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Cited by 15Genomics and Rare DiseasesPancreatic function and diabetesMetabolism, Diabetes, and Cancer

Deep mutational scanning of the human insulin receptor ectodomain to inform precision therapy for insulin resistance

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Aslanzadeh, Vahid, Brierley, Gemma V., Kumar, Rupa, Çubuk, Hasan, Vigouroux, Corinne, Matreyek, Kenneth A., Kudla, Grzegorz, Semple, Robert K.·2025·Nature Communications
DOI 10.1038/s41467-025-64178-4PMC PMC12528385
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Abstract

The insulin receptor entrains tissue growth and metabolism to nutritional conditions. Complete loss of function in humans leads to extreme insulin resistance and infantile mortality, while loss of 80-90% function permits longevity of decades. Even low-level activation of severely compromised receptors, for example by anti-receptor monoclonal antibodies, thus offers the potential for decisive clinical benefit. A barrier to genetic diagnosis and translational research is the increasing identification of variants of uncertain significance in the INSR gene, encoding the insulin receptor. By coupling saturation mutagenesis to flow-based assays, we stratified approximately 14,000 INSR extracellular missense variants by cell surface expression, insulin binding, and insulin- or monoclonal antibody-stimulated signalling. Resulting function scores correlate strongly with clinical syndromes, offer insights into dynamics of insulin binding, and reveal novel potential gain-of-function variants. This INSR sequence-function map has biochemical, diagnostic and translational utility, aiding rapid identification of variants amenable to activation by non-canonical INSR agonists.

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  • Some protocols reduce error by employing barcoding strategies, where variants are tagged with two or more unique barcodes, enabling noise reduction through averaged fitness scores across technical replicates.

    “Some protocols further reduce error by employing barcoding strategies (59), where variants are tagged with two or more unique barcodes, enabling noise reduction through averaged fitness scores across technical replicates.”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [59]

    The Methods (Quantification and statistical analysis; page unavailable) describe averaging barcode scores for each variant and then averaging variant scores across replicates. The source calls these biological replicates, not technical replicates, and allows variants to have any number of barcodes rather than requiring two or more. It does not explicitly establish that this averaging reduces error. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • By amplifying and sequencing barcodes from each bin and using a predetermined barcode-variant index, a functional score can be computed for each variant.

    “By amplifying barcodes and sequencing them from each bin, then using the predetermined barcode-variant index to determine the distribution of each variant across bins, a functional score can be computed for each variant (e.g. (59, 92, 93)).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [59]

  • The INSR MAVE yielded novel findings related to INSR structure-function relationships.

    “As well as yielding novel findings related to INSR structure–function relationships, and addressing the ‘VUS problem’ in genetic diagnosis of severe insulin resistance, this study, like the MC4R MAVE above, will help to stratify patients with newly identified INSR variants for potential targeted therapies with non-canonical INSR ligands including monoclonal antibodies.”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [59]

  • 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)).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [59]

  • In all published receptor MAVEs, discriminatory performance was high, with areas under ROC curves for identifying pathogenic mutations generally in excess of 0.90.

    “Although different performance metrics were reported for each receptor MAVE, illustrating the evolving approaches taken in the field, in all cases discriminatory performance was high, with areas under ROC curves for identifying pathogenic mutations generally in excess of 0.90 (43, 59).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [59]

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Authors
Aslanzadeh, V., Brierley, G. V., Kumar, R., Çubuk, H., Vigouroux, C., Matreyek, K. A., Kudla, G., & Semple, R. K.
Affiliations
  • Institute of Genetics and Cancer (GB)
  • Royal Veterinary College (GB)
  • University of Cambridge (GB)
  • Inserm (FR)
  • Case Western Reserve University (US)
  • Cambridge University Hospitals NHS Foundation Trust (GB)
Corresponding author
Semple, Robert K.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-10-15
Volume
16
Issue
1
Pages
9143-9143
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-64178-4
PMC
PMC12528385
OpenAlex
W4415207571
Cited by
15
References
2
Citations by year
2026: 13 · 2025: 2
FWCI
7.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
  • Apollo (University of Cambridge) (submittedVersion)
Funders
  • Wellcome Trust (210752, 207507)
  • British Heart Foundation (RE/18/5/34216)
  • Diabetes UK (22/0006407)
  • French Ministry of Health and Assistance-Publique Hôpitaux de Paris PRISIS rare disease reference centre European Reference Network on Rare Endocrine Conditions
  • RCUK | Medical Research Council (MC_UU_00035/8)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
insulin receptor, insulin resistance, deep mutational scanning, missense mutations, variants of uncertain significance, insulin binding, receptor signaling, monoclonal antibodies, gain-of-function mutations, cell surface expression, precision therapy, genetic diagnosis
MeSH terms
Protein Domains, Antibodies, Monoclonal, DNA Mutational Analysis, Humans, Insulin, Insulin Resistance, Receptor, Insulin, Signal Transduction, Antigens, CD, Mutation, Missense, Precision Medicine, HEK293 Cells

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