38
  • May combine two claims. 26
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Many computational variant effect predictors (VEPs) have been described, with variable diagnostic performance.

    “Many computational variant effect predictors (VEPs) have been described, with variable diagnostic performance.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [29] Livesey, 2025, Variant effect predictor correlation with functional assays is reflective of clinical classification performance, Genome Biol source
    • cites [30] Dawood, 2024, Using multiplexed functional data to reduce variant classification inequities in underrepresented populations, Genome Med source

      The Discussion names several commonly used computational predictors—BayesDel no-AF, aGVGD, and REVEL—supporting the claim that multiple predictors exist. The Results section on computational predictor evidence codes reports different contributions to VUS reclassification across ancestry groups, but does not compare the predictors’ diagnostic performance. No page numbers are available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [31] Tejura, 2024, Calibration of variant effect predictors on genome-wide data masks heterogeneous performance across genes, Am J Hum Genet source

  • May combine two claims.

    Premisecitation

    Each pegRNA directs editing to the target locus and provides the reverse-transcription template needed to install the programmed variant at the endogenous site.

    “Each pegRNA directs editing to the target locus and provides the reverse-transcription template needed to install the programmed variant at the endogenous site (56, 57) (Fig. 2B).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [56] Anzalone, 2019, Search-and-replace genome editing without double-strand breaks or donor DNA, Nature source
    • cites [57] Erwood, 2022, Saturation variant interpretation using CRISPR prime editing, Nat Biotechnol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Yeast surface display has been used to screen thousands of protein variants for binding affinity and function.

    “Yeast surface display, in particular, has been used to screen thousands of protein variants for binding affinity and function (65, 66).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [65] Adams, 2016, Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves, Elife source

      The Results (Proof-of-principle Tite-Seq experiments; no page available) describe yeast-displayed libraries totaling thousands of antibody variants, assayed for antigen binding and surface expression. This supports the scale and binding-affinity portion of the claim, but the paper does not report screening those variants for a separate measure of protein function. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [66] Starr, 2020, Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding, Cell source

  • May combine two claims.

    Findingcitation

    Documented VUS have rapidly outstripped pathogenic variants in widely used clinical genomic databases such as ClinVar and now pose a major challenge in genetic diagnosis.

    “Documented VUS have rapidly outstripped pathogenic variants in widely used clinical genomic databases such as ClinVar (Fig. 1) and now pose a major challenge in genetic diagnosis (24).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Landrum, 2025, ClinVar: updates to support classifications of both germline and somatic variants, Nucleic Acids Res source

      The Introduction identifies ClinVar as a routinely used clinical variant database with more than 3 million variants. But neither the Introduction nor the passages found by searching for VUS and uncertain significance compare VUS counts or growth with pathogenic variants, or state that VUS pose a major challenge in genetic diagnosis. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Error-prone PCR tends to produce single-nucleotide substitutions, limiting its utility as it cannot produce the full repertoire of amino acid substitutions at each codon.

    “However, this tends to produce single-nucleotide substitutions, limiting its utility as this cannot produce the full repertoire of amino acid substitutions at each codon (45, 46).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [45] Copp, 2014 source
    • cites [46] Kircher, 2019, Saturation mutagenesis of twenty disease-associated regulatory elements at single base-pair resolution, Nat Commun source

      The Results section ('Construction of saturation mutagenesis libraries'; page unavailable) says error-prone PCR introduced fewer than one change per 100 bp and produced libraries covering nearly all possible single-nucleotide variants. It does not address amino-acid substitutions at codons or say that error-prone PCR cannot produce their full repertoire; the experiments concern noncoding regulatory elements. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    The second key design consideration is whether or not the functional readout of the chosen downstream assay is relevant to the clinical condition or phenotype being studied.

    “The second key design consideration is whether or not the functional readout of the chosen downstream assay is relevant to the clinical condition or phenotype being studied (38).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Gelman, 2019, Recommendations for the collection and use of multiplexed functional data for clinical variant interpretation, Genome Med source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MAVEs provide a functional readout for large numbers of variants in genes of interest simultaneously, often on a scale of many thousands of variants.

    “MAVEs provide a functional readout for large numbers of variants in genes of interest simultaneously, sometimes referred to as fitness landscapes (34), often on a scale of many thousands of variants (35, 36).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [34] de Visser, 2014, Empirical fitness landscapes and the predictability of evolution, Nat Rev Genet source
    • cites [35] Fowler, 2014, Deep mutational scanning: a new style of protein science, Nat Methods source
    • cites [36] Findlay, 2018, Accurate classification of BRCA1 variants with saturation genome editing, Nature source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    New VEPs, such as AlphaMissense, now utilising deep learning techniques offer significant improvements over earlier heuristic-based approaches.

