Folami Y. Ideraabdullah

Vitamin D insufficiency (VDI) is primarily determined by serum levels of calcidiol, which serves as a biomarker for the less abundant but most potent bioactive metabolite, calcitriol. However, population studies often show discordance between calcidiol and calcitriol. Here, a genetically diverse population of 7 inbred mouse strains was used to investigate the role of interindividual genetic differences in driving calcidiol-to-calcitriol discordance under vitamin D sufficient (VDS) vs depleted (VDD) conditions. We found high interstrain variability in calcitriol that was discordant with calcidiol under VDS and VDD conditions. However, under VDS conditions, stratification by calcitriol level revealed that strains with serum calcitriol >60 pM (HighC) exhibited the expected positive calcidiol-to-calcitriol association, whereas strains with low calcitriol (<60 pM, LowC) did not. Thus, discordance under VDS was driven by genetically divergent strains with LowC. Discordance under VDD was not associated with LowC. LowC was not caused by increased calcitriol degradation or by transcriptional dysregulation of canonical vitamin D metabolism enzymes. Instead, LowC strains exhibited low renal expression of Lrp2 (megalin), the primary transporter required for renal calcitriol production. LowC strains also exhibited reduced renal expression of the vitamin D receptor (Vdr) and several target genes, demonstrating impaired vitamin D signaling. These findings reveal novel, naturally occurring genetic determinants of VDI that function by disrupting calcitriol production and signaling in a manner that cannot be predicted by calcidiol levels. Cross-species conservation of this phenomenon would have important implications for clinical management of VDI and related disease risks across genetically diverse populations.

Standard chow diets influence reproducibility in animal model experiments because chows have different nutrient compositions, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here we measured the impact of different diets (5V5M, 5V0G, 2920X and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE) and developmental exposure (DE)) on growth and development, metabolic health indicators and gut bacterial microbiota profiles across genetically identical C57BL/6J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on phenotypic outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences only from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. However, compared with AE, LE effects on beta diversity were sex dependent, and LE mice exhibited nine times more differentially abundant bacterial genera, the majority of which were inversely affected by 2920X and 5058 diets. Our findings demonstrate that LE to different chow diets has the greatest impact on the reproducibility of several experimental measures commonly used in preclinical mouse model studies. Importantly, weaning mice from different diets onto the same diet for maturation may be an effective way to reduce unwanted phenotypic variability among experimental models.

Standard chow diet contributes to reproducibility in animal model experiments since chows differ in nutrient composition, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here, we measured the impact of diet (5V5M, 5V0G, 2920X, and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE), and developmental exposure (DE)) on growth & development, metabolic health indicators, and gut bacterial microbiota profiles across genetically identical C57BL6/J mice. Diet drove differences in macro-and micronutrient intake for all exposure models. AE had no effect on measured outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight, and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. Diet-induced beta diversity profiles were sex-dependent for LE mice. Compared to AE, LE drove 9X more diet-driven differentially abundant genera, majority of which were the result of inverse effects of 2920X and 5058. Our findings demonstrate that lifetime exposure to different chow diets has the greatest impact on reproducibility of experimental measures that are common components of preclinical mouse model studies. Importantly, weaning DE mice onto a uniform diet is likely an effective way to reduce unwanted phenotypic variability among experimental models.

Genomic imprinting results in gene expression bias caused by parental chromosome of origin and occurs in genes with important roles during human brain development. However, the cell-type and temporal specificity of imprinting during human neurogenesis is generally unknown. By detecting within-donor allelic biases in chromatin accessibility and gene expression that are unrelated to cross-donor genotype, we inferred imprinting in both primary human neural progenitor cells and their differentiated neuronal progeny from up to 85 donors. We identified 43/20 putatively imprinted regulatory elements (IREs) in neurons/progenitors, and 133/79 putatively imprinted genes in neurons/progenitors. Although 10 IREs and 42 genes were shared between neurons and progenitors, most putative imprinting was only detected within specific cell types. In addition to well-known imprinted genes and their promoters, we inferred novel putative IREs and imprinted genes. Consistent with both DNA methylation-based and H3K27me3-based regulation of imprinted expression, some putative IREs also overlapped with differentially methylated or histone-marked regions. Finally, we identified a progenitor-specific putatively imprinted gene overlapping with copy number variation that is associated with uniparental disomy-like phenotypes. Our results can therefore be useful in interpreting the function of variants identified in future parent-of-origin association studies.

Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental development. We conducted an epigenome-wide association study of 42 placentas from pregnancies in the South African Soweto First 1000 days cohort (S1000). Using genome-wide bisulfite sequencing, we compared non-GDM placentas to GDM placentas with similar proportions from obese and non-obese mothers. Compared to non-GDM, GDM placentas exhibited a distinct methylation profile consisting of 12,210 differentially methylated CpGs (DMCs) that mapped to 3,875 genes. Epigenetically altered genes were enriched in Wnt and cadherin signalling pathways, both critical in placentation and embryogenesis. We also defined regional DNA methylation perturbation in GDM placentas at 11 placental development genes. These findings reveal extensive changes to the placental epigenome of GDM pregnancies and highlight perturbation enriched at important placental development genes. These molecular changes represent potential mechanisms for GDM-induced placental effects that may serve as candidate biomarkers for placental, maternal, and foetal health. Using a study design that used similar proportions of obese and non-obese mothers in our case and control pregnancies, we minimized the detection of changes due to obesity alone. Further work will be necessary to investigate the extent of the influence of obesity on these GDM-related placental epigenetic changes.

