Endocrine Abstracts

Pathogenic extracellular mutations in the insulin receptor (INSR) produce recessive severe insulin resistance (IR), while intracellular mutations confer autosomal dominant IR explained by trans inhibition of wild-type (WT) INSR by mutant. Few recurrent mutations are described, however, and large-scale sequencing has revealed many “Variants of Unknown Significance” (VUS) that confound genetic diagnosis and stratification for personalised therapies. We recently applied multiplexed assays of variant effects (MAVEs) to evaluate expression, insulin binding and signalling by ~14,000 extracellular INSR variants. We now seek to interrogate ~8,500 INSR variants in the intracellular tyrosine kinase-containing domain essential for signal transmission. Most such variants are classified as VUS. To do this we have adapted our prior MAVE approach. First, we have enhanced the cell line used, an Igf1r knockout mouse embryo fibroblast (MEF) line in which endogenous mouse Insr is knocked down potently, with concomitant expression of mutant human INSR under the control of doxycycline. We have now introduced a separate transposon-encoded WT INSR allele tagged with a bromotag, whose transcription is induced by cumate and whose protein level can be modulated by a small molecule PROTAC (Proteolysis Targeting Chimera). This system has been validated and permits controlled testing of mutant receptor in isolation, and as a heterodimer with WT receptor. As a final step in assay optimisation, we have used RNA-seq to identify a promoter to use in an insulin dose-responsive transcriptional reporter currently being constructed. This will be added to a saturating, plasmid-based barcoded INSR variant library already made before A. barcode-variant phasing by long read sequencing, B. incorporation via Bxb1 at a unique landing pad in target MEFS, and C. MAVEs. This powerful new system will enable functional stratification of all intracellular INSR variants, identifying residues essential for INSR function and dominant negative activity, and panning of candidate therapies against all variants.