    “New VEPs continue rapidly to be developed, with many, like AlphaMissense, now utilising deep learning techniques that offer significant improvements over earlier heuristic-based – that is, involving computational ‘trial and error’ (27, 28) – approaches.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [27] Rentzsch, 2019, CADD: predicting the deleteriousness of variants throughout the human genome, Nucleic Acids Res source

      In “The Impact of CADD Scores in Human Genetics” (page unavailable), the paper says that CADD’s feature set has been used to train deep neural networks, supporting the point that deep-learning variant predictors were being developed. It does not mention AlphaMissense or establish significant improvements over earlier heuristic-based approaches; it says it knows of no competing tool that consistently outperforms CADD across diverse tests. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [28] Cheng, 2023, Accurate proteome-wide missense variant effect prediction with AlphaMissense, Science source

      The paper describes AlphaMissense as an AlphaFold-based model (p. 2) and reports improved performance over earlier predictors on clinical benchmarks (Improved pathogenicity classification, p. 3). It does not establish that the approaches it outperforms are heuristic-based, so that comparison is not fully supported. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MC4R is the most commonly mutated gene in monogenic obesity.

    “MC4R is the most commonly mutated gene in monogenic obesity (110).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [110] Wade, 2021, Loss-of-function mutations in the melanocortin 4 receptor in a UK birth cohort, Nat Med source

      The Introduction describes MC4R mutations in people with obesity and says obesity-associated mutations in leptin and its receptor are very rare. The Discussion estimates MC4R deficiency at about 1 in 340, but the paper does not compare MC4R with all other monogenic obesity genes or establish that it is the most commonly mutated. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A pragmatic solution to the problem has been proposed based on proactively generating benign variant lists based on factors such as population frequencies, disease rarity, and ACMG/AMP criteria.

    “A pragmatic solution to the problem has been proposed based on proactively generating benign variant lists based on factors such as population frequencies, disease rarity, and ACMG/AMP criteria (105).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [105] Rowlands, 2025, Availability of benign missense variant ‘truthsets’ for validation of functional assays: current status and a systematic approach, Am J Hum Genet source

      The Workflow for systematic generation of benign missense truthsets proposes a proactive approach using ACMG/AMP benignity criteria and population-frequency codes. The Assignation of putative pathogenicity and benignity flags section describes gene-phenotype-specific frequency thresholds, but the passages in our copy do not explicitly identify disease rarity as a factor in generating the lists. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The PCA approach is powerful and generalisable and has been used at scale.

    “This approach, although more contrived and indirect, is powerful and generalisable and has been used at scale (90).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [90] Beltran, 2025, Site-saturation mutagenesis of 500 human protein domains, Nature source
    • Rests on an assumption: Cellular survival assays can provide a powerful platform for identifying damaging variants in disease genes.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TileSeqMave v1.0 is commonly used in MAVEs employing a direct/tile sequence approach.

    “Examples include TileSeqMave v1.0 (47), commonly used in MAVEs employing a direct/tile sequence approach, and Enrich2 (100), widely adopted for MAVEs utilising barcode sequencing (89).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [47] Weile, 2017, A framework for exhaustively mapping functional missense variants, Mol Syst Biol source

      The Results section ('A tiled-region strategy for mapping functional variation'; page unavailable) describes direct sequencing of tiled amplicons for DMS-TileSeq, and 'Code and data availability' lists a TileSeq sequence analysis pipeline. Our copy does not identify that pipeline as 'TileSeqMave v1.0' or establish that it is commonly used across MAVEs. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    Two independent MAVEs of Parkin using different cellular systems and assay designs showed strong agreement (R = 0.77) and high diagnostic performance (ROC AUC 0.92 and 0.94), despite differences in library design, functional readout, and model system.

    “Two independent MAVEs of Parkin using different cellular systems and assay designs showed strong agreement (R = 0.77) and high diagnostic performance (ROC AUC 0.92 and 0.94), despite differences in library design, functional readout, and model system (101, 102).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [101] Sigmarsdóttir, 2026, Comprehensive variant effect map of Parkin-mediated mitophagy in Parkinson’s disease, bioRxiv source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [102] Aslanzadeh, 2026, A functional genetic atlas of Parkin resolves variants of uncertain significance and predicts Parkinson’s disease age at onset, bioRxiv source

  • May combine two claims.

    Premisecitation

    MaveDB includes application programming interfaces and visualisation tools that simplify data analysis, making it a valuable resource for the genomics community.

    “The inclusion of application programming interfaces and visualisation tools further simplifies data analysis, making MaveDB a valuable resource for the genomics community (41).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] Rubin, 2025, MaveDB 2024: a curated community database with over seven million variant effects from multiplexed functional assays, Genome Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    VAMP-seq has been optimised and validated across multiple targets.

    “Common strategies involve fusion of the gene of interest to a gene encoding a fluorescent protein, with fluorescence used as a surrogate for expression of the target protein, in the assay subtype known as VAMP-seq (variant abundance by massively parallel sequencing), which has been optimised and validated across multiple targets (70).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [70] Matreyek, 2018, Multiplex assessment of protein variant abundance by massively parallel sequencing, Nat Genet source

      The Results section validates VAMP-seq abundance measurements for PTEN and TPMT against individual experiments and western blots. It also reports preliminary applicability tests for five additional proteins, but not comparable validation or optimisation across those targets; two other proteins tested were unsuitable. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Yeast models have been used to screen human disease genes in complementation assays, where the human gene functionally replaces a homologous yeast gene essential for growth, as well as protein-protein and protein-DNA interactions.

