Endocrine Abstracts

JOINT1906

Introduction: Monogenic diabetes (MD) makes up up to 5% of diabetes cases, with over 40 genes confirmed to cause it, though many remain unidentified. A Lithuanian-Swiss study identified a novel variant in the RFX2 gene, suggesting its potential role in diabetes. The study aimed to explore how RFX2 gene knockdown impacts insulin production and its clinical implications in a patient with this variant.

Methods: A novel c. 1894G>A (p. Ala632Thr) variant in the RFX2 gene was confirmed by PCR and Sanger sequencing. Functional analysis was performed by knocking down the gene in INS-1 832/13 cells, and insulin production was assessed using ELISA after glucose stimulation at 0 mM, 2. 5 mM, and 15 mM concentrations. Statistical analysis was conducted using IBM SPSS software 23. 0, and data are presented as median (min. -max. values).

Results: A 9-year-old boy diagnosed with diabetes and treated for type 1 diabetes was later found to have a novel c. 1894G>A (p. Ala632Thr) variant in the RFX2 gene at age 16. Autoantibodies for glutamic acid decarboxylase and tyrosine phosphatase-like protein were negative, though insulin autoantibodies were at the cut-off range for positivity, considering the ongoing insulin treatment, patient was suspected to have genetic diabetes. Co-segregation analysis showed no family members with diabetes signs or the same variant, prompting functional analysis of RFX2 in cell culture. Insulin production of siRFX2 cells vs. wild-type cells after stimulation with glucose (2. 5 mM) was lower, 1. 5 × 10^-5 (1. 4 × 10^-5 – 1. 7 × 10^-5) vs. 1. 6 × 10^-5 (1. 3 × 10^-5 – 1. 8 × 10^-5), respectively, P = 0. 049. Stimulation with 15 mM glucose concentration triggered the same insulin production from both, wild-type and knocked-down cells. Insulin secretion in siRFX2 cell after using different glucose solutions, showed higher response with higher glucose concentration, 0 nM glucose - 0. 5 × 10^-5 (0. 4 × 10^-5 – 0. 8 × 10^-5); 2. 5 mM glucose –1. 5 × 10^-5 (1. 4 × 10^-5 – 1. 7 × 10^-5), 15 mM glucose - 6. 6 × 10^-5 (6. 1 × 10^-5 – 7. 6 × 10^-5), respectively, P < 0. 001.

Conclusion: This experiment investigated the effect of RFX2 gene knockdown on insulin production In vitro. While the findings did not confirm RFX2 as a causative gene for monogenic diabetes, they suggested an altered glucose threshold in mutant cells, with impaired insulin production at low glucose and normal secretion at high glucose concentrations. This highlights the complexity of diabetes classification and underscores the need for further research into its etiology and pathogenesis.