Genomic imprinting results in gene expression biased by parental chromosome of origin and occurs in genes with important roles during human brain development. However, the cell-type and temporal specificity of imprinting during human neurogenesis is generally unknown. By detecting within-donor allelic biases in chromatin accessibility and gene expression that are unrelated to cross-donor genotype, we inferred imprinting in both primary human neural progenitor cells (phNPCs) and their differentiated neuronal progeny from up to 85 donors. We identified 43/20 putatively imprinted regulatory elements (IREs) in neurons/progenitors, and 133/79 putatively imprinted genes in neurons/progenitors. Though 10 IREs and 42 genes were shared between neurons and progenitors, most imprinting was only detected within specific cell types. In addition to well-known imprinted genes and their promoters, we inferred novel IREs and imprinted genes. We found IREs overlapped with CpG islands more than non-imprinted regulatory elements. Consistent with DNA methylation-based regulation of imprinted expression, some putatively imprinted regulatory elements also overlapped with differentially methylated regions on the maternal germline. Finally, we identified a progenitor-specific putatively imprinted gene overlap with copy number variation that is associated with uniparental disomy-like phenotypes. Our results can therefore be useful in interpreting the function of variants identified in future parent-of-origin association studies.

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

Liver metabolite concentrations have the potential to be key biomarkers of systemic metabolic dysfunction and overall health. However, for most conditions we do not know the extent to which genetic differences regulate susceptibility to metabolic responses. This limits our ability to detect and diagnose effects in heterogeneous populations. Here, we investigated the extent to which naturally occurring genetic differences regulate maternal liver metabolic response to vitamin D deficiency (VDD), particularly during perinatal periods when such changes can adversely affect maternal and fetal health. We used a panel of 8 inbred Collaborative Cross (CC) mouse strains, each with a different genetic background (72 dams, 3–6/treatment group, per strain). We identified robust maternal liver metabolic responses to vitamin D depletion before and during gestation and lactation using a vitamin-D-deficient (VDD; 0 IU vitamin D₃/kg) or -sufficient diet (1000 IU vitamin D₃/kg). We then identified VDD-induced metabolite changes influenced by strain genetic background. We detected a significant VDD effect by orthogonal partial least squares discriminant analysis (Q2 = 0.266, pQ2 = 0.002): primarily, altered concentrations of 78 metabolites involved in lipid, amino acid, and nucleotide metabolism (variable importance to projection score ≥1.5). Metabolites in unsaturated fatty acid and glycerophospholipid metabolism pathways were significantly enriched [False Discovery Rate (FDR) <0.05]. VDD also significantly altered concentrations of putative markers of uremic toxemia, acylglycerols, and dipeptides. The extent of the metabolic response to VDD was strongly dependent on genetic strain, ranging from robustly responsive to nonresponsive. Two strains (CC017/Unc and CC032/GeniUnc) were particularly sensitive to VDD; however, each strain altered different pathways. These novel findings demonstrate that maternal VDD induces different liver metabolic effects in different genetic backgrounds. Strains with differing susceptibility and metabolic response to VDD represent unique tools to identify causal susceptibility factors and further elucidate the role of VDD-induced metabolic changes in maternal and/or fetal health for ultimately translating findings to human populations.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well annotated genome, wealth of genetic resources and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost effective, informative and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole genome sequencing. Here we describe the content and performance of a new Mouse Universal Genotyping Array (MUGA). MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: 1) chromosomal sex determination, 2) discrimination between substrains from multiple commercial vendors, 3) diagnostic SNPs for popular laboratory strains, 4) detection of constructs used in genetically engineered mice, and 5) an easy to interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA we genotyped 6,899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived and recombinant inbred lines. Here we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, the extension of the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. There is preliminary but striking evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the main stock for a significant action of the genotyped inbred samples. We conclude that MiniMUGA is a valuable platform for genetic QC and important new tool to the increase rigor and reproducibility of mouse research.

Deficiency of methyl donor nutrients folate, choline, and methionine (methyl deficiency) during gestation can impair fetal development and perturb DNA methylation. Here, we assessed genetic susceptibility to methyl deficiency by comparing effects in wildtype C57BL/6J (B6) mice to mutant mice carrying a 1.3 kb deletion at the H19/Igf2 Imprinting Control Region (ICR) ( H19^ICRΔ2,3). The H19^ICRΔ2,3 mutation mimics microdeletions observed in Beckwith-Wiedemann syndrome (BWS) patients, who exhibit epimutations in cis that cause loss of imprinting and fetal overgrowth. Dams were treated during pregnancy with 1 of 4 methyl sufficient (MS) or methyl deficient (MD) diets, with or without the antibiotic commonly used to deplete folate producing gut microbes. As expected, after ~9 weeks of treatment, dams in MD and MD + antibiotic groups exhibited substantially reduced plasma folate concentrations. H19^ICRΔ2,3 mutant lines were more susceptible to adverse pregnancy outcomes caused by methyl deficiency (reduced birth rate and increased pup lethality) and antibiotic (decreased litter size and litter survival). Surprisingly, pup growth/development was only minimally affected by methyl deficiency, while antibiotic treatment caused inverse effects on B6 and H19^ICRΔ2,3 lines. B6 pups treated with antibiotic exhibited increased neonatal and weanling bodyweight, while both wildtype and mutant pups of heterozygous H19^ICRΔ2,3/+ dams exhibited decreased neonatal bodyweight that persisted into adulthood. Interestingly, only antibiotic-treated pups carrying the H19^ICRΔ2,3 mutation exhibited altered DNA methylation at the H19/Igf2 ICR, suggesting ICR epimutation was not sufficient to explain the altered phenotypes. These findings demonstrate that genetic mutation of the H19/Igf2 ICR increases offspring susceptibility to developmental perturbation in the methyl deficiency model, maternal and pup genotype play an essential role, and antibiotic treatment in the model also plays a key independent role.