    “Yeast models have also been used to screen human disease genes in complementation assays, where the human gene functionally replaces a homologous yeast gene essential for growth (47), as well as protein–protein and protein–DNA interactions (67, 68).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [47] Weile, 2017, A framework for exhaustively mapping functional missense variants, Mol Syst Biol source
    • cites [67] Diss, 2018, The genetic landscape of a physical interaction, Elife source

      The Results (no page listed) describe a yeast assay that measures how mutations in human FOS and JUN affect their protein–protein interaction, supporting that part of the clause cited to this paper. The paper does not report screening protein–DNA interactions. The earlier claim about replacing an essential yeast gene is attached to citation 47, not this source; this paper’s complementation assay instead joins enzyme fragments when two proteins interact. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [68] McDonnell, 2024, Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants, Mol Syst Biol source

  • May combine two claims.

    Premisecitation

    HEK293 cells have been modified to optimise variant library integration, rendering them particularly useful for generic functional assays such as VAMP-seq, which determines variant effects on protein abundance.

    “HEK293 cells have moreover been modified to optimise variant library integration, rendering them particularly useful for generic functional assays such as VAMP-seq, which determines variant effects on protein abundance (70, 71).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [70] Matreyek, 2018, Multiplex assessment of protein variant abundance by massively parallel sequencing, Nat Genet source
    • cites [71] Matreyek, 2020, An improved platform for functional assessment of large protein libraries in mammalian cells, Nucleic Acids Res source

      The Introduction and Results (‘Lentiviral landing pads expedite cell engineering’; page unavailable) describe improved landing pads in HEK 293T cells that increase recombination and enable variant-library expression for multiplex assays. Our copy does not identify VAMP-seq as an application or describe its protein-abundance readout, so that part of the sentence is not supported by this source. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingcitation

    Some MAVE data analysis tools can analyse experiments with multiple time points or conditions, while some are limited to analysing single pre-selected and post-selected cell populations.

    “At least seven different MAVE data analysis tools have been released (89); some are able to analyse experiments with multiple time points or conditions and some are limited to analysing single pre-selected and post-selected cell populations.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [89] Çubuk, 2025, Variant scoring tools for deep mutational scanning, Mol Syst Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    MAVEs in Hap1 cells have been semi-automated for large-scale deployment in functional genomics.

    “Together, these factors have led to MAVEs in Hap1 cells being semi-automated for large-scale deployment in functional genomics (74).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [74] Obolenski, 2025, Protocol for the functional evaluation of genetic variants using saturation genome editing, STAR Protoc source

      The ‘Before you begin’ section describes saturation genome editing in HAP1-A5 cells to study variant effects at scale, and ‘Expected outcomes’ describes high-throughput mutational data. The copy does not say the workflow has been semi-automated for large-scale deployment; a listed automated cell counter is optional and does not establish that claim. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    These challenges are being circumvented, and the power and versatility of iPSCs in MAVEs are now beginning to be revealed.

    “Nevertheless, these challenges are being circumvented, and the power and versatility of iPSCs in MAVEs are now beginning to be revealed (88).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [88] Fayer, 2025, Editing stem cell genomes at scale to measure variant effects in diverse cell and genetic contexts, MedRxiv source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Enrich2 is widely adopted for MAVEs utilising barcode sequencing.

    “Examples include TileSeqMave v1.0 (47), commonly used in MAVEs employing a direct/tile sequence approach, and Enrich2 (100), widely adopted for MAVEs utilising barcode sequencing (89).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [100] Rubin, 2017, A statistical framework for analyzing deep mutational scanning data, Genome Biol source
    • cites [89] Çubuk, 2025, Variant scoring tools for deep mutational scanning, Mol Syst Biol source

      The Enrich2 section describes Enrich2 as a commonly used DMS scoring tool and says it can process barcode-sequencing data. It does not establish that Enrich2 is widely adopted specifically among MAVEs using barcode sequencing; that is more specific than the usage claim in the source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Mutations skewing MC4R signalling and mutations selectively disrupting agonist binding were both identified.

    “Mutations skewing MC4R signalling and mutations selectively disrupting agonist binding were both identified, informing on mechanisms of ligand binding and signal transduction.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [109] Howard, 2025, High-resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery, Elife source

      The public review in our copy (section labeled “28 citations”; no page given) says the authors identified variants that bias MC4R signaling and variants that disrupt activation by one agonist but not another. It describes an effect on agonist binding as a hypothesis, not an established finding that the mutations selectively disrupt binding. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    At least seven different MAVE data analysis tools have been released.

