GENERATING HUMAN iPSC-BASED MODELS TO STUDY THE MOLECULAR MECHANISMS UNDERLYING THE DEVELOPMENT OF DIABETES DUE TO GLUCOKINASE MUTATIONS

Abstract

Glucokinase (GCK, hexokinase IV) is a member of the hexokinase family that phosphorylates glucose to glucose-6-phosphate during glycolysis process in pancreatic β-cells and hepatocytes. This is the rate limiting step in glucose metabolism and enables pancreatic β-cells and hepatocytes to respond accordingly to blood glucose level. Mutations in GCK gene could alter glucose homeostasis causing either hyperglycemia or hypoglycemia. Heterozygous loss of function mutation in GCK gene causes maturity‐onset diabetes of the young 2 (MODY2), a monogenic form of diabetes that is characterized by mild fasting hyperglycemia, while homozygous inactivating mutation leads to permanent neonatal diabetes mellitus (PNDM), which is characterized by severe hyperglycemia. Understanding the development of diabetes due to GCK mutations is not fully understood due to lack of human models. In the current work, we were able to generate induced pluripotent stem cells (iPSCs) from blood cells of two patients diagnosed with heterozygous and homozygous mutations in GCK. In patient’s samples, the heterozygosity and homozygosity were confirmed using whole exome sequencing (WES) followed by Sanger sequencing. Three iPSC lines were established from each patient, in which the mutation was confirmed using Sanger sequencing. All iPSC lines were extensively characterized using different approaches. The generated cell lines expressed pluripotency markers at both mRNA and protein levels and had the ability to differentiate into all three germ layers upon spontaneous differentiation. To generate isogenic controls, we used CRISPR/Cas9 knock-in approach to correct the mutation in iPSCs with homozygous and heterozygous mutations. One corrected patient-specific iPSC line was established from each cell line. Mutated and corrected iPSCs had been differentiated into pancreatic ß-cells using a well-established protocol. The generated iPSCs will be used to understand the role of GCK mutations in MODY2 and neonatal diabetes development.

Date of Award	2021
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences