Deciphering Silence: Functional Studies of GCK Synonymous and Nonsense Variants and Their Importance in Understanding Diabetes

<b>Background:</b> The most common form of monogenic diabetes is maturity onset diabetes of the young (MODY). This study investigates the molecular basis of MODY type 2 (<i>GCK</i>-MODY) in a group of Italian patients, focusing on the functional characterization of a synonymous variant, c.579G>T (p.Gly193Gly), in the glucokinase gene (<i>GCK</i>). <b>Methods:</b> Clinical evaluation and genetic analysis, including whole exome sequencing and Sanger sequencing, were used to identify the variant in <i>GCK</i>, then functional studies using a minigene approach allowed the functional characterization. <b>Results:</b> This study identified the synonymous variant, along with a nonsense mutation, c.859C>T (p.Gln287Ter), in <i>GCK</i> in two Italian patients. Minigene approach demonstrated that the synonymous variant disrupts splicing at the exon 5 boundary, leading to a frameshift and premature stop codon. Similarly, the nonsense mutation also altered splicing, exacerbating the molecular defect. <b>Conclusions:</b> These findings highlight the importance of functional assays, particularly minigene studies, in interpreting the pathogenicity of synonymous and nonsense variants, especially in genes like <i>GCK</i> where splicing alterations can significantly impact protein function. This study underscores the clinical utility of targeted genetic screening for personalized diabetes management.