Clinical and functional characteristics of the HNF1a-Q125ter variant. (A) Clinical and biochemical indices of subject carrying the HNF1a-Q125ter variant. Urine glucose and urine ketosis levels suggested the patient had diabetic ketosis. Meanwhile, a high level of LAC indicated lactic acid metabolism disorder: +, positive; −, negative. (B) Pedigree. Circle, female; square, male; black slash segment, death; black arrow, proband. Those affected with diabetes are shaded black. Corresponding variant status, if known. Carriers of the mutation are marked with a (+). (C) Sanger sequencing map of HNF1a-Q125ter variant. Black arrow indicates heterozygous mutation for HNF1a: Exon2, c.373C>T, p.(Gln125*). (D) A diagram showing the HNF1a protein and domains. (E) Protein structure modeling of WT and HNF1a-Q125ter. White arrow indicates Gln125. HbA1c, hemoglobin A1c; LAC, lactic acid; Ket, ketosis.

Similar variant impaired β-cell function in zebrafish. (A) Schematic representation of a similar HNF1a-Q125ter variant in zebrafish generated by CRISPR/Cas9. Mutation was caused via a two-base-pair deletion at the CRISPR target site for HNF1a. (B) The transcription level of hnf1a in WT and hnf1a^+/−; n = 50 larvae for each genotype. (C) Survival curves of WT and hnf1a^+/− zebrafish from 0 h to 24 h. (D,E) Representative confocal images (D) and quantification (E) of the β-cell number from Tg(−1.2ins:H2BmCherry) and hnf1a^+/−; Tg(−1.2ins:H2BmCherry) zebrafish larvae. Scale bar indicates 25 μm; n = 20–35 larvae for each genotype. (F) Total free glucose level of WT and hnf1a^+/− larvae at 6 dpf. (G) qRT-PCR analysis of the expression of insa and insb mRNA levels in WT and hnf1a^+/− larvae at 6 dpf. (H) Representative confocal images of Tg(ins:H2BmCherry); Tg(pdx1:GFP) at 6 dpf for WT and hnf1a^+/−. β-cells expressed insulin (mCherry⁺) without pdx1 (GFP⁻) are shown by white arrows. Scale bar: 10 μm. (I) Quantification of fluorescence intensity for insulin in WT and hnf1a^+/−. (J) Quantification of double-positive cells’ (mCherry⁺/GFP⁺) rate in WT and hnf1a^+/− larvae at 6 dpf; n = 3 larvae for each genotype. (K) Representative images of GCaMP6s response in β cells of Tg(Ins:H2BmCherry); Tg(Ins:GCaMP6s) and hnf1a^−/−; Tg(Ins:H2BmCherry); Tg(Ins:GCaMP6s) by 5 or 20 mM glucose ECS solution; the green signal is GCaMP6s. Scale bar: 10 μm. (L) RT-qPCR quantification of mRNA levels for insulin-secretion markers in WT and hnf1a^+/− larvae at 6 dpf: abcc8, scl2a2, gck, kcnj11, and kcnh6. (M) RT-qPCR quantification of mRNA levels for β-cell maturation and differentiation markers in WT and hnf1a^+/− larvae at 6 dpf: mafa, pdx1, nkx6.1, and pax6b. (N,O) Representative electron micrographs of β cells (N) and quantification of insulin granules (O) in β cells from WT and hnf1a^+/− larvae at 6 dpf. (N,O) Transmission electron microscopy for insulin granules. Islet isolation from WT and hnf1a^+/− larvae at 6 dpf. The dotted blue lines represent intact individual β cells. Bar scale: 2 μm. n = 3 intact individual β cells for each genotype. Results are represented as means with standard errors; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Student’s t-test. All experiments were performed at least three times, unless otherwise indicated. WT, wild type.

The β cells of hnf1a^+/− zebrafish displayed ER stress. (A) Transmission electron microscopy analysis of ER in WT and hnf1a^+/− β cells at 6 dpf zebrafish. Blue arrows indicated magnified ER morphology. Scale bar: 3 μm. (B) Quantification of ER lumen width for WT and hnf1a^+/− β cells. At least 15 different locations of the same cell were measured; n = 3 β cells for each genotype. (C) RT-qPCR analysis of the mRNA expression levels for ER-stress-related marker genes in WT and hnf1a^+/− larvae at 6 dpf: bip, atf4, chop, xbp1, sXBP1, and atf6b. (D) Immunofluorescence analysis of WT and hnf1a^+/− larvae at 6 dpf with atf4 staining (Green). β cells are indicated by the red fluorescence with Tg(ins:H2BmCherry). Scale bar indicates 20 μm. (E). Quantification of fluorescence intensity for atf4 in WT and hnf1a^+/−; n = 3 larvae for each genotype. (F) RT-qPCR analysis of the mRNA expression levels for nrf2a, nrf2b, gstp1, and hmoxla in WT and hnf1a^+/− larvae; n = 3 larvae for each genotype. Results are represented as means with standard errors; * p < 0.05, ** p < 0.01, and *** p < 0.001, and **** p < 0.0001. Student’s t-test. All experiments were performed in at least three biological repeats. WT, wild type.

