Role for TBC1D30 in Secretion of Mature Insulin

Genome-wide association studies (GWAS) have identified loci associated with the processing and secretion of mature insulin. Increased proinsulin (PI) secretion due to incomplete processing of insulin precursors has been linked to pancreatic beta-cell stress. At one PI GWAS locus, rs150781447-T is associated with increased PI secretion in Finns (p=5.5X10-11) and causes an amino acid (AA) change (R279C) at a conserved position in exon 4 of TBC1D30, a putative Rab GTPase Activating Protein (GAP), while no GWAS proxy variants (r2>0.8, 1000G Finns) are located in transcribed or enhancer regions. We hypothesized that TBC1D30 is involved in PI processing and that missense variant rs150781447, predicted to be damaging, has a negative impact on gene function. We used genome editing to generate four sets of TBC1D30 mutants in 832/13-INS1 cells. Compared to mock-edited control cells, apparent loss-of-function in frame insertion-deletion cells (106% expression) secreted 2.1-fold more PI (p=0.0001), and heterozygous truncation knockout cells (44% expression) secreted 1.9-fold more PI (p=0.005). Cells heterozygous for rs150781447, generated by prime editing, secreted 1.2-fold more PI (p=0.89), suggesting that the heterozygous variant has subtle or no effect on gene function. Unexpectedly, when exon 4 (42 AA) was deleted in frame, the cells secreted 3.2-fold less PI (p=0.02), consistent with a gain-of-function effect and suggesting that exon 4, part of the conserved Rab GAP protein domain, has a critical function. Preliminary insulin secretion data, while variable, demonstrate trends similar to proinsulin secretion, consistent with a stronger effect of TBC1D30 on processing than secretion. Together, these data demonstrate TBC1D30 as the likely target of the nearby PI GWAS locus and suggest that Rab GAP TBC1D30 plays a critical role in the processing of mature insulin. Disclosure V. A. Parsons: None. S. Vadlamudi: None. A. H. Moxley: None. K. L. Mohlke: None. Funding National Institutes of Health (1T32GM135128-01)