Modelling genetic risk of β-cell dysfunction in human induced pluripotent stem cells from patients carrying theMTNR1Brisk variant

Abstract Disruptions in circadian rhythm, partly controlled by the hormone melatonin, increase the risk of type 2 diabetes (T2D). Accordingly, a variant of the gene encoding the melatonin receptor 1B ( MTNR1B ) is robustly associated with increased risk of T2D. This single nucleotide polymorphism (SNP; rs10830963; G-allele) is an expression quantitative trait locus (eQTL) in human pancreatic islets, conferring increased expression of MTNR1B , which is thought to perturb pancreatic β-cell function. To understand this pathogenic mechanism in detail, we utilized human induced pluripotent stem cells (hiPSC), derived from individuals with T2D carrying the MTNR1B G-allele. Patient-derived fibroblasts were reprogrammed to hiPSC and single-base genome editing by CRISPR/Cas9 was employed to create isogenic lines of either the C/C or G/G genotypes (non-risk and risk, respectively). In addition, the human embryonic stem cell (hESC) line (HUES4) was subjected to genome editing to create isogenic lines of either the C/C or G/G genotypes. hiPSC and hESC were differentiated into β-cells, using a 50-day 2D protocol. Single-base genome editing generated cells with the desired genotype at a success rate of >90%. Expression of stage-specific markers confirmed differentiation of both hiPSC and hESC into β-cells. MTNR1B mRNA levels were consistently low in differentiated β-cells, precluding quantitative analysis of gene expression. However, Western blot analysis showed higher levels of MTNR1B in differentiated β-cells carrying the risk allele, consistent with rs10830963 (G-allele) being an eQTL in β-cells. Insulin secretion in response to glucose and IBMX was similar between the genotypes, whereas addition of melatonin reduced secretion in G-allele carriers. We conclude that the stem cell-derived β-cells are not sufficiently mature to allow determination of eQTL status at the mRNA level. However, we did observe increased MTNR1B protein and increased sensitivity of β-cells from risk allele carriers (G-allele) to melatonin with regard to insulin secretion, thus supporting a functional role for the rs10830963 SNP in β-cell dysfunction.