HNF1a-Q125ter impaired β-cell function in vitro. (A) Western blot analysis of HNF1a in INS-1 cells after transfection with control, HNF1a-OE, or HNF1a-Q125ter plasmids for 24 h. (B) The cell-proliferation analysis of control, HNF1a-WT, or HNF1a-Q125ter plasmid transfected Ins-1 cells at 0, 24, and 48 h. (C,D) RT-qPCR analysis of the expression levels of insulin genes Ins1 (C) and Ins2 (D) in Ins-1 cells after plasmid transfection for 24 h. (E) RT-qPCR quantification of insulin-secretion markers in control, HNF1a-WT, and HNF1a-Q125ter after plasmid transfection for 24 h: Slc2a2, GCK, Abcc8, Kcnj11, and Kcnh6. (F,G) Representative immunofluorescence images (F) and quantification of insulin fluorescence (G) of insulin content from control, HNF1a-WT, and HNF1a-Q125ter transfected Ins-1 cell. Insulin was stained with red, and HNF1a was stained with Magenta; GFP indicates the plasmid-transfected positive cells and DAPI-stained nuclei. Scale bar indicates 20 μm. (H) RT-qPCR quantification of β-cell maturation and differentiation markers (Mafa, Pdx1, and Pax6) in control, HNF1a-WT, and HNF1a-Q125ter after transfection for plasmids at 24 h. (I,J) Immunofluorescence analysis of insulin granules in different plasmid-transfected Ins-1 cells. The representative images (I) and quantification (J) of insulin granules are displayed. Insulin stained with red, HNF1a stained with magenta, GFP indicates the plasmid-transfected positive cells, and DAPI is stained nuclei. Scale bar indicates 40 μm. (K) Glucose-stimulated insulin secretion analysis in Ins-1 cells transfected with different plasmids. Results are represented as means with standard errors: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. One-way ANOVA. All experiments were performed at least three times, unless otherwise indicated. WT, wild type.

HNF1a-Q125ter induced ER stress through PERK/eIF2a/ATF4 signaling pathway. (A) Western blot of HNF1a in Ins-1 cells transfected with shHNF1a or control construct for 24 h. (B) Quantification of ER lumen width for control, HNF1a-WT, HNF1a-Q125ter, shScramble, and shHNF1a transfected Ins-1 cells, at least 15 different locations of the same cell were measured, n = 5 individual cells for each group. (C,D) Representative Western blot images (C) and quantification analysis (D) of p-PERK and PERK in different plasmids transfected Ins-1 cell. (E,F) Representative Western blot images (E) and quantification analysis (F) of p-eIF2a and eIF2a in different plasmids transfected Ins-1 cell. (G,H) Representative Western blot images (G) and quantification analysis (H) of ATF4 in different plasmid-transfected Ins-1 cell. Protein levels were normalized to β-actin. (I) RT-qPCR analysis of the expression levels of Atf4 mRNA level in different plasmids transfected Ins-1 cell. The tunicamycin treatment (5 μg/mL) was used as a positive control for ER stress, and chemical chaperone 4-PBA (300 μm/L) was used as an ER-stress reliever. (J) RT-qPCR analysis of the expression levels of Nrf2a, Nrf2b, and Gstp1 mRNA levels in different plasmids transfected Ins-1 cell. (K) Working model for the molecular mechanism of new HNF1a variant resulted in β-cell dysfunction. A schematic that showed how the new variant of HNF1a resulted in MODY3. HNF1a-Q125ter induced ER stress through upregulated p-PERK/p-eIF2a/ATF4 and resulted in β-cell dysfunctions, including decreased β-cells numbers, decreased insulin expression, reduced insulin granules, and impaired insulin secretion. Results are represented as means with standard errors; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. One-way ANOVA. All experiments were conducted at least three times. WT, wild type; ER, endoplasmic reticulum; PERK, protein kinase R-like ER kinase; eIF2a, eukaryotic initiation factor 2; ATF4, activating transcription factor 4.