Pleiotropic ZIP8 A391T implicates abnormal manganese homeostasis in complex human disease

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.


Introduction
A nonsynonymous single nucleotide polymorphism (SNP) in SLC39A8 (rs13107325) is one of the most pleiotropic variants in the human genome, ranking 9 th of 341 genomic regions associating with more than one human disease or trait in genome wide association studies (GWAS) (1). In silico modeling predicts rs13107325 to be in the top 1.4% of deleterious substitutions in the human genome (2). The major allele associates with an increased risk of Parkinson's disease, hypertension (3), and alcohol misuse disorders (4) and the minor allele associates with increased risk of schizophrenia (5), Crohn's disease (6), obesity (7), dyslipidemia (8), and scoliosis (9). The minor allele frequency (MAF) is approximately 0.05 in American populations, 0.08 in Northern European populations, and increases to 0.14-0.25 in the Ashkenazi Jewish population; the major allele is monomorphic in African, East and South Asian populations (10)(11)(12). Understanding how and why this pleiotropic SNP associates with disease offers the potential to uncover commonalities of pathogenesis and establish therapeutic strategies.
SLC39A8 encodes ZIP8, a member of the SLC39 family of transporters that increases cytoplasmic metals. The functional characterization of ZIP8 has been focused on induced zinc transport during inflammation (13)(14)(15), however emerging data suggest a constitutive role for ZIP8 in manganese (Mn) homeostasis. In particular, individuals with private loss-of-function mutations in SLC39A8 (distinct from rs13107325) have Mn deficiency and congenital disorders of glycosylation attributed to impaired activity of Mn-utilizing glycosyltransferases (16)(17)(18).
Zip8 inducible knockout (iKO) mice have reductions in total Mn in liver, kidney, brain, and heart (19). Specific to ZIP8 391-Thr, a GWAS studying SNPs associated with blood metal levels found that ZIP8 391-Thr was the lead SNP associated with reduced blood Mn (9,20), and small studies have found N-glycosylation defects in carriers of ZIP8 391-Thr (19,21). Therefore, the hypothesis is that the pleiotropic effects of ZIP8 391-Thr could, at least in part, be driven by perturbations in Mn homeostasis and Mn-dependent processes, including N-glycosylation, with under-recognized clinical relevance in human disease.
To test this hypothesis, we utilized CRISPR/Cas9 in vivo gene editing to generate the Zip8 393T knock-in (KI) mouse taking advantage of the 84% homology between human and mouse Slc39a8 gene (10); mouse amino acid 393 corresponds with human amino acid 391 (Supplemental Figure 1). At baseline, Zip8 393T-KI mice showed no anatomical abnormalities despite abnormal Mn homeostasis with reduced whole blood Mn (male and female mice), reduced liver and kidney Mn (male mice) and increased biliary Mn excretion (male and female mice). Both heterozygous and homozygous male KI mice showed enhanced susceptibility to epithelial injury in a chemical model of colitis, consistent with the SNP-disease association with Crohn's disease (6); female KI mice did not exhibit a phenotype in the chemical model of colitis suggesting possible sex-specific effects of Mn homeostasis and/or limitations of the DSS colitis model in female mice. Sex-stratified analysis of the genetic association between ZIP8 391-Thr and Crohn's disease supports a stronger association in males. Finally, we demonstrated an association between the plasma N-glycan profile and ZIP8 391-Thr in the largest populationbased study to date, specifically implicating reduced triantennary plasma N-glycan species. The accumulating human data, supported by the Zip8 393T-KI mouse model, support the relevance of abnormal Mn homeostasis and dysregulation of Mn-utilizing processes, including Nglycosylation, in complex human disease.

