Neonatal diabetes affects 1 in 90,000-200,000 live births. Permanent neonatal diabetes mellitus (PNDM) presents itself in the first few months of life, and patients require treatment henceforth. PNDM can be caused by mutations in the insulin gene, INS, most of which are heterozygous. This study involves two INS gene mutations: C109Y and G32V. The first mutation was reported in a Singaporean family, and the latter has not been reported yet. Patients with either mutation are unable to secrete insulin despite having one wild type (WT) allele. We hypothesize that both C109Y and G32V mutations result in misfolding of the proinsulin protein, leading to β-cell dysfunction. The misfolded proinsulin is then able to exert its dominant negative effect on WT proinsulin, either directly through heteromerization, or indirectly through altering endoplasmic reticulum (ER) integrity, preventing insulin secretion and eventually insulin synthesis and/or even β-cell death. We computationally modelled both C109Y and G32V proinsulin structures, and found that both mutations result in conformational changes that likely affect further insulin processing. We also overexpressed C109Y and G32V preproinsulin in a mouse insulinoma cell line, MIN6. Analysis of electron microscopy images revealed differences in the size and number of insulin granules. In addition, we obtained skin fibroblasts from patients with the C109Y mutation and their family members, reprogrammed them into induced pluripotent stem cells (iPSCs), and characterized them. We differentiated the iPSCs into β-like cells and saw no difference in size between WT and mutant cells. Current efforts are underway to determine the impact of the mutant insulin gene in the iPSC-derived β-like cells. Our findings will reveal mechanistic insights into this neonatal diabetes condition caused by the mutant insulin gene and potentially identify therapeutic approaches to reinstate the function of the existing wild type insulin gene.
N. Amirruddin: None. Y. Tan: None. C.S. Verma: Other Relationship; Self; sinopsee therapeutics. D. Gardner: None. Y. Bee: None. S. Hoon: None. A. Teo: None.
Institute of Molecular and Cell Biology; Khoo Teck Puat Hospital; National University Health System; Agency for Science, Technology and Research; National Medical Research Council; Lee Foundation; Skin Research Institute of Singapore