title: Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies.
abstract: Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2 CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20878 dab68@cam.ac.uk.|Institute of Metabolic Science, University of Cambridge, CB2 0QQ, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Bulger
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20878.
authors.forename: Tetsunari
authors.initials: T
authors.lastname: Fukushige
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20878.
authors.forename: Sijung
authors.initials: S
authors.lastname: Yun
authors.affiliation: Institute of Metabolic Science, University of Cambridge, CB2 0QQ, UK.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Semple
authors.affiliation: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20878.
authors.forename: John A
authors.initials: JA
authors.lastname: Hanover
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20878.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Krause
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D008136:Longevity / Q000235:genetics*; D009154:Mutation; D011972:Receptor, Insulin / Q000235:genetics*
chemical_list: D029742:Caenorhabditis elegans Proteins; D007328:Insulin; C107483:DAF-2 protein, C elegans; D011972:Receptor, Insulin
keywords: CRISPR; DAF-2; INSR; Million Mutation Project; dauer