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  • One of the patients in this

    2019-07-24

    One of the patients in this study was observed to have a mutation in the GCK gene. GCK phosphorylates glucose, producing glucose-6-phosphate and facilitating glucose storage (as glycogen) as well as its disposal via glycolysis in hepatocytes. Glycolysis mediates an increase in ATP and a decrease in Mg-ADP concentrations, leading to the closure of KATP channels and membrane depolarization. These changes subsequently trigger the opening of calcium channels and promote calcium entry into β (−)-Apomorphine and insulin exocytosis [24]. Therefore, GCK and KATP channels play a key role in glucose sensing and insulin secretion in β cells [25]. Mutations in the GCK gene could cause significant disruption of these processes [9]. In order to further investigate this particular GCK mutation, we used cloned liver GCK cDNA and constructed mutant GCK-T432K DNA to generate wild-type GST-GCK and mutant GST-GCK-T432 K recombinant proteins. The activity of these proteins was then analyzed, and the mutant appears to have only 2.5% of the enzymatic activity of the wild-type GST-GCK. This explains why the PND developed early in this patient, who was diagnosed at 0.1 year after birth and was homozygous for this GCK-T423K mutation. Notably, an E. coli expression system was used to express the GST-GCK and GST-GCK-T432 K recombinant proteins in this study. Unfortunately, proteins expressed with this particular system may have misfolding, impaired disulfide bonds, and/or incorrect post-translation modifications [26]. However, the GST-GCK activity measured here appears to be similar to that of purified rat liver GCK [19,27], rat islet GCK [19,28], and tag-free GCK [19,29]. This suggests that our GST-GCK recombinant protein may be similar to native GCK with regards to conformation and function. GST-GCK and GST-GCK-T432K were also expressed in the same system for comparison. Therefore, while this expression system has some limitations, our results are reliable and comparable to previous studies for the wild-type. Other limitations also exist. First, we used a human liver cell cDNA library to clone the GCK gene. Human GCK has three isoforms (one in the pancreas and two in the liver). It is, therefore, unclear whether the T432K mutation similarly reduces GCK kinase activity in β cells. Second, we only focused on the GCK mutation and did not confirm the novel mutations in the GLIS3 and INSR genes in other patients. While additional work is necessary to evaluate the causative nature and function of these other mutations, the present study did identify multiple novel gene mutations involved in PND or T1BD and provides a foundation for further exploration. Furthermore, our focus on the novel GCK mutation may also directly influence current treatment options for PND. Glibenclamide, a well-known sulfonylurea antidiabetic drug, was previously reported to be only partially effective in a child bearing a homozygous GCK T168A mutation [30]. This mutation had reduced glucokinase activity down to 2%, thus causing disease-related issues that could not be remedied by glibenclamide treatment. It is likely that the efficacy of glibenclamide in patients with GCK mutations depends on the residual activity of mutant GCK. Therefore, baseline GCK activity should be measured before sulfonylurea treatment. In conclusion, we utilized WES to detect genetic mutations in both PND and T1BD patients. Our analysis indicates that WES is a robust technique that can be used to unravel the etiologies of genetically heterogeneous diseases and may lead to more effective personalized therapy for the patients. Using our WES data, we also identified a novel homozygous inactivating mutation in the GCK gene and evaluated the mutation-mediated changes in protein function that may be related to PND pathogenesis in the affected patient. Taken together, while the molecular diagnosis of monogenic diabetes still presents numerous challenges, this study provides insight into the diagnostic power of WES and how the identified mutations can be studied and individually treated.