    “At least seven different MAVE data analysis tools have been released (89); some are able to analyse experiments with multiple time points or conditions and some are limited to analysing single pre-selected and post-selected cell populations.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [89] Çubuk, 2025, Variant scoring tools for deep mutational scanning, Mol Syst Biol source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationcitation

    There is a pressing need to characterise the functional consequences of as many VUS as possible experimentally, to guide both research and clinical decision-making.

    “It follows from these observations that there is a pressing need to characterise the functional consequences of as many VUS as possible experimentally, to guide both research and clinical decision-making (33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Starita, 2017, Variant interpretation: functional assays to the rescue, Am J Hum Genet source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given

    Eukaryotic cellular models have the greatest relevance to endocrine scientists.

    “MAVEs have also been used extensively in bacterial models, often to interrogate mechanisms of antibiotic resistance (64); however, it is eukaryotic cellular models that have the greatest relevance to endocrine scientists.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Directed evolution has been powerfully applied to developing and maturing monoclonal antibodies and was recognised as part of the 2018 Nobel Prize for Chemistry.

    “This form of directed evolution has been powerfully applied to developing and maturing monoclonal antibodies and was recognised as part of the 2018 Nobel Prize for Chemistry (62).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [62] Barderas, 2019, The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies, Anal Bioanal Chem source

  • May combine two claims.

    Interpretationreasoning

    Genes with multifunctional proteins often exhibit distinct genotype-phenotype associations, with different mutations sometimes yielding diseases affecting different developmental or physiological processes.

    “This is clinically important as such genes often exhibit distinct genotype–phenotype associations, with different mutations sometimes yielding diseases affecting different developmental or physiological processes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Customised site saturation variant libraries eliminate codon bias, allow equal representation of each amino acid substitution, and substantially reduce the time required to optimise and implement saturation mutagenesis experiments.

    “These libraries eliminate codon bias, allow equal representation of each amino acid substitution, and substantially reduce the time required to optimise and implement saturation mutagenesis experiments.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Clausen, 2024, A mutational atlas for Parkin proteostasis, Nat Commun source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An acellular cDNA display proteolysis system has been used to measure the thermodynamic folding stability of nearly a million protein domains in a single experiment.

    “More recently, an acellular cDNA display proteolysis system has been used to measure the thermodynamic folding stability of nearly a million protein domains in a single experiment (63).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Tsuboyama, 2023, Mega-scale experimental analysis of protein folding stability in biology and design, Nature source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    To reduce sampling noise, multiple cells expressing each variant, ideally encompassing multiple clones, should be measured in a single experiment.

    “Moreover, to reduce sampling noise, multiple cells expressing each variant, ideally encompassing multiple clones, should be measured in a single experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Even where there is only a single gene function relevant to disease, VEPs fail at present to predict the molecular mechanism underpinning predicted loss-of-function.

    “Finally, even where there is only a single gene function relevant to disease, VEPs fail at present to predict the molecular mechanism underpinning predicted loss-of-function.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The well-established approach of (bacterio)phage display, developed in the 1980s, can be regarded as an in vitro MAVE prototype.

    “The well-established approach of (bacterio)phage display, developed in the 1980s, can be regarded as an in vitro MAVE prototype (60, 61).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [60] Smith, 1985, Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, Science source
    • cites [61] Fowler, 2010, High-resolution mapping of protein sequence-function relationships, Nat Methods source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    For a 100 amino acid protein, there are 2,000 potential variants, which increases when insertions and deletions are included in the mutational library.

    “For each position in a protein, there are 20 possible amino acids plus a potential stop codon, so for a 100 amino acid protein, there are 2,000 potential variants, which increases when insertions and deletions are included in the mutational library.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: For each position in a protein, there are 20 possible amino acids plus a potential stop codon.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The first key consideration in assay design is choosing a model system in which the desired scale of the library can be accommodated and expressed.

    “The first key consideration in assay design is thus choosing a model system in which the desired scale of the library can be accommodated and expressed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given

    Sequencing depth must be tailored to variant abundance.

    “Finally, sequencing depth must be tailored to variant abundance.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Customised site saturation variant libraries are now favoured by many experienced MAVE laboratories.

    “Such libraries are now favoured by many experienced MAVE laboratories (53); however, limited target length and cost considerations still impede global adoption in the field (52).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Feng, 2022, A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRASG12C inhibitors, Proc Natl Acad Sci U S A source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Investigating each new variant discovered individually is resource-intensive and not feasible.

    “Investigating each new variant discovered individually is resource-intensive, and not feasible, however.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    MAVEs have been used extensively in bacterial models, often to interrogate mechanisms of antibiotic resistance.

    “MAVEs have also been used extensively in bacterial models, often to interrogate mechanisms of antibiotic resistance (64); however, it is eukaryotic cellular models that have the greatest relevance to endocrine scientists.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [64] Dewachter, 2023, Deep mutational scanning of essential bacterial proteins can guide antibiotic development, Nat Commun source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationreasoning

    For a 1,000 amino acid protein, there would be 20,000 protein variants, as well as a large number of wild-type protein copies derived from a sequence with synonymous variants.

