Building a vertically integrated genomic learning health system: The biobank at the Colorado Center for Personalized Medicine.

Precision medicine initiatives across the globe have led to a revolution of repositories linking large-scale genomic data with electronic health records, enabling genomic analyses across the entire phenome. Many of these initiatives focus solely on research insights, leading to limited direct benefit to patients. We describe the biobank at the Colorado Center for Personalized Medicine (CCPM Biobank) that was jointly developed by the University of Colorado Anschutz Medical Campus and UCHealth to serve as a unique, dual-purpose research and clinical resource accelerating personalized medicine. This living resource currently has more than 200,000 participants with ongoing recruitment. We highlight the clinical, laboratory, regulatory, and HIPAA-compliant informatics infrastructure along with our stakeholder engagement, consent, recontact, and participant engagement strategies. We characterize aspects of genetic and geographic diversity unique to the Rocky Mountain region, the primary catchment area for CCPM Biobank participants. We leverage linked health and demographic information of the CCPM Biobank participant population to demonstrate the utility of the CCPM Biobank to replicate complex trait associations in the first 33,674 genotyped individuals across multiple disease domains. Finally, we describe our current efforts toward return of clinical genetic test results, including high-impact pathogenic variants and pharmacogenetic information, and our broader goals as the CCPM Biobank continues to grow. Bringing clinical and research interests together fosters unique clinical and translational questions that can be addressed from the large EHR-linked CCPM Biobank resource within a HIPAA- and CLIA-certified environment.

MicroRNA-146a is induced by inflammatory stimuli in airway epithelial cells and augments the anti-inflammatory effects of glucocorticoids.

BACKGROUND: MicroRNAs (miRNAs) are emerging as central regulators of inflammation, but their role in asthma and airway epithelial cells is not well studied. Glucocorticoids are the cornerstone of therapy in asthma and other inflammatory disease, yet their mechanisms of action are not completely elucidated, and it is not clear whether miRNAs modulate their effects. OBJECTIVE: We aimed to identify miRNAs that regulate cytokine and chemokine expression in airway epithelial cells and whether these miRNAs are subject to the effects of glucocorticoids. METHODS AND RESULTS: MicroRNAomic analyses of immortalized, normal human bronchial epithelial cells identified 7 miRNAs that were altered by inflammatory cytokine treatment and 22 that were regulated by glucocorticoids (n = 3 for each treatment condition). MiR-146a emerged as a central candidate, whose expression was induced by TNF-α and repressed by glucocorticoids. Its role as a candidate in asthmatic inflammation was supported by expression profiling in human asthmatics, which showed that plasma miR-146a expression was elevated in asthma and associated with measures related to worse asthma outcomes, including elevated blood eosinophil counts, higher asthma control questionnaire scores, and need for higher doses of inhaled glucocorticoids. However, transfection of miR-146a in A549 cells treated with TNF-α +/- glucocorticoids produced an anti-inflammatory effect and increased efficacy of glucocorticoids. CONCLUSIONS: We propose a model whereby miR-146a is induced by inflammatory conditions as a feedback mechanism to limit inflammation. Exogenous administration of miR-146a augmented the effects of glucocorticoids and could be a novel therapeutic strategy to enhance efficacy of these medications.

Effects of allergic stimulation and glucocorticoids on miR-155 in CD4+ T-cells.

RATIONALE: MicroRNAs (miRNAs) are emerging as important regulators of allergic inflammation and potential therapeutic targets. We sought to identify which miRNAs are expressed in CD4+ T-cells and determine whether allergic stimuli or glucocorticoids alter their expression. METHODS: After IRB approval, blood was collected from dust mite (DM) allergic rhinitis subjects (n=20), non-allergic controls (n=8), and asthmatics (n=16). Peripheral blood mononuclear cells were incubated with dust mite extract (DME), diluent control, or DME + dexamethasone (0.1 µM). CD4+ T-cells were collected by magnetic bead column, and RNA was isolated by guanidinium/phenol-chloroform extraction. MicroRNA expression was measured using Nanostring microarray and quantitative real time PCR (qPCR). RESULTS: We identified 196 miRNAs that were stably expressed in circulating CD4+ T-cells. Allergen stimulation of CD4+ T-cells with DME differentially induced miR-155 expression in cells of DM-allergic subjects as compared to non-allergic subjects. Induction of miR-155 expression was also observed with anti-CD3/anti-CD28 simulation and phorbol-12-Myristate-13-Acetate (PMA) treatment, and further augmented by calcium inophore and bromocyclic AMP in the latter treatment. The level of miR-155 expression was positively associated with expression of the TH2 cytokines IL-5 and IL-13. Inhibition of miR-155 in Jurkat T-cells inhibited the production of these cytokines. Glucocorticoids attenuated the effects of dust mite allergen, raising the possibility that inhibition of this miRNA could be a mechanism through which glucocorticoids exhibit their anti-inflammatory effects. The CD4+ T-cells had a higher level of miR-155 expression in asthma compared to in allergic rhinitis and non-asthmatics. The inhibitory effects of glucocorticoids on CD4+ T-cell miR-155 expression were lost in severe asthmatics. CONCLUSION: Mir-155 is differentially expressed in allergic T-cells exposed to DM extract compared to in non-allergic cells and it is inhibited by glucocorticoids. MiR-155 may play a role in mediating allergic inflammation in T-cells and could be an anti-inflammatory target of steroids. This pathway may be de-regulated in severe asthma.