Zip8 393T-KI mice exhibit abnormal Mn homeostasis
Regulation of Mn homeostasis in humans is an interplay between import and export proteins involved in enteric absorption, transport to the liver and other tissues, and excretion primarily via hepatobiliary route (22). In the liver, Mn homeostasis is regulated by ZNT10 (SLC30A10) at the apical membrane of hepatocytes to excrete Mn (23), ZIP8 (SLC39A8) at the apical membrane of hepatocytes and bile canalicular cells to reclaim Mn from bile (19), and ZIP14 (SLC39A14) at the basolateral membrane of hepatocytes to mediate liver uptake of Mn from blood (24). Loss-of-function of ZNT10 and ZIP14 result in Mn excess (23,24), whereas loss-of-function of ZIP8 leads to Mn deficiency (18,19). In vitro data support that ZIP8 391-Thr results in hypomorphic function of ZIP8 (9,25). We therefore hypothesized that Zip8 393T-KI mice would exhibit abnormal Mn homeostasis. It is well appreciated that Mn homeostasis exhibits age-and sex-specificity: Males require more dietary Mn, absorb less dietary Mn, and have lower blood Mn levels compared to females (26,27). We observe these trends in the Zip8 393T-KI mice and present data for both sexes.
Additional findings relevant to known transport activities of ZIP8 included a trend towards reduced liver Zn in heterozygous and homozygous mice (p=0.0502), but not kidney Zn; there were no differences in plasma Zn levels (Supplemental Figure 2). Liver Cd was reduced in heterozygous (-25.0%, p=0.0450) and homozygous mice (-27.3%, p=0.0441), but not kidney Cd (Supplemental Figure 3A, B). There were no differences in plasma or tissue Fe (Supplemental Figure 3C, D).
To test the hypothesis that hepatic Mn was reduced due to increased biliary losses (19), we measured biliary Mn. Consistent with the hypothesis, biliary Mn was increased 2-fold in Zip8 393T-KI homozygous mice compared to WT (p=0.027) ( Figure 1E). These findings are the most conclusive in vivo evidence that Zip8 393-Thr results in hypomorphic ZIP8 function as suggested by prior in vitro studies (9,19).
Like the male KI mice, female Zip8 393T-KI homozygous mice exhibited reduced blood Mn and increased biliary Mn compared to WT mice. Liver Mn was reduced in female Zip8 393T-KI homozygous mice with nominal significance (p=0.056). Notably, when comparing male and female mice of all genotypes, blood Mn was greater in the female mice, consistent with human data (22), while biliary Mn excretion was less in the female mice compared to male mice (Supplemental Figure 4).

Slc39a8 mRNA expression and Zip8 protein localization is unchanged in Zip8 393T-KI mice
There are no reported expression quantitative trait loci associations between rs13107325 and any gene in GTEx (28). Most consistent with these data, there was no genotype-dependent effect on Slc39a8 mRNA ( Figure 1F). Zip8 localized to the apical membrane of bile canalicular cells, in a pattern consistent with Mdr1, independent of genotype ( Figure 1G). These data suggest that ZIP8 391-Thr affects metal transport by mechanisms other than transcriptional regulation or protein localization.

Male Zip8 393T-KI mice exhibit enhanced susceptibility to chemical-induced colitis
We next sought to challenge Zip8 393T-KI mice using a disease-relevant model to test if the Zip8 393T-KI mouse could recapitulate a disease phenotype implicated by the GWAS associations with rs13107325. Given the association with Crohn's disease (6)  At baseline, mice of all genotypes (and sexes) showed no spontaneous intestinal inflammation. Male Zip8 393T-KI homozygous mice used for these experiments had higher baseline body weights when analyzed in aggregate compared to WT (Figure 2A). Mice were treated with 3.5% DSS for five days ( Figure 2B). Weight loss in the WT mice nadired at day 7, while weight loss in the Zip8 393T-KI heterozygous and homozygous mice nadired at day 9 with a greater mean total percentage loss consistent with a more severe and sustained inflammatory response. Rectal bleeding developed in numerically more Zip8 393T-KI heterozygous and homozygous mice at days 5-6 ( Figure 2C, non-significant, chi-square test). Histopathology analyses confirmed Zip8 393T-KI heterozygous and homozygous mice exhibited more severe inflammation extending into the muscularis mucosa with epithelial cell loss ( Figure 2D).
Colonic Il-6 mRNA was elevated in the Zip8 393T-KI heterozygous and homozygous mice compared to WT consistent with ongoing inflammation at Day 14 ( Figure 2E).
An expanded transcriptomic panel of immune-relevant genes was performed on bulk mRNA isolated from the distal colon for sham, Day 8 and Day 14 treated WT and homozygous mice to study possible genotype-specific alterations to the mucosal immune microenvironment.
These data are limited after adjustment for multiple comparison testing, but as exploratory data, the most significantly differentially expressed genes at Day 8 are Prkca (log2 fold decrease of -

