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bioRxiv · 10.1101/2024.07.19.603674

Modelling genetic risk of β-cell dysfunction in human induced pluripotent stem cells from patients carrying the MTNR1B risk variant.

Singh, T.·Kalamajski, S.·Cunha, J. P. M. C. M.·Hladkou, S.·Roberts, F.·Gheibi, S.·Soltanian, A.·Yektay Farahmand, K.·Ekstrom, O.·Mamidi, A.·Franks, P. W.·Rosengren, A.·Semb, H.·Mulder, H.·Fex, M.
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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 {beta}-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 {beta}-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 {beta}-cells. MTNR1B mRNA levels were consistently low in differentiated {beta}-cells, precluding quantitative analysis of gene expression. However, Western blot analysis showed higher levels of MTNR1B in differentiated {beta}-cells carrying the risk allele, consistent with rs10830963 (G-allele) being an eQTL in {beta}-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 {beta}-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 {beta}-cells from risk allele carriers (G-allele) to melatonin with regard to insulin secretion, thus supporting a functional role for the rs10830963 SNP in {beta}-cell dysfunction.

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Singh, T., Kalamajski, S., Cunha, J. P. M. C. M., Hladkou, S., Roberts, F., Gheibi, S., Soltanian, A., Yektay Farahmand, K., Ekstrom, O., Mamidi, A., Franks, P. W., Rosengren, A., Semb, H., Mulder, H., Fex, M.. 2024-07-22. Modelling genetic risk of β-cell dysfunction in human induced pluripotent stem cells from patients carrying the MTNR1B risk variant.. https://doi.org/10.1101/2024.07.19.603674

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