Identifying diabetes genes regulating beta cell function using a pooled insulin secretion assay

Type 2 diabetes (T2D) is characterized by impaired insulin action and the failure of compensatory insulin secretion. Determining the genetic pathways that regulate insulin secretion would bring us closer to a cure. Recently, >85 common DNA variants have been identified through genome-wide association studies (GWAS) that increase risk for T2D, many of which appear to affect beta cell function. While genes near these variants are likely to play a role in insulin secretion, for most implicated regions, the variants are noncoding, and the causal genes remain undefined.

Understanding which candidate T2D genes affect insulin secretion requires a systematic and comprehensive approach, in which the expression of each gene is perturbed in a beta cell model,and the effect on glucose response quantified. As the number of potential causal genes near disease-associated variants is large (i.e., >1100 using conservative thresholds), attempts to assay the role of each gene in an arrayed format are impractical.

To overcome this bottleneck, we recently developed a fluorescent reporter of insulin secretion that enables pooled assays of beta-cell function. Fluorescence of each beta cell expressing the reporter tracks closely> with the amount of insulin secreted per cell, providing a potential means to identify and isolate those cells with altered secretion resulting from genetic manipulation.

Apply the assay to test the effects of silencing a small subset of candidate T2D genes harboring variants linked to insulin secretion in humans; Perform a pooled CRISPR/Cas9 nuclease screen of all 1169 candidate T2D genes within a 500kb radius of disease-associated variants.

Should the assay prove useful for testing the effect of genetic perturbations on insulin secretion, I intend to apply for an ADA Junior Faculty Award to comprehensively screen all candidate diabetes genes in human induced pluripotent stem (iPS) cell-derived beta cells. As I have no current NIH funding, support from the BADERC to complete this pilot study will be will be vital to establishing sufficient preliminary data to be competitive for the ADA grant.

Relevance to public health

Identification of the genes that control insulin secretion in type 2 diabetes will help us to develop new targeted preventative and therapeutic measures for the disease.