1.35) and
Il-11 (log2 fold increase of 5.71) in homozygous Zip8 393T-KI mice compared to WT (Supplemental Table 1). Protein kinase C alpha (Prkca) is a Th17-cell-selective kinase regulating IL-17A production (30) and acts as a tumor suppressor in the intestine (31); reduced expression is associated with other immune-mediated phenotypes, including multiple sclerosis (32). Mouse models support Il-11 as a profibrotic cytokine (33,34). Recent single-cell RNAseq data have shown an association between inflammatory-type fibroblasts (IL13RA2 + -IL11 + ), IL-11 and Crohn's disease (35) and ulcerative colitis (36). Given the specific association of ZIP8 391-Thr with stricturing and penetrating Crohn's disease (6), the enhanced expression of a profibrotic cytokine is of particular interest, however, this will require further confirmatory mouse and human studies.

Human genetic association between ZIP8 391-Thr and Crohn's disease is more significant in males compared to females
The enhanced susceptibility to DSS-induced injury in male KI mice compared to female KI mice prompted us to ask if the association between ZIP8 391-Thr and Crohn's disease exhibits a sex-specific signal. Sex-stratified analysis was performed in a Crohn's disease casecontrol cohort (female, N = 7378; male, N = 7927). There is an association between ZIP8 391-Thr and Crohn's disease in both sexes; however, notably, the association is more significant in males (p=4.15E-09) than females (p=1.93E-05) (Supplemental Table 2). It is possible that the results are biased towards greater significance in males because the cohort includes more male individuals. However, we also speculate that these data may support differential, sex-specific effects of ZIP8 391-Thr in Crohn's disease and reinforce the role and impact of sex-specific mechanisms in Mn homeostasis.

N-glycosylation in human population-based samples
Finally, we sought to reinforce the relevance of abnormal Mn homeostasis associated with ZIP8 391-Thr in human data. Two small cohort studies have shown an association between ZIP8 391-Thr homozygous carriers and increased abundance of monosialo-monogalactobiantennary plasma glycans hypothesized to be secondary to reduced activity of Mn-dependent β-1,4-galactosyltransferase (19,21). Population-based studies of the plasma N-glycome were needed. Further, given the impact of alternative N-glycosylation at Asn297 site in the Fc domain of IgG on antibody effector function (37) and the initial observations of hypogalactosylation associated with ZIP8 391-Thr (19,21), we hypothesized that there would be an association between ZIP8 391-Thr and N-glycosylation of IgG. To study associations between ZIP8 391-Thr and the N-glycome, we used genome-wide association studies of human blood plasma (N=2763) (38) and IgG N-glycome (N=8080) (39), where N-glycome profiles were analyzed using ultrahigh performance liquid chromatography (UHPLC). The frequency of ZIP8 391-Thr was 8% and 8.3% respectively; notably, ZIP8 391-Thr (rs13107325) was not included in the initial analyses as it was not included on the original genotyping arrays. In these cohorts, ZIP8 391-Thr was associated with reduced plasma triantennary traits, including trisyalated and trigalactosylated structures ( Table 1; Supplemental Table 3). However, there were no associations between ZIP8 391-Thr and N-glycosylation of IgG (Supplemental Table 3).

