Modelling Evolutionarily Conserved Human Insulin Receptor Mutations in the Worm

Authors

• D Bulger
• T Fukushige
• R Semple
• J Hanover
• M Krause

Abstract

Rare monogenic disorders of severe insulin resistance can be traced to non-synonymous single nucleotide polymorphisms (nsSNPs), often affecting evolutionarily conserved insulin signalling components. Similar mutations in many of the homologous proteins in C. elegans also have phenotypes indicative of decreased signalling, including increased dauer diapause formation and increased longevity. The sole insulin-like receptor in the worm is DAF-2 that shares key structural features with human insulin regulated receptors. A semi-nonbiased exploration of viable alleles of DAF-2 without any previously recognized phenotype was carried out. Our results add 40 new alleles to the list of 26 previously reported daf-2 alleles identified in large scale genetic screens. Twelve of the new daf-2 alleles had changes in conserved amino acids that in humans cause severe disorders of insulin resistance. Using a sensitive dauer assay to measure signalling in the worm, 7 new alleles have been identified that result in phenotypes at high temperature. CRISPR-Cas9 genome editing was used to recreate and confirm three alleles with positive phenotypes and two alleles without any apparent phenotype. Additionally, one conserved allele with WT phenotype was re-engineered along with two human alleles at that amino acid, which remarkably showed the same gradient of phenotypes observed in humans. This study highlights the importance of modelling evolutionarily conserved human disease alleles in one of the simplest organisms with this pathway.

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