Genetic and environmental influences on plasma vitamin D binding protein concentrations.

Recent studies suggest that low vitamin D-binding protein (VDBP aka group-specific complement or Gc) concentrations may be linked with inflammatory-mediated conditions, including asthma, chronic obstructive pulmonary disease, and cancer. However, these studies may be confounded by substantial racial and ethnic or genetic differences. The purpose of this study was to test the hypothesis that circulating VDBP concentrations are significantly associated with genetic ancestry. We used a validated high-performance liquid chromatography tandem mass spectrometry assay of 25-hydroxyvitamin D3 and its downstream metabolite 24,25-dihydroxyvitamin D3. VDBP concentrations (milligrams per liter) were measured in duplicate using a commercial enzyme-linked immunosorbent assay among healthy African American (n = 56) and Caucasian American (n = 60) participants. Ancestry informative markers across the genome were used to estimate individual genetic ancestry proportions, designed to robustly distinguish between West African and European ancestry. Genotype-defined Gc isoforms were defined using rs7041 and rs4588 combination groups. VDBP concentration was correlated with both Gc isoform (r = 0.93, P < 0.001) and West African genetic ancestry (r = -0.66, P < 0.001). In the final model, Gc isoform, the catabolic ratio of serum vitamin D, oral contraceptive use, and body mass index remained significantly associated with VDBP concentration, after adjustment for genetic ancestry. Failure to adjust for Gc isoform may lead to spurious associations in studies of VDBP concentration and disease risk, particularly when the condition of interest may also be associated with genetic ancestry. The higher circulating VDBP concentrations and higher vitamin D catabolic rate among Caucasian Americans observed here appear to be consistent with lower bone mineral density and racial and ethnic differences in vitamin D-inducing cytokines.

MicroRNA-570-3p regulates HuR and cytokine expression in airway epithelial cells.

Asthma is a chronic lung disease that affects people of all ages and is characterized by high morbidity. The mechanisms of asthma pathogenesis are unclear, and there is a need for development of diagnostic biomarkers and greater understanding of regulation of inflammatory responses in the lung. Post-transcriptional regulation of cytokines, chemokines, and growth factors by the action of microRNAs and RNA-binding proteins on stability or translation of mature transcripts is emerging as a central means of regulating the inflammatory response. In this study, we demonstrate that miR-570-3p expression is increased with TNFα stimuli in normal human bronchial epithelial cells (2.6 ± 0.6, p = 0.01) and the human airway epithelial cell line A549 (4.6 ± 1.4, p = 0.0068), and evaluate the functional effects of its overexpression on predicted mRNA target genes in transfected A549 cells. MiR-570-3p upregulated numerous cytokines and chemokines (CCL4, CCL5, TNFα, and IL-6) and also enhanced their induction by TNFα. For other cytokines (CCL2 and IL-8), the microRNA exhibited an inhibitory effect to repress their upregulation by TNFα. These effects were mediated by a complex pattern of both direct and indirect regulation of downstream targets by miR-570-3p. We also show that the RNA-binding protein HuR is a direct target of miR-570-3p, which has implications for expression of numerous other inflammatory mediators that HuR is known regulate post-transcriptionally. Finally, expression of endogenous miR-570-3p was examined in both serum and exhaled breath condensate (EBC) from asthmatic and healthy patients, and was found to be significantly lower in EBC of asthmatics and inversely correlated to their lung function. These studies implicate miR-570-3p as a potential regulator of asthmatic inflammation with potential as both a diagnostic and therapeutic target in asthma.

Post-transcriptional regulation of meprin α by the RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP).

Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3'-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease.

Lead, calcium uptake, and related genetic variants in association with renal cell carcinoma risk in a cohort of male Finnish smokers.

BACKGROUND: Lead is classified as a probable human carcinogen. However, its role in renal cell cancer (RCC) has not been established. Calcium and vitamin D may off-set toxicity in vivo. METHODS: In this nested case-control study, whole blood lead, total serum calcium, and serum 25-hydroxyvitamin D levels were measured in blood drawn prior to diagnosis among male smokers participating in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Single-nucleotide polymorphisms (SNP) in five genes (CALB1, TRPV5, TRPV6, VDR, and ALAD) related to lead toxicity or calcium transport were genotyped. Logistic and linear regressions were used to determine RCC risk and time to diagnosis (respectively), adjusting for other risk factors. RESULTS: Among 154 newly diagnosed cases and 308 matched controls, RCC was associated with higher whole blood lead [OR = 2.0; 95% confidence interval (CI), 1.0-3.9; quartile 4 (Q4) vs. Q1, P(trend) = 0.022] and CALB1 rs1800645 (P(trend) = 0.025, minor 'T' allele frequency = 0.34). Higher total serum calcium (P(trend) ≤ 0.001) was associated with reduced RCC risk. Total serum calcium and 25-hydroxyvitamin D levels did not alter the association observed with lead. Time from enrollment to RCC diagnosis was positively associated with serum calcium (P(trend) = 0.002) and 25-hydroxyvitamin D (P(trend) = 0.054) among cases. CONCLUSIONS: Higher blood lead concentrations, below the 10 µg/dL level of concern, were associated with RCC, independent from serum calcium and CALB1 promoter polymorphism. IMPACT: Increased risk of RCC is associated with lower serum calcium and higher whole blood lead in smokers. The clinical prognostic value of serum calcium and vitamin D in RCC should be further investigated.

Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5.

Asthma is a chronic inflammatory disease that can be difficult to manage due to a lack of diagnostic biomarkers and an incomplete understanding of the molecular pathogenesis. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs with increasing importance in regulation of immune function and as biomarkers. We profiled miRNAs in the serum of asthmatics and non-asthmatic controls to identify miRNAs that could serve as diagnostic markers and potential regulators of allergic inflammation. Differential expression of miR-1248, miR-26a, Let-7a, and Let-7d were observed in asthmatic patients compared to controls. Predictive algorithm analyses of these miRNAs revealed their specificity for different Th2 cytokines, including IL-5, which has not previously been shown to be post-transcriptionally regulated. Using multiple approaches, we showed that miR-1248 physically interacts with the IL-5 transcript in the 3' untranslated region and serves as a positive regulator to increase IL-5 expression. Collectively, our results demonstrate a previously uncharacterized mode of regulation of IL-5 expression and potential use for miRNAs in the diagnosis and clinical management of asthma.

Genetic Ancestry, Skin Reflectance and Pigmentation Genotypes in Association with Serum Vitamin D Metabolite Balance.

BACKGROUND: Lower serum vitamin D (25(OH)D) among individuals with African ancestry is attributed primarily to skin pigmentation. However, the influence of genetic polymorphisms controlling for skin melanin content has not been investigated. Therefore, we investigated differences in non-summer serum vitamin D metabolites according to self-reported race, genetic ancestry, skin reflectance and key pigmentation genes (SLC45A2 and SLC24A5). MATERIALS AND METHODS: Healthy individuals reporting at least half African American or half European American heritage were frequency matched to one another on age (+/- 2 years) and sex. 176 autosomal ancestry informative markers were used to estimate genetic ancestry. Melanin index was measured by reflectance spectrometry. Serum vitamin D metabolites (25(OH)D3, 25(OH)D 2 and 24,25(OH)2D3) were determined by high performance liquid chromatography (HPLC) tandem mass spectrometry. Percent 24,25(OH)2D3 was calculated as a percent of the parent metabolite (25(OH)D3). Stepwise and backward selection regression models were used to identify leading covariates. RESULTS: Fifty African Americans and 50 European Americans participated in the study. Compared with SLC24A5 111Thr homozygotes, individuals with the SLC24A5 111Thr/Ala and 111Ala/Ala genotypes had respectively lower levels of 25(OH)D3 (23.0 and 23.8 nmol/L lower, p-dominant=0.007), and percent 24,25(OH)2D3 (4.1 and 5.2 percent lower, p-dominant=0.003), controlling for tanning bed use, vitamin D/fish oil supplement intake, race/ethnicity, and genetic ancestry. Results were similar with melanin index adjustment, and were not confounded by glucocorticoid, oral contraceptive, or statin use. CONCLUSIONS: The SLC24A5 111Ala allele was associated with lower serum vitamin 25(OH)D3 and lower percent 24,25(OH)2D3, independently from melanin index and West African genetic ancestry.

A novel SNP in a vitamin D response element of the CYP24A1 promoter reduces protein binding, transactivation, and gene expression.

The active form of vitamin D (1alpha,25(OH)(2)D(3)) is known to have antiproliferative effects and has been implicated in cancers of the colon, breast, and prostate. These cancers occur more frequently among African Americans than Caucasians, and individuals with African ancestry are known to have approximately twofold lower levels of serum vitamin D (25(OH)D) compared with individuals of European ancestry. However, epidemiological studies of the vitamin D receptor (VDR) have shown inconsistent associations with cancer risk, suggesting that differences in other genes in the pathway may be important. We sought to identify functionally significant polymorphic variants in CYP24A1, a gene that is highly inducible by 1alpha,25(OH)(2)D(3) and that encodes the primary catabolic enzyme in the pathway. Here we report the identification of six novel SNPs in the human CYP24A1 promoter, including one at nucleotide -279 occurring within the distal vitamin D response element (VDRE2). Our experiments demonstrate that the VDRE2 variant results in decreased protein binding and transactivation in vitro, and reduced expression of CYP24A1 in cultured primary human lymphocytes provides evidence for an effect in vivo. This variant was only observed in our African American population, and represents a first step toward understanding differences in disease risk among racial/ethnic groups.