The ZIP8 391-Thr-associated plasma N-glycan profile defines a distinct glycophenotype in patients with Crohn's disease
Plasma and IgG N-glycan profiles of patients with inflammatory bowel disease (IBD) have been recently studied by UHPLC (40,41). Increased triantennary and tetra-antennary glycan species are features of the IBD-associated plasma N-glycome compared to healthy individuals, therefore we next studied if there was an interaction between ZIP8 391-Thr and the plasma and IgG N-glycome in a cohort of patients with Crohn's disease (N=313). The frequency of ZIP8 391-Thr was 18% (6). Consistent with the genotype-specific plasma N-glycan profile found in the population data, three triantennary glycan species were decreased in patients with Crohn's disease with ZIP8 391-Thr compared to non-carriers ( Table 2; Supplemental Table 4), and there were no associations with IgG N-glycosylation. These data are distinct from the published analysis of patients with Crohn's disease (agnostic to genotype) compared to controls where there were no differences in two of the glycan species (A3F0GE, A3GE), and H6N5E3 was increased (40). Therefore, the ZIP8 391-Thr-associated glycophenotype defines a distinct patient subset within Crohn's disease. In addition to providing in vivo evidence of hypomorphic Zip8 function in the setting of 393T-KI, the Zip8 393T-KI mouse model importantly exhibits a more limited distribution of tissue sites with relative reductions in Mn compared to the inducible ZIP8 knockout mouse model (19). The two mouse models were not tested in parallel in this report, yet benchmarking our findings off the published literature (Supplemental Table 5), shows that male Zip8 iKO and liver-specific knockout results in 69%-77% reduction in liver Mn compared to matched controls, while the Zip8 393-TI KI male mice exhibit a 19% (heterozygous) and 28% (homozygous) reduction in liver Mn. Whole blood Mn is only reported in the LSKO mouse with a 65% reduction, whereas we observe a 18.9% reduction in the homozygous Zip8 393T-KI male mice. Therefore, we estimate the function of Zip8 in the context of Zip8 393T-KI to be approximately 70% compared to the wild-type allele, and because heterozygous Zip8 393T-KI mice also exhibit relative, albeit smaller reductions, in liver and kidney Mn, we speculate that Zip8 393T may act as a dominant negative mutation, although this remains to be tested.
The limited tissue distribution of relative Mn reductions in the Zip8 393T-KI compared to the Zip8 iKO model underscores an incomplete understanding of possible compensatory mechanisms at play when ZIP8 function is perturbed. As demonstrated by the lethality of Zip8 global knockout mouse models (42,43), Zip8 is required for embryogenesis and early life.
Potential interactions between ZIP8, ZIP14, and ZNT10 (as key regulators of Mn homeostasis) (44) or other compensatory mechanisms will require dedicated study across the mouse models. The additional variable of dietary Mn will also require testing. Together, these experiments will afford a more complete appreciation of the biological impact of ZIP8 391-Thr, mechanisms of Mn homeostasis, and interaction with dietary Mn. The DSS colitis model experiments provide important evidence that male Zip8 393T-KI homozygous and heterozygous mice, although phenotypically indistinguishable from WT at baseline, exhibited a disease-relevant phenotype once challenged. We acknowledge that the relevance of the DSS colitis model to ileal Crohn's disease, the subphenotype of Crohn's disease with the strongest association with ZIP8 391-Thr (6), can be debated, yet with this limitation, we use DSS colitis as a challenge of intestinal homeostasis in the setting of intestinal injury, and both the heterozygous and homozygous Zip8 393T-KI male mice exhibited a more severe phenotype than WT mice. We note a recent report of enhanced susceptibility to DSS-induced injury in mice fed Mn-deficient chow (45). We acknowledge that our experiments do not directly test that abnormal Mn homeostasis specifically drives the colitis phenotype, and future experiments will test variable Mn dietary content. Impairment of other activities of ZIP8, like zinc-mediated regulation of NF-κB signaling (14) or the zinc-ZIP8-MTF1 transcriptional cascade (15), may participate. The impact of inflammation on Mn homeostasis has not been tested.
Finally, the physiologic effects of increased biliary Mn are unknown, but may have particular importance when considering how dysregulation of Mn in the liver impacts the gut: A single study in a rodent model supports an interaction between increased taurohyodeoxycholic acid and low biliary Mn (46), however differential bile salt composition in the context of elevated biliary Mn has not been reported.
Coupled with understanding the role of Mn homeostasis on the colitis phenotype in the Zip8 393T-KI mice is studying the underlying sex-specific mechanisms of Mn handling and why the female Zip8 393T-KI fail to show a phenotype. Human studies have shown that blood Mn is reduced in males compared to females (22), a phenotype recapitulated in the Zip8 393T-KI mouse model. Further, males are known to require more dietary Mn than females (22). To our knowledge, sex-specific differential biliary Mn levels has not been previously explored. Is this sexual dimorphism with biological relevance linked to the sex-specific Mn homeostasis? Is the lack of phenotype in the female mice using the DSS-induced colitis model related to an inherent limitation of the DSS model (29) or, again, sex-specific Mn homeostasis? Are females more tolerant of Mn dyshomeostasis? Systematic assessment of the absorption, distribution and excretion of radiolabeled Mn, characterization of the mucosal immune microenvironment, and additional models of intestinal inflammation, such as bacterial infection models, may provide useful approaches to study these questions. Further, there are recent gene-interactions studies in children examining neurodevelopment and polymorphisms in Mn transporters, including ZIP8 391-Thr; these studies reinforce genotype, sex, age and timing of Mn exposures influence phenotype (47,48), therefore these factors will require study. We position the Zip8 393-Thr KI mice to provide a critical model with direct human relevance for further such studies.
The SNP-N-glycome analyses clarify that ZIP8 391-Thr-associated abnormal Mn homeostasis associates with reduced triantennary N-glycan branching in plasma (but not IgG Nglycome species) and implicates Mn-utilizing, N-acetylglucosaminyltransferases and/or downstream Mn-utilizing galactosyltransferases and sialotransferases. We speculate that the reduced abundance of trisylated and trigalactosylated glycans may be secondary to a critical limitation of Golgi N-acetylglucosaminyltransferasesour data implicate Nacetylglucosaminyltransferase IV (MGAT4) that catalyzes the formation of triantennary glycans.
In support of this hypothesis and because all N-glycan species on IgG are biantennary, bigalactosylated, and bisyalated in their most complex form (49), we found no association between ZIP8 391-Thr and the IgG N-glycome. Finally, if generation of triantennary glycans is impaired, we would expect reduced abundance of downstream tetra-antennary glycans; we found a trend in this direction, however it did not meet statistical significance after FDR correction.
This may reflect the overall low abundance of tetra-antennary glycans in plasma (<5.6% of total glycosylated proteins) impairing the power to detect genotype-dependent differential abundance (50).
Clinically, the association between ZIP8 391-Thr and reduced triantennary N-glycans begs the question, what is the significance of the ZIP8 391-Thr glycophenotype? Alpha-1-acid glycoprotein (AGP) is the most abundant plasma protein with triantennary and tetra-antennary N-glycans (50) and the leading protein to prioritize for study of the ZIP8 391-Thr glycophenotype. AGP is an acute phase reactant described as an immunomodulator and a major binding protein for endogenous ligands and drugs (51). AGP has five predicted N-glycosylation sites with over 150 glycoforms that affect the AGP-ligand-binding site and change protein dynamics (52,53). We speculate further study of differential biological function of AGP glycoforms, driven in part by ZIP8 391-Thr genotype and abnormal Mn homeostasis, may reveal mechanistic underpinnings of the pleiotropic disease associations of ZIP8 391-Thr. We acknowledge that a limitation to our current work is failing to demonstrate that the Zip8 393T-KI mice recapitulate the human glycophenotype; these studies are underway.
Mn status is not routinely considered in clinical practice, however we position our data to postulate that abnormal Mn homeostasis and dysregulation of Mn-dependent processes have under-appreciated clinical implicationspotentially with sex-specific effects. This central hypothesis will require further translational studies to understand how abnormal Mn homeostasis and impact on Mn-utilizing pathways (like N-glycosylation) contribute to pathogenesis of the diverse set of human diseases associated with ZIP8 391-Thr.