    “For a 1,000 amino acid protein, there would thus be 20,000 protein variants, as well as a large number of wild-type protein copies derived from a sequence with synonymous variants.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: For each position in a protein, there are 20 possible amino acids plus a potential stop codon.

  • The quote only partly states this claim.

    Interpretationreasoning

    The failure of VEPs to predict molecular mechanism limits attempts to develop precision therapies for rare endocrine diseases.

    “This limits attempts to develop precision therapies for rare endocrine diseases.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    MAVEs should not be regarded simply as competition for VEPs, but rather as complementary to them, with huge potential for synergy between experimental and computational approaches.

    “Importantly, MAVEs should not be regarded simply as competition for VEPs, but rather as complementary to them, with huge potential for synergy between experimental and computational approaches.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationcitation

    Similar strategies could be adapted and integrated into future MAVE experimental designs.

    “iPSCs have already been employed in large-scale CRISPR screens, suggesting that similar strategies could be adapted and integrated into future MAVE experimental designs (75, 76, 77).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationnone given

    There remains a pressing need for more replication studies of the same genes across models and laboratories.

    “Such efforts to standardise data analysis and reporting are major steps towards the realisation of the clinical diagnostic potential of MAVEs; however, there remains a pressing need for more replication studies of the same genes across models and laboratories.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Few examples, and none for hormone receptors, exist of genes studied in different model systems or laboratories.

  • May combine two claims.

    Premisecitation

    Fluorophore-labelled ligand or antibody binding to the relevant gene product or downstream upregulated protein works best for cell surface proteins, but can sometimes be used with fixation and permeabilisation for intracellular proteins.

    “Another strategy involves the use of a fluorophore-labelled ligand or antibody that binds specifically to the relevant gene product itself (e.g. (59)) or to a downstream upregulated protein (e.g. (84)). This works best for cell surface proteins, but can sometimes be used with fixation and permeabilisation for intracellular proteins (e.g. (59)).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [59] Aslanzadeh, 2025, Deep mutational scanning of the human insulin receptor ectodomain to inform precision therapy for insulin resistance, Nat Commun source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [84] Bi, 2020, Decellularized tissue matrix enhances self-assembly of islet organoids from pluripotent stem cell differentiation, ACS Biomater Sci Eng source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Assigning a score to each variant in a MAVE is based on enrichment of the variant or its tagging barcode in the cell population after selection, across survival, FACS or other assay paradigms.

    “Assigning a score to each variant in a MAVE is based on their enrichment, or enrichment of their tagging barcode, in the cell population after selection, whether this be in a survival, FACS or other assay paradigm.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisereasoning

    scRNA-seq is particularly powerful for studying a gene with significant effects on gene expression, such as ligand-activated transcription factors like nuclear hormone receptors, where selective modulation of function by different ligands is well documented.

    “This approach is particularly powerful for studying a gene which has significant effects on gene expression, for example ligand-activated transcription factors such as nuclear hormone receptors, where selective modulation of function by different ligands is well documented.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    To date, some of the best examples of scRNA-seq MAVEs have studied oncogenes or tumour suppressor genes, such as p53.

    “To date, however, some of the best examples have studied oncogenes or tumour suppressor genes, such as p53 (99).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [99] Ursu, 2022, Massively parallel phenotyping of coding variants in cancer with Perturb-seq, Nat Biotechnol source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisereasoning

    Intermediate loss-of-function variants distribute between null and wild-type bins and can be captured by sampling intermediate bins.

    “Intermediate loss-of-function variants will distribute between null and wild-type bins, which can be captured by sampling cells also from intermediate bins.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hap1 cells are widely used in MAVEs.

    “Also widely used in MAVEs are spontaneously haploid mammalian cells such as the myeloid lineage Hap1 line (36).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Findlay, 2018, Accurate classification of BRCA1 variants with saturation genome editing, Nature source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The need to develop high-confidence, benign truthsets has been identified as a key need for the community.

    “The need to develop high-confidence, benign truthsets has thus been identified as a key need for the community.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Relatively few benign variants have been functionally validated.
    • Rests on an assumption: Curation of pathogenicity in ClinVar is not based on rigorous functional data.

  • May combine two claims.

    Interpretationcitation

    Assays measuring protein function directly generally provide larger effect sizes and improved variant discrimination compared to stability or abundance assays.

    “In contrast, abundance-based assays such as VAMP-seq showed lower classification performance (ROC AUC ∼0.70–0.78), suggesting that assays measuring protein function directly generally provide larger effect sizes and improved variant discrimination compared to stability or abundance assay (70).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Matreyek, 2018, Multiplex assessment of protein variant abundance by massively parallel sequencing, Nat Genet source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    MAVEs of hormone receptors present particularly appealing translational opportunities.

    “As well as the general aim of mitigating the VUS problem in diagnostic sequencing of endocrine disease genes, MAVEs of hormone receptors present particularly appealing translational opportunities.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Attempts to develop standards and a statistical framework for assessment of diagnostic performance of functional assays lagged behind efforts to curate and assess gene variants, but a rigorous approach has been proposed.

