Structural and biophysical characterization of HNF-1A as a tool to study MODY3 diabetes variants

Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating expression of numerous target genes. Pathogenic variants in the HNF1A gene cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), characterized by dominant inheritance, age of onset before 25-35 years of age, and pancreatic β-cell dysfunction. A precise diagnosis alters management as insulin can be exchanged with sulfonylurea tablets and genetic counselling differs from polygenic forms of diabetes. More knowledge on mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants identified by exome sequencing is required for precise diagnostics. Here, we have structurally and biophysically characterized an HNF-1A protein containing both the DNA binding domain and the dimerization domain. We also present a novel approach to characterize HNF-1A variants. The folding and DNA binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S) were determined. All three variants showed reduced functionality compared to the wild-type protein. While the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for all investigated variants and allow for the dissection of structurally unstable and DNA binding defective variants. This points towards structural and biochemical investigation of HNF-1A being a valuable aid in reliable variant classification needed for precision diagnostics and management.