Generation of the Zip8 393T-KI mouse
We designed two single-guide RNA targeting Zip8 393Ala and a single-strand donor oligonucleotide for homology-directed repair harboring the three point mutations to knock-in our allele of interest, mutate the PAM site, and insert a restriction enzyme site for genotyping ( Figure S1A). Pronuclear injection of one-cell C57BL/6J embryos (Jackson Laboratories) was performed by the JHU Transgenic Core using standard microinjection techniques (54) using a mix of Cas9 protein (30ng/ul, PNABio), tracrRNA (0.6µM, Dharmacon), crRNA (0.6µM, IDT) and ssDNA oligo (10ng/ul, IDT) diluted in RNAse free injection buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA). Injected embryos were transferred into the oviducts of pseudopregnant ICR females (Envigo) using the technique described in Nagy, et al (55). Of 29 pups obtained, five had the desired modified alleles. These founder male mice were bred to Jackson B6 female mice to generate the heterozygous F2 generation that were used for breeding thereafter.
Genotyping was accomplished using a nested PCR approach and restriction enzyme digestion ( Figure S1B) and confirmed by Sanger sequencing (Figure S1C). Heterozygous mating yielded pups following an expected Mendelian genetics distribution. Mice were fed a standard mouse chow (Harlan Teklad Global 18% Protein Extruded Diet 2018SX, Mn 100pm); of note, 100ppm Mn is considered a Mn supplemented diet whereas Mn 'replete diets' most commonly include only 10ppm Mn.

