Pleiotropic ZIP8 A391T implicates abnormal manganese homeostasis in complex human disease

Abstract

ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic SNPs identified in GWAS; A391T has associations with an increased risk of schizophrenia, obesity, Crohn's disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knockin (KI) to generate a mouse model of ZIP8 A391T (Zip8 393T-KI mice). Recapitulating the SNP association with blood Mn, blood Mn was reduced in Zip8 393T-KI mice. There was restricted abnormal tissue Mn homeostasis, with decreases in liver and kidney Mn and a reciprocal increase in biliary Mn, providing in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemically induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of patients with Crohn's disease. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in complex human disease.

Keywords: Gastroenterology; Genetic variation; Genetics; Glycobiology; Inflammatory bowel disease.

Figure 1. Zip8 393T-KI mice exhibit abnormal Mn homeostasis.

(A) Whole blood Mn was reduced in Zip8 393T-KI homozygous mice (–18.9%, P = 0.0395). n = 11–13 male mice/genotype, 9–14 weeks of age, fasted. (B) Liver Mn was reduced in Zip8 393T-KI heterozygous and homozygous mice. (C) Kidney Mn was reduced in Zip8 393T-KI heterozygous and homozygous mice. (D) Relative Mn levels were comparable between WT and Zip8 393T-KI mice across additional tissue types. n = 3–7 male mice/genotype, 8–10 weeks of age (B–D). (E) Biliary Mn was increased in Zip8 393T-KI homozygous mice compared with WT mice. n = 4 male mice/genotype, 8–10 weeks of age, fasted. (F) There was no difference in liver Slc39a8 mRNA across genotypes. Relative expression, normalized to Gapdh mRNA. n = 3 male mice/genotype, 8–10 weeks of age. (G) Zip8 localized to the bile canalicular membrane in a pattern similar to that of Mdr1 in WT and Zip8 393T-KI homozygous mice. Confocal images are representative of at least 3 animals/genotype. Scale bars: 5 μm. Whole blood (A) and bile Mn (E) were measured by atomic absorption spectroscopy; tissue Mn was measured by ICP-MS and normalized to wet tissue weight (B–D). Mean ± SEM. Statistical analysis by 1-way ANOVA with Kruskal-Wallis and Dunn’s multiple comparisons tests when 3 or more groups were compared (A–C and F); unpaired, 1-sided t test was used for comparison of 2 groups (D and E).

Figure 2. Zip8 393T-KI male mice exhibit increased susceptibility to chemically induced colitis.

(A) Initial body weight of Zip8 393T-KI homozygous mice at 11.5–13 weeks of age was higher compared with that of WT and heterozygous mice. n = 16–20 male mice/genotype. (B) Zip8 393T-KI heterozygous and homozygous mice exhibited increased and more sustained weight loss in DSS-induced colitis model. Statistical analysis by linear regression (day 7–14). Slopes are equal between groups, but intercepts were significantly different between WT and Zip8 393T-KI heterozygous and homozygous mice (P < 0.0001), with no difference between Zip8 393T-KI heterozygous and homozygous mice (P = 0.9498). (C) Percentage of mice with rectal bleeding at day 5 and/or 6 was numerically higher in the heterozygous and homozygous mice (nonsignificant, χ² test). (D) Day 14 histology was consistent with more inflammation in Zip8 393T-KI homozygous mice, with crypt elongation, crypt branching (top right), and expansion of lamina propria immune infiltrate and squamous metaplasia (bottom right). Scale bar: 400 μm; 200 μm (right). Images are representative of n = 5–7 mice/genotype. (E) Zip8 393T-KI heterozygous and homozygous mice had increased Il6 mRNA, consistent with ongoing inflammation. Relative expression was graphed and normalized to Gapdh mRNA. n = 4–7 male mice/genotype. Statistical analysis by 1-way ANOVA with Kruskal-Wallis and Dunn’s multiple comparisons tests when 3 or more groups compared (A and E).