Multiplexed assessment of human glucokinase variant activity

Diabetes is a complex genetic disease affecting millions of people worldwide. 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 catalyzes the phosphorylation of glucose and is known as the pancreatic glucose sensor. Patients with GCK-MODY, in contrast to other diabetics, 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 genetic variants. To address this challenge, we generated a comprehensive map of human GCK variant activity. The activity map includes 97% of the possible missense and nonsense variants and correlate with in vitro catalytic efficiency, fasting glucose levels in patients and evolutionary conservation analysis. Activity scores include both hyper- and hypoactive variants. The codon-optimized human GCK sequence was divided into three regions (aa 2-171, 172-337, 338-466) that were separately mutagenized, assayed and sequenced. Regional plasmid libraries were transformed into yeast (hxk1Δhxk2Δglk1Δ). Yeast cells were used for plasmid extraction following 3 days of selection on 0.2% glucose medium. The GCK ORF was amplified in 14 tiles spanning the ORF before and after selection. The relative frequency of variants in each tile was determined using Illumina sequencing.