Inductively coupled plasma mass spectrometry (ICP-MS)
Tissues were harvested promptly after sacrifice, placed in metal-free conical tubes (VWR), and wet weight recorded. To each sample, 200 µL of nitric acid (67 to 70% (w/w), TraceMetal

Atomic absorption spectroscopy
Metal analysis of whole blood and bile was carried out by atomic absorption spectroscopy (AAS) performed on a PerkinElmer Life Sciences AAnalyst 600 graphite furnace atomic absorption spectrometer. For whole blood assay, the blood was digested with nitric acid (1:2) (Fisher Chemical, TraceMetal Grade) and further diluted in double distilled water for AAS.
The sections were washed with TBST and incubated with anti-ZIP8 (Sigma-Aldrich, HPA038832) (1:50), MDR1 (Santa Cruz) and E-cadherin (1:100) (BD Transduction Laboratories) overnight at 4°C. The following day, sections were washed with TBST and stained for 1 hour at room temperature with anti-rabbit Alexa Fluor 488 (1:100) and anti-mouse Alexa Fluor 568 as the secondary antibodies, while protected from light and exposed to counterstains for DAPI. The sections were imaged on the Olympus FV3000RS confocal microscope with the 40X oil immersion objective as indicated.

mRNA extraction and quantitative PCR
RNA was extracted using Qiagen RNeasy mini kit per protocol. Purified RNA concentrations were measured using wavelengths of 260/280 nm on the Take3 micro-volume plate system (Biotek). From equal amounts of RNA, cDNA was generated using iSCRIPT. Real time quantitative PCR was performed and analyzed using SYBRGreen reagents on the QuantStudio 12K flex platform. Primer sets used included in Supplemental Table 6; Glyceraldehyde 3phosphate dehydrogenase (Gapdh) was included as internal control, and relative expression was calculated using the 2(-delta delta Ct) method.

Dextran sodium sulfate-induced colitis
Mice of all three genotypes were co-housed for the experiment. Mice at 11.5-13 weeks of age were fed 3.5% (w/v) dextran sodium sulfate (DSS, MP Biomedicals; MW=36,000-50,000) dissolved in sterile water ab libitum for 5 days. The 3.5% DSS water was replenished after 2 days and continued until the 5 th day. After five days, the mice were switched to sterile drinking water and followed for up to 9 days. The mice were monitored daily to assess survival, weight loss, and presence of rectal bleeding. At the end of the experiment, the mice were sacrificed by CO2 inhalation and terminal bleeding. Body weight, colon, liver and spleen weight, and colon length were recorded. The cecum and colon as a "swiss roll" were fixed in formalin and prepared for histology. The distal 1cm of colon was reserved for mRNA. Raw reads were normalized by background thresholding, geometic mean of positive controls and codeset content normalization with housekeeping genes. Data analysis was performed using R (v3.3.2) and NanoString Advanced Analysis platform for differential gene expression.