    “Attempts to develop standards, and a statistical framework, for assessment of the diagnostic performance of functional assays lagged somewhat behind efforts to curate and assess gene variants, but a rigorous approach to this has been proposed (38).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Gelman, 2019, Recommendations for the collection and use of multiplexed functional data for clinical variant interpretation, Genome Med source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    MaveDB follows strict data and documentation standards, mandating HGVS nomenclature for variant representation, embodying FAIR principles for data sharing, and complying with GA4GH frameworks.

    “This follows strict data and documentation standards, mandating HGVS nomenclature for variant representation and embodying FAIR (findable, accessible, interoperable, and reusable) principles for data sharing, and compliance with GA4GH (Global Alliance for Genomics and Health) frameworks.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Rubin, 2025, MaveDB 2024: a curated community database with over seven million variant effects from multiplexed functional assays, Genome Biol source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Single-cell RNA sequencing simultaneously determines expression of thousands of genes at the individual cell level.

    “Single-cell RNA sequencing (scRNA-seq) simultaneously determines expression of thousands of genes at the individual cell level (98).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [98] Replogle, 2022, Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq, Cell source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Panning agonists against mutational libraries may stratify all possible mutations a priori in terms of different ligand responsiveness, obviating the need to study every mutation functionally as it is identified.

    “may also stratify all possible mutations a priori in terms of different ligand responsiveness, obviating the need to study every mutation functionally as it is identified”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    By sequencing barcodes in situ, registration of barcodes to images is ensured.

    “By sequencing barcodes in situ, registration of barcodes to images is ensured.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    The application of MAVEs to endocrine disorders is in its infancy.

    “The application of MAVEs to endocrine disorders is in its infancy, yet the potential is immense.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A major challenge across MAVE applications is that non-receptor endocrine genes span diverse functional classes and require different assay systems, unlike receptors where generalisable multiplexed assay platforms are available.

    “A major challenge, which applies broadly across MAVE applications, is that unlike receptors – where generalisable and well-established multiplexed assay platforms are available (for example, FACS antibodies to quantify surface expression or cAMP-responsive reporters to measure GPCR signalling) – non-receptor endocrine genes span diverse functional classes and require different assay systems.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The challenge in applying MAVEs to secreted peptide hormones lies in retaining the coupling of expressed, secreted mutant peptides to the tagging barcode in the cell of origin.

    “so the challenge lies in retaining the coupling of expressed, secreted mutant peptides to the tagging barcode in the cell of origin.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Making a mutational library of a secreted peptide would be straightforward.
60 of 60

  • The paper relies on this without support.

    NNS and NNK codons encode all possible amino acid changes at the target codon while minimising stop codons.

    “NNS and NNK codons encode all possible amino acid changes at the target codon while minimising stop codons.”

  • The paper relies on this without support.

    CRISPR prime editing enables in situ mutagenesis without requiring homologous donor templates.

    “Unlike SGE, CRISPR prime editing enables in situ mutagenesis without requiring homologous donor templates.”

  • The paper relies on this without support.

    Endogenous genome-editing approaches preserve the native genomic context, including introns and regulatory elements.

    “Endogenous genome-editing approaches preserve the native genomic context, including introns and regulatory elements.”

  • The paper relies on this without support.

    For each position in a protein, there are 20 possible amino acids plus a potential stop codon.

    “For each position in a protein, there are 20 possible amino acids plus a potential stop codon”

  • The paper relies on this without support.

    The simplest widely used eukaryotic models are yeasts.

    “The simplest widely used eukaryotic models are yeasts, which offer a simple, cost-effective, and powerful system for high-throughput studies.”

  • The paper relies on this without support.

    Mammalian cells provide a closer approximation to human tissue than bacteriophages, bacteria, and yeasts, and have been used most widely for endocrinology-relevant MAVEs.

    “Although less experimentally tractable than bacteriophages, bacteria, and yeasts, mammalian cells provide a closer approximation to human tissue, and have been used most widely for endocrinology-relevant MAVEs.”

  • The paper relies on this without support.

    The complexity and higher costs involved in working with mammalian cells constrain scalability.

    “The complexity and higher costs involved in working with mammalian cells’ constrain scalability, and strategies both for cellular library creation and downstream assay need to be developed carefully, mindful of the required variant library size.”

  • The paper relies on this without support.

    Where in situ editing is used, endogenous expression of the target gene is a prerequisite for an effective MAVE.

    “Where in situ editing is used, endogenous expression of the target gene is a prerequisite for an effective MAVE.”

  • The paper relies on this without support.

    Where the gene of interest is expressed transgenically, endogenous expression can be a major confounder, usually dealt with by endogenous gene knockout or silencing.

    “In contrast, where the gene of interest is expressed transgenically, endogenous expression can be a major confounder, usually dealt with by endogenous gene knockout or silencing.”

  • The paper relies on this without support.

    HEK293 cells are the workhorse mammalian cell model for MAVE given their ease of culture and transfection, rapid growth, and ready adaptation to pooled, high-throughput assays, and provide a human cellular context that better captures protein folding, post-translational modification and intracellular signalling than bacteria or yeast.

