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In vitro modeling of GCK mutation-associated monogenic diabetes using human iPSC and gene-editing technologies

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submitted on 2023-05-11, 05:57 and posted on 2023-05-17, 11:40 authored by Yasmin Abu Aqel, Racha Chouaib, Aldana I. Alnesf, Idil Ahmed, Gowher Ali, Ahmed K. Elsayed, Zeyaul Islam, Sara Al-Khawaga, Prasanna R. KolatkarV, Paul J. Thornalley, Khalid Hussain, Essam M. Abdelalim

Poster by Yasmin Abu Aqel (Hamad Bin Khalifa University), Racha Chouaib (Hamad Bin Khalifa University), Aldana I. Alnesf (Hamad Bin Khalifa University), Idil Ahmed (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Ahmed K. Elsayed (Hamad Bin Khalifa University), Zeyaul Islam (Hamad Bin Khalifa University), Sara Al Khawaga (Hamad Bin Khalifa University), Prasanna R. Kolatkar (Hamad Bin Khalifa University), Paul J. Thornalley (Hamad Bin Khalifa University), Khalid Hussain (Sidra Medicine), and Essam M. Abdelalim (Hamad Bin Khalifa University)

Background: Glucokinase (GCK, hexokinase IV) 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 those cells to respond appropriately to blood glucose level. Mutations in the GCK gene can either result in hyperglycemia or hypoglycemia. Heterozygous loss of function mutation causes maturity‐onset diabetes of the young 2 (MODY2), while homozygous inactivating mutation leads to permanent neonatal diabetes mellitus (PNDM). The mechanism of diabetes development due to GCK mutations is not fully understood due to the lack of human models

Objective: This study aims to understand the effect of GCK mutations on both pancreatic β-cells and mature hepatocytes to identify the molecular mechanisms underlying the defects associated with GCK mutations by using human iPSCs models.

Methods: We generated induced pluripotent stem cells (iPSCs) from blood cells of two patients diagnosed with heterozygous and homozygous mutations in the GCK gene. The mutations were confirmed in patient’s samples using whole exome sequencing (WES) and Sanger sequencing. The iPSC lines were extensively characterized using different approaches. Several experiments were done to understand the effect of this mutation. CRISPR/Cas9 knock-in approach was used to generate isogenic controls by correcting the GCK mutation in the generated iPSCs. Mutated and corrected iPSC lines were differentiated into pancreatic and hepatic lineages to understand the effect of GCK mutations in the development of MODY2 and PNDM. in silico study was done to capture the protein’s 3D structure of GCK and predict the effect of this GCK mutation on protein level.

Results: Our findings showed that the generated iPSC lines displayed pluripotency characteristics, were able to differentiate into the three germ layers spontaneously and showed normal karyotypes. Our enzymatic preliminary results showed that the mutated GCK protein is less stable compared to the wild type (WT) controls although they are both well folded. Furthermore, it showed higher binding ability and affinity to glucose. Moreover, in silico analysis suggested that this GCK mutation may affect the binding affinity of GCK with glucokinase regulatory protein (GKRP). All generated iPSCs and their isogenic controls were successfully differentiated into pancreatic β-cells and mature hepatocytes. Additional functional studies were performed on both pancreatic β-cells and mature hepatocytes to understand the molecular mechanisms underlying the defects associated with GCK mutations.

Conclusion: In conclusion, these human iPSC models can provide valuable insights into the underlying mechanisms of monogenic diabetes, which will pave the way towards personalized treatment.


QBRI-HSCI collaboration project QB09



  • English

Publication Year

  • 2023

Institution affiliated with

  • Hamad Bin Khalifa University
  • Sidra Medical and Research Center
  • Qatar Biomedical Research Institute

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