Sex-stratified analysis of association between rs13107325 and Crohn's disease
rs13107325 genotyping was available from the Illumina HumanExome+ array as previously described (6). Sex-stratified logistic regression, adjusting for population sub-stratification with 4 principal components, was performed to determine strength of association with Crohn's Disease and direction of effect in each sex separately (PLINK v1.9) (56,57). Sex-combined regression was performed for comparison.
Sex-and age-corrected total plasma (n=141 traits) and IgG (n=81 traits) N-glycomes for inflammatory bowel disease patients were measured or derived as previously published (40,41).
Rs13107325 genotyping was available from the HumanExome+ array (Illumina) (6) or Taqman SNP genotyping assay using a predesigned functionally tested probe (Applied Biosystems/ThermoFisher Scientific) and a 7900HT real-time PCR System (Applied Biosystems). SNP-N-glycome associations were analyzed using an additive genetic model and an FDR of <0.05.

Statistics. Statistical tests and sample numbers included in figure legends. Unpaired, one-sided
Student's t test was used for comparison of two groups. One-way ANOVA with multiple comparison testing with Kurskal-Wallis test with p-values for intergroup comparisons determined by Mann-Whitney test was used for three group comparisons. Linear regression was used to analyze the weight curves for the DSS experiments. A p value of <0.05 was considered statistically significant. Note additional statistical considerations for the N-glycome analyses.

Mouse experiments approved by the Johns Hopkins University Animal Care and Use
Committee. De-identified patient data, rs13107325 genotype, and plasma and IgG N-glycome data were analyzed with approvals as published (38)  Mn levels are comparable between WT and Zip8 393T-KI mice across additional tissue types. N=3-7 males/genotype, 8-10 weeks (Fig. 1B-D). (E) Biliary Mn is increased in Zip8 393T-KI homozygous mice compared to WT. N=4 males/genotype, 8-10 weeks, fasted. (F) There is no difference in liver Slc39a8 mRNA across genotypes. Relative expression, normalized to Gapdh mRNA. N=3 males/genotype, 8-10 weeks. (G) Zip8 localizes to the bile canalicular membrane like Mdr1 in WT and Zip8 393T-KI homozygous mice. Confocal images representative of at least 3 animals/genotype. Scale bars = 5um. Whole blood (Fig. 1A) and bile Mn (Fig. 1E) measured by atomic absorption spectroscopy; tissue Mn measured by ICP-MS and normalized to wet tissue weight (Fig. 1B-D). Mean and S.E.M. are graphed (Fig. 1A-1F). Statistical analysis by one-way ANOVA with Kruskal-Wallis and Dunn's multiple comparisons tests when 3 or more groups compared (Fig. 1A-C, 1F); unpaired, one-sided t-test used for comparison of 2 groups (Fig. 1D-E). (A) Initial body weight of Zip8 393T-KI homozygous mice at 11.5-13 weeks of age is higher compared to WT and heterozygous mice. N=16-20 males/genotype. (B) Zip8 393T-KI heterozygous and homozygous mice exhibit increased and more sustained weight loss in DSSinduced colitis model. Statistical analysis by linear regression (day [7][8][9][10][11][12][13][14]. Slopes equal between groups, but intercepts are 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 (non-significant, Chi-square test). (D) Day 14 histology is consistent with more inflammation in Zip8 393T-KI homozygous mice with crypt elongation, crypt branching (inset i), expansion of lamina propria immune infiltrate and squamous metaplasia (inset ii). Scale bar = 400um, inset 200 um. Images representative of n=5-7 mice/genotype. (E) Zip8 393T-KI heterozygous and homozygous mice have increased Il-6 mRNA, consistent with ongoing inflammation. Relative expression graphed, normalized to Gadph mRNA. N = 4-7 males/genotype. Statistical analysis by one-way ANOVA with Kruskal-Wallis and Dunn's multiple comparisons tests when 3 or more groups compared ( Fig. 2A, 2E).  Table 2. Associations between ZIP8 391-Thr and plasma N-glycome traits in patients with Crohn's disease reveal a distinct glycophenotype. Beta defines the effect size of ZIP8 391-Thr allele on plasma glycan trait. STAT is the T-statistics of association. T = beta / se, where sestandard error of the effect size.