    “Kidney-derived HEK293 cells are the ‘workhorse’ mammalian cell model for MAVE given their ease of culture and transfection, rapid growth, and ready adaptation to pooled, high-throughput assays – all while providing a human cellular context that better captures protein folding, post-translational modification and intracellular signalling than bacteria or yeast.”

  • The paper relies on this without support.

    Use of haploid cell lines eliminates the confounding effects of a second allele, and such lines are particularly amenable to genome editing approaches.

    “Use of such lines eliminates the confounding effects of a second allele, and such lines are particularly amenable to genome editing approaches.”

  • The paper relies on this without support.

    iPSCs have inherent capacity for directed differentiation into a large number of mature cell types, offering vast scope for MAVEs in different lineage contexts.

    “iPSCs, with their inherent capacity for directed differentiation into a large number of mature cell types, excite particular interest because of the vast scope they offer for MAVEs in different lineage contexts.”

  • The paper relies on this without support.

    Significant barriers currently exist to wide-scale development of iPSC-based MAVEs, including high cost of iPSC culture, challenge of efficient gene editing, and silencing of many promoters in iPSCs, especially in context of viral sequences.

    “Despite the huge potential of MAVEs based on iPSCs, significant barriers currently exist to their wide scale development. These include the high cost of iPSC culture, the challenge of efficient gene editing of iPSCs, the silencing of many promoters in iPSCs, especially in the context of viral sequences, although recent progress has been made towards overcoming these problems (87).”

  • The paper relies on this without support.

    iPSC differentiation protocols are frequently long and labour-intensive, often with bottlenecks that attenuate library diversity.

    “A further challenge is that iPSC differentiation protocols are frequently long and labour-intensive, often with bottlenecks that attenuate library diversity.”

  • The paper relies on this without support.

    The likely statistical robustness of the MAVE, and thus the requisite number of replicates and controls, can only fully be modelled once the performance characteristics and disease relevance of the downstream assay are understood.

    “The likely statistical robustness of the MAVE, and thus the requisite number of replicates and controls, can only fully be modelled once the performance characteristics and disease relevance of the downstream assay are understood.”

  • The paper relies on this without support.

    Core requirements of the functional assay chosen are that it must allow linking of phenotypic outcome to mutation, be relevant to disease, and offer sufficient dynamic range.

    “Core requirements of the functional assay chosen are that i) it must allow for linking of the phenotypic outcome for each cell to the mutation harboured by that cell, ii) it must be relevant to the disease or in vivo process of interest, and iii) it must offer sufficient dynamic range to discriminate signal from noise and ideally different degrees of loss of function.”

  • The paper relies on this without support.

    Linking outcomes to mutations is generally achieved through next-generation sequencing approaches.

    “Linking outcomes to mutations is generally achieved through next-generation sequencing approaches.”

  • The paper relies on this without support.

    In MAVEs based on in situ editing, sequencing the whole gene of interest (exon by exon) is the most common approach.

    “This is the most common approach in MAVEs based on in situ editing, where targeting exon by exon is employed, and the exon concerned can be sequenced.”

  • The paper relies on this without support.

    For many MAVEs based on transgenic expression sequencing, sequencing the whole open reading frame is precluded by cost, especially for large genes.

    “For many MAVEs based on transgenic expression sequencing, the whole open reading frame is precluded by cost, especially for large genes.”

  • The paper relies on this without support.

    In such MAVEs, incorporation of scrambled barcode DNA sequences in cis with each point mutation is commonly employed in library creation.

    “In such MAVEs, the incorporation of scrambled ‘barcode’ DNA sequences in cis with each point mutation is thus commonly employed in library creation.”

  • The paper relies on this without support.

    In library validation, long-read DNA sequencing can be used to phase barcodes and variants.

    “In the process of library validation, long-read DNA sequencing can be used to ‘phase’ barcodes and variants – that is, to create an index listing the barcode(s) associated with every variant.”

  • The paper relies on this without support.

    The assay employed and its relevance to disease depend exquisitely on the gene and disease concerned.

    “The assay employed and its relevance to disease depend exquisitely on the gene and disease concerned.”

  • The paper relies on this without support.

    The exacting requirement to study vast numbers of cells has practically constrained assays to several broad types.

    “However, the exacting requirement to study vast numbers of cells – often numbering many millions – has practically constrained assays to several broad types (Fig. 3).”

  • The paper relies on this without support.

    Cellular survival assays can provide a powerful platform for identifying damaging variants in disease genes.

    “Cellular survival assays, whether in yeast or in mammalian cells, can provide a powerful platform for identifying damaging variants in disease genes.”

  • The paper relies on this without support.

    Diploid cells require biallelic knockout of the gene of interest to demonstrate its essentiality.

    “as diploid cells require biallelic knockout of the gene of interest to demonstrate its essentiality”

  • The paper relies on this without support.

    Persistence of cells harbouring gene variants under selective pressure indicates that function is preserved.

    “the ability of cells harbouring gene variants to persist in a population under this selective pressure indicates that function is preserved”

  • The paper relies on this without support.

    Relatively few benign variants have been functionally validated.

    “Relatively few such benign variants have been functionally validated”

  • The paper relies on this without support.

    Curation of pathogenicity in ClinVar is not based on rigorous functional data.

    “curation of pathogenicity in ClinVar is not based on rigorous functional data”

  • The paper relies on this without support.

    The MAVE field has tended to prioritise novel MAVE designs and/or MAVEs of hitherto unstudied genes.

    “The rapidly expanding MAVE field has tended to prioritise novel MAVE designs and/or MAVEs of hitherto unstudied genes to date.”

  • The paper relies on this without support.

    Individual studies have taken a variety of approaches to statistical analysis of within-study variability.

    “Individual studies have taken a variety of approaches to statistical analysis of within-study variability”

  • The paper relies on this without support.

    Few examples, and none for hormone receptors, exist of genes studied in different model systems or laboratories.

    “few examples – and none for hormone receptors – exist of genes studied in different model systems or laboratories”

  • The paper relies on this without support.

    No receptors for canonical nuclear hormone receptor hormones have yet been subjected to a MAVE.

    “No receptors for such canonical hormones have yet been subjected to a MAVE; however, another important member of the nuclear hormone receptor family that is of endocrine relevance, namely PPARγ, has.”

  • The paper relies on this without support.

    Other important hormone receptors from different structural classes (cytokine-type and TGF-family receptors) have yet to be reported in MAVEs.

    “Other important hormone receptors from different structural classes (e.g. cytokine-type receptors (exemplified by receptors for growth hormone and leptin) and TGF-family receptors (e.g. for GDF15) have yet to be reported.”

  • The paper relies on this without support.

    The PPARγ variant pathogenicity classifier has been used subsequently to support clinical genetic diagnosis of familial partial lipodystrophy type 3.

    “This has been used subsequently to support clinical genetic diagnosis of familial partial lipodystrophy type 3.”

  • The paper relies on this without support.

    PPARγ plays a crucial role in metabolic homeostasis and is the master transcriptional regulator of adipocyte differentiation.

    “PPARγ plays a crucial role in metabolic homeostasis and is the master transcriptional regulator of adipocyte differentiation.”

  • The paper relies on this without support.

    The beta-2 adrenergic receptor mainly acts via Gαs G-proteins to increase intracellular cAMP and thus protein kinase A activity.

    “The first of these, the beta-2 adrenergic receptor (β2AR), which mediates some actions of epinephrine in particular, mainly acts via Gαs G-proteins to increase intracellular cAMP and thus protein kinase A (PKA) activity.”

  • The paper relies on this without support.

    Making a mutational library of a secreted peptide would be straightforward.

    “Making a mutational library of a secreted peptide would be straightforward”
37 of 37

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • ClinVar glossary entry cites a sequencing review

    Minor

    The glossary identifies reference 21 as its citation for ClinVar, but the listed reference 21 is a review of next-generation sequencing technologies. Elsewhere, the paper cites its ClinVar reference as 24.

  • MaveMD is cited to a paper about benign-variant truthsets

    Minor

    The text cites reference 105 for MaveMD, but reference 105 is about benign missense-variant truthsets. The paper's MaveMD reference is numbered 104.

  • Adrenal iPSC-organoid claim cites an adult-stem-cell study

    Minor

    The text lists reference 86 as an example of an iPSC-derived adrenal organoid, while the cited reference describes differentiation of adult bone-marrow-derived stem cells into Leydig or adrenocortical cells.

  • PPARG disease claim cites the truthsets paper

    Minor

    The citation attached to the claim about PPARG mutations points to reference 105, whose listed subject is benign-variant truthsets rather than PPARG or lipodystrophy.

  • GnomAD glossary entry cites a sequencing review

    Minor

    The glossary attaches reference 22 to GnomAD, but reference 22 is about the history and future of DNA sequencing. The paper elsewhere cites reference 103 for gnomAD.

5 of 5

Cites

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Related

Authors
Xu, J., Aslanzadeh, V., Kudla, G., & Semple, R. K.
Affiliations
  • 1
  • 2
Corresponding author
Semple, Robert K
ORCID
Venue
Journal of Molecular Endocrinology
Publisher
Bioscientifica
Published
2026-05-01
Volume
76
Issue
4
ISSN
  • 0952-5041 (ISSN-L)
  • 1479-6813
DOI
10.1530/jme-25-0134
PMC
PMC13250644
OpenAlex
W7161584916
Cited by
0
References
1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Molecular Endocrinology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
Diabetes UK (23/0006652)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
MAVE, variants of uncertain significance, saturation mutagenesis, hormone receptors, rare variants, structure-function relationship, patient stratification
MeSH terms
Animals, Endocrine System Diseases, Endocrinology, Humans, Mutation, Genetic Variation, Genomics, High-Throughput Nucleotide Sequencing

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