C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.

An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference.

Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to ∼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.

Predicting exon criticality from protein sequence.

Alternative splicing is frequently involved in the diversification of protein function and can also be modulated for therapeutic purposes. Here we develop a predictive model, called Exon ByPASS (predicting Exon skipping Based on Protein amino acid SequenceS), to assess the criticality of exon inclusion based solely on information contained in the amino acid sequence upstream and downstream of the exon junctions. By focusing on protein sequence, Exon ByPASS predicts exon skipping independent of tissue and species in the absence of any intronic information. We validate model predictions using transcriptomic and proteomic data and show that the model can capture exon skipping in different tissues and species. Additionally, we reveal potential therapeutic opportunities by predicting synthetically skippable exons and neo-junctions arising in cancer cells.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

Warm nights disrupt transcriptome rhythms in field-grown rice panicles.

In rice, a small increase in nighttime temperature reduces grain yield and quality. How warm nighttime temperatures (WNT) produce these detrimental effects is not well understood, especially in field conditions where the typical day-to-night temperature fluctuation exceeds the mild increase in nighttime temperature. We observed genome-wide disruption of gene expression timing during the reproductive phase in field-grown rice panicles acclimated to 2 to 3 °C WNT. Transcripts previously identified as rhythmically expressed with a 24-h period and circadian-regulated transcripts were more sensitive to WNT than were nonrhythmic transcripts. The system-wide perturbations in transcript levels suggest that WNT disrupt the tight temporal coordination between internal molecular events and the environment, resulting in reduced productivity. We identified transcriptional regulators whose predicted targets are enriched for sensitivity to WNT. The affected transcripts and candidate regulators identified through our network analysis explain molecular mechanisms driving sensitivity to WNT and identify candidates that can be targeted to enhance tolerance to WNT.

Efficacy of Cochleated Amphotericin B in Mouse and Human Mucocutaneous Candidiasis.

Candida albicans causes debilitating, often azole-resistant, infections in patients with chronic mucocutaneous candidiasis (CMC). Amphotericin B (AMB) resistance is rare, but AMB use is limited by parenteral administration and nephrotoxicity. In this study, we evaluated cochleated AMB (CAMB), a new oral AMB formulation, in mouse models of oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC) and in patients with azole-resistant CMC. OPC and VVC were modeled in Act1-/- mice, and mucosal tissue fungal burden was assessed after once-daily treatment with CAMB, vehicle, or AMB-deoxycholate (AMB-d). Four patients with azole-resistant CMC enrolled in a phase 2 CAMB dose-escalation study. The primary endpoint was clinical improvement at 2 weeks followed by optional extension for long-term CMC suppression to assess safety and efficacy. CAMB-treated mice had significantly reduced tongue and vaginal fungal burdens compared to vehicle-treated mice and exhibited comparable fungal burden reduction relative to AMB-d-treated mice. All CAMB-treated patients reached clinical efficacy by 2 weeks, three at 400 mg twice daily and one at 200 mg twice-daily dosing. All patients continued to the extension phase, with three having sustained clinical improvement of OPC and esophageal candidiasis (EC) for up to 60 months. One patient had a relapse of esophageal symptoms at week 24 and was withdrawn from further study. Clinical responses were not seen for onychomycosis or VVC. CAMB was safe and well-tolerated, without any evidence of nephrotoxicity. In summary, oral CAMB reduced tongue and vaginal fungal burdens during murine candidiasis. A proof-of-concept clinical trial in human CMC showed efficacy with good tolerability and safety. This study has been registered at ClinicalTrials.gov under identifier NCT02629419.

Optimisation of momelotinib with improved potency and efficacy as pan-JAK inhibitor.

Dysregulated JAK-STAT signaling has been proven to be involved in several immune-mediated diseases. Several janus kinase (JAK) inhibitors have been approved for the treatment of various inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), plaque psoriasis, psoriatic arthritis, inflammatory bowel disease (IBD). Here, we report the design, optimisation, synthesis and biological evaluation of momelotinib analogues (a pyrimidine based JAK inhibitor), to get pan-JAK inhibitors. Systematic structure activity relationship studies led to the discovery of compound 32, which potently inhibited JAK1, JAK2 and JAK3. The in vivo investigation indicated that compound 32 possessed favourable pharmacokinetic properties and displayed superior anti-inflammatory efficacy than momelotinib 1. Accordingly, compound 32 was advanced into preclinical development.

Analysis of differential gene expression and alternative splicing is significantly influenced by choice of reference genome.

