A multiplexed assay of human glucokinase reveals thousands of potential disease variants with both decreased and increased activity

Diabetes is a complex disease spanning from the heterogeneous etiology of type 1 and type 2 diabetes to monogenic diabetes. A common monogenic form of diabetes is glucokinase (GCK) maturity-onset diabetes of the young (GCK-MODY), which is caused by heterozygous inactivating variants in the gene encoding GCK. GCK is known as the pancreatic glucose sensor, as it regulates insulin secretion to maintain appropriate blood glucose levels. Accordingly, variants that alter GCK activity can cause hypo- and hyperglycemia, associated with hyperinsulinemic hypoglycemia (HH) and GCK-MODY, respectively, affecting up to 10 million people worldwide. Patients with GCK-MODY, in contrast to other people with diabetes, often do not require treatment but are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. To address this, we generated a comprehensive map of GCK variant activity in yeast. The activity map includes 97% of the possible missense and nonsense variants and correlates with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and evolutionary conservation analysis. Activity scores include both hyper- and hypoactive variants. We found that some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. As expected, hypoactive variants were concentrated at buried positions, near the active site, and at a partially surface-exposed region involved in GCK conformational dynamics. In conclusion, we provide a comprehensive assessment of GCK variant activity to facilitate variant interpretation and diagnosis, and we expand the mechanistic understanding of hyperactive variants to support development and refinement of drugs targeting GCK. ### Competing Interest Statement The authors have declared no competing interest.