RNA-seq analysis has enabled the evaluation of transcriptional changes in many species including nonmodel organisms. However, in most species only a single reference genome is available and RNA-seq reads from highly divergent varieties are typically aligned to this reference. Here, we quantify the impacts of the choice of mapping genome in rice where three high-quality reference genomes are available. We aligned RNA-seq data from a popular productive rice variety to three different reference genomes and found that the identification of differentially expressed genes differed depending on which reference genome was used for mapping. Furthermore, the ability to detect differentially used transcript isoforms was profoundly affected by the choice of reference genome: Only 30% of the differentially used splicing features were detected when reads were mapped to the more commonly used, but more distantly related reference genome. This demonstrated that gene expression and splicing analysis varies considerably depending on the mapping reference genome, and that analysis of individuals that are distantly related to an available reference genome may be improved by acquisition of new genomic reference material. We observed that these differences in transcriptome analysis are, in part, due to the presence of single nucleotide polymorphisms between the sequenced individual and each respective reference genome, as well as annotation differences between the reference genomes that exist even between syntenic orthologs. We conclude that even between two closely related genomes of similar quality, using the reference genome that is most closely related to the species being sampled significantly improves transcriptome analysis.

Discovery of novel, potent and orally efficacious inhibitor of neutral amino acid transporter B0AT1 (SLC6A19).

Amino acid restriction by inhibition of neutral amino acid transporter, B0AT1 (SLC6A19) activity has been recently shown to improve glyceamic control by upregulating glucagon like peptide (GLP1) and fibroblast growth factor (FGF21) in mice. Hence, pharmacological inhibition of B0AT1 is expected to treat type-2 diabetes and related disorder. In this study, rationally designed trifluoromethyl sulfonyl derivatives were identified as novel, potent and orally bioavailable B0AT1 inhibitors. Compound 39 was found to be nanomolar potent (IC50: 0.035 µM) B0AT1 inhibitor with excellent pharmacokinetic profile (%F: 66) in mice and efficacious in vivo in diet induced obese (DIO) mice model.

CASE (CemiplimAb-rwlc Survivorship and Epidemiology) study in advanced cutaneous squamous cell carcinoma.

In 2018, cemiplimab-rwlc became the first systemic treatment approved by the US FDA for patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation. In 2019, conditional approvals were granted by Health Canada and the European Commission for the same indications. Limited data exist pertaining to the clinical characteristics, disease progression and survivorship of patients with advanced CSCC in real-world clinical practice. CemiplimAb-rwlc Survivorship and Epidemiology (CASE) is a prospective Phase IV, noninterventional, survivorship and epidemiology study that will enroll patients with advanced CSCC who have recently initiated or who plan to receive cemiplimab in a real-world setting. Trial registration number: NCT03836105.

Discovery of diaminopyrimidine-carboxamide derivatives as JAK3 inhibitors.

Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound 11i showed good anti-arthritic activity, which could be correlated with its improved oral bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of rheumatoid arthritis.

Novel transcriptional responses to heat revealed by turning up the heat at night.

KEY MESSAGE: The circadian clock controls many molecular activities, impacting experimental interpretation. We quantify the genome-wide effects of time-of-day on the heat-shock response and the effects of "diurnal bias" in stress experiments. Heat stress has significant adverse effects on plant productivity worldwide. Most experiments examining heat stress are performed during daytime hours, generating a 'diurnal bias' in the pathways and regulatory mechanisms identified. Such bias may confound downstream interpretations and limit our understanding of the full response to heat stress. Here we show that the transcriptional and physiological responses to a sudden heat shock in Arabidopsis are profoundly sensitive to the time of day. We observe that plant tolerance and acclimation to heat shock vary throughout the day and are maximal at dusk. Consistently, over 75% of heat-responsive transcripts show a time of day-dependent response, including many previously characterized heat-response genes. This temporal sensitivity implies a complex interaction between time and temperature where daily variations in basal transcription influence thermotolerance. When we examined these transcriptional responses, we uncovered novel night-response genes and cis-regulatory elements, underpinning new aspects of heat stress responses not previously appreciated. Exploiting this temporal variation can be applied to most environmental responses to understand the underlying network wiring. Therefore, we propose that using time as a perturbagen is an approach that will enhance our understanding of plant regulatory networks and responses to environmental stresses.

New candidate loci and marker genes on chromosome 7 for improved chilling tolerance in sorghum.

Sorghum is often exposed to suboptimal low temperature stress under field conditions, particularly at the seedling establishment stage. Enhancing chilling tolerance will facilitate earlier planting and so minimize the negative impacts of other stresses experienced at later growth stages. Genome-wide association mapping was performed on a sorghum association panel grown under control (30/20 °C; day/night) and chilling (20/10 °C) conditions. Genomic regions on chromosome 7, controlling the emergence index and seedling (root and shoot) vigor, were associated with increased chilling tolerance but they did not co-localize with undesirable tannin content quantitative trait loci (QTLs). Shoot and root samples from highly contrasting haplotype pairs expressing differential responses to chilling stress were used to identify candidate genes. Three candidate genes (an alpha/beta hydrolase domain protein, a DnaJ/Hsp40 motif-containing protein, and a YTH domain-containing RNA-binding protein) were expressed at significantly higher levels under chilling stress in the tolerant haplotype compared with the sensitive haplotype and BTx623. Moreover, two CBF/DREB1A transcription factors on chromosome 2 showed a divergent response to chilling in the contrasting haplotypes. These studies identify haplotype differences on chromosome 7 that modulate chilling tolerance by either regulating CBF or feeding back into this signaling pathway. We have identified new candidate genes that will be useful markers in ongoing efforts to develop tannin-free chilling-tolerant sorghum hybrids.

A VIGS screen identifies immunity in the Arabidopsis Pla-1 accession to viruses in two different genera of the Geminiviridae.

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus-resistant genes that derive from alterations in essential host factors. We used a virus-induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus-host interactions in 190 Arabidopsis accessions. Silencing of CH-42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla-1, lacked both symptoms and silencing, and was immune to wild-type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla-1 X Col-0 F2 population was used to detect a major peak on chromosome 1, which is designated gip-1 (geminivirus immunity Pla-1-1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip-1 locus suggest that a novel resistance gene(s) confers immunity.

VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans.

BACKGROUND: Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. OBJECTIVES: To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. METHODS: MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. RESULTS: Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. CONCLUSIONS: VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.

VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice.

Background: Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives: To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods: The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results: Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions: VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.

Axitinib, cabozantinib, or everolimus in the treatment of prior sunitinib-treated patients with metastatic renal cell carcinoma: results of matching-adjusted indirect comparison analyses.

BACKGROUND: In the absence of head-to-head trials comparing axitinib with cabozantinib or everolimus, the aim of this study was to conduct an indirect comparison of their relative efficacy in patients with metastatic renal cell carcinoma (mRCC), using data from the AXIS and METEOR trials. METHODS: Progression-free survival (PFS) and overall survival (OS) in prior sunitinib-treated patients with mRCC were compared by conducting matching-adjusted indirect comparison (MAIC) analyses, including base-case and sensitivity analyses. Individual patient-level data from prior sunitinib-treated patients who received axitinib in AXIS were weighted to match published baseline characteristics of prior sunitinib-treated patients who received either cabozantinib or everolimus in METEOR. RESULTS: There was no statistically significant difference in PFS (aHR [adjusted hazard ratio] = 1.15 [CI: 0.82-1.63]) and OS (aHR = 1.00 [CI: 0.69-1.46]) between axitinib versus cabozantinib in the base-case analysis. In the sensitivity analysis, PFS (aHR = 1.39 [CI: 1.00-1.92]) and OS (aHR = 1.35 [CI: 0.95-1.92]) were shorter for axitinib compared with cabozantinib; however, the OS difference was not statistically significant. Axitinib was associated with significantly longer PFS compared with everolimus in the base-case (aHR = 0.53 [CI: 0.36-0.80]) and sensitivity analyses (aHR = 0.63 [CI: 0.45-0.88]), respectively. Results suggested an OS benefit for axitinib versus everolimus in base-case analyses (aHR = 0.63 [CI: 0.42-0.96]); however, the difference in OS in the sensitivity analysis was not statistically significant (aHR = 0.84 [CI: 0.59-1.18]). CONCLUSIONS: MAIC analyses suggest PFS and OS for axitinib and cabozantinib are dependent on the Memorial Sloan Kettering Cancer Center definition used; in the base-case analysis, there was no significant difference in PFS and OS between axitinib and cabozantinib. In the sensitivity analysis, PFS in favour of cabozantinib was significant; however, the trend for prolonged OS with cabozantinib was not significant. For axitinib and everolimus, MAIC analyses indicate patients treated with axitinib may have an improved PFS and OS benefit when compared to everolimus. Disparities between the base-case and sensitivity analyses in this study underscore the importance of adjusting for the differences in baseline characteristics and that naïve indirect comparisons are not appropriate.

VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae.

When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during 'uranium-shock' implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Utilization of Positive and Negative Controls to Examine Comorbid Associations in Observational Database Studies.

BACKGROUND: Opportunities to leverage observational data for precision medicine research are hampered by underlying sources of bias and paucity of methods to handle resulting uncertainty. We outline an approach to account for bias in identifying comorbid associations between 2 rare genetic disorders and type 2 diabetes (T2D) by applying a positive and negative control disease paradigm. RESEARCH DESIGN: Association between 10 common and 2 rare genetic disorders [Hereditary Fructose Intolerance (HFI) and α-1 antitrypsin deficiency] and T2D was compared with the association between T2D and 7 negative control diseases with no established relationship with T2D in 4 observational databases. Negative controls were used to estimate how much bias and variance existed in datasets when no effect should be observed. RESULTS: Unadjusted association for common and rare genetic disorders and T2D was positive and variable in magnitude and distribution in all 4 databases. However, association between negative controls and T2D was 200% greater than expected indicating the magnitude and confidence intervals for comorbid associations are sensitive to systematic bias. A meta-analysis using this method demonstrated a significant association between HFI and T2D but not for α-1 antitrypsin deficiency. CONCLUSIONS: For observational studies, when covariate data are limited or ambiguous, positive and negative controls provide a method to account for the broadest level of systematic bias, heterogeneity, and uncertainty. This provides greater confidence in assessing associations between diseases and comorbidities. Using this approach we were able to demonstrate an association between HFI and T2D. Leveraging real-world databases is a promising approach to identify and corroborate potential targets for precision medicine therapies.