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BackgroundHeterogeneity exists in type 1 diabetes (T1D) development and presentation. Islet autoantibodies form the foundation for T1D diagnostic and staging efforts. We hypothesized that autoantibodies can be used to identify heterogeneity in T1D before, at, and after diagnosis, and in response to disease modifying therapies. at clinically relevant timepoints throughout T1D progression.MethodsWe performed a systematic review assessing 10 years of original research studies examining relationships between autoantibodies and heterogeneity during disease progression, at the time of diagnosis, after diagnosis, and in response to disease modifying therapies in individuals at risk for T1D or within 1 year of T1D diagnosis.Results10,067 papers were screened. Out of 151 that met data extraction criteria, 90 studies characterized heterogeneity before clinical diagnosis. Autoantibody type/target was most commonly examined, followed by autoantibody number, titer, order of seroconversion, affinity, and novel islet autoantibodies/epitopes. Recurring themes included positive relationships of autoantibody number and specific types and titers with disease progression, differing clinical phenotypes based on the order of autoantibody seroconversion, and interactions with age and genetics. Overall, reporting of autoantibody assay performance was commonly included; however, only 43% (65/151) included information about autoantibody assay standardization efforts. Populations studied were almost exclusively of European ancestry.ConclusionsCurrent evidence most strongly supports the application of autoantibody features to more precisely define T1D before clinical diagnosis. Our findings support continued use of pre-clinical staging paradigms based on autoantibody number and suggest that additional autoantibody features, particularly when considered in relation to age and genetic risk, could offer more precise stratification. Increased participation in autoantibody standardization efforts is a critical step to improving future applicability of autoantibody-based precision medicine in T1D.Plain Language SummaryWe performed a systematic review to ascertain whether islet autoantibodies, biomarkers of autoimmunity against insulin-producing cells, could aid in stratifying individuals with different clinical presentations of type 1 diabetes. We found existing evidence most strongly supporting the application of these biomarkers to the period before clinical diagnosis, when certain autoantibody features (number, type) and the age when they develop, can provide important information for patients and care providers on what to expect for future type 1 diabetes progression.

BackgroundType 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing beta cells. Efforts to prevent T1D have focused on modulating immune responses and supporting beta cell health; however, heterogeneity in disease progression and responses to therapies have made these efforts difficult to translate to clinical practice, highlighting the need for precision medicine approaches to T1D prevention.MethodsTo understand the current state of knowledge regarding precision approaches to T1D prevention, we performed a systematic review of randomized-controlled trials from the past 25 years testing disease-modifying therapies in T1D and/or identifying features linked to treatment response, analyzing bias using a Cochrane-risk-of-bias instrument.ResultsWe identified 75 manuscripts, 15 describing 11 prevention trials for individuals with increased risk for T1D, and 60 describing treatments aimed at preventing beta cell loss in individuals at disease onset. Seventeen agents tested, mostly immunotherapies, showed benefit compared to placebo (only two prior to T1D onset). Fifty-seven studies employed precision analyses to assess features linked to treatment response. Age, measures of beta cell function and immune phenotypes were most frequently tested. However, analyses were typically not prespecified, with inconsistent methods reporting, and tended to report positive findings.ConclusionsWhile the quality of prevention and intervention trials was overall high, low quality of precision analyses made it difficult to draw meaningful conclusions that inform clinical practice. Thus, prespecified precision analyses should be incorporated into the design of future studies and reported in full to facilitate precision medicine approaches to T1D prevention.Plain Language SummaryType 1 diabetes (T1D) results from the destruction of insulin-producing cells in the pancreas, necessitating lifelong insulin dependence. T1D prevention remains an elusive goal, largely due to immense variability in disease progression. Agents tested to date in clinical trials work in a subset of individuals, highlighting the need for precision medicine approaches to prevention. We systematically reviewed clinical trials of disease-modifying therapy in T1D. While age, measures of beta cell function, and immune phenotypes were most commonly identified as factors that influenced treatment response, the overall quality of these studies was low. This review reveals an important need to proactively design clinical trials with well-defined analyses to ensure that results can be interpreted and applied to clinical practice.

Type 1 diabetes is a complex, chronic disease in which the insulin-producing beta cells in the pancreas are sufficiently altered or impaired to result in requirement of exogenous insulin for survival. The development of type 1 diabetes is thought to be an autoimmune process, in which an environmental (unknown) trigger initiates a T cell-mediated immune response in genetically susceptible individuals. The presence of islet autoantibodies in the blood are signs of type 1 diabetes development, and risk of progressing to clinical type 1 diabetes is correlated with the presence of multiple islet autoantibodies. Currently, a “staging” model of type 1 diabetes proposes discrete components consisting of normal blood glucose but at least two islet autoantibodies (Stage 1), abnormal blood glucose with at least two islet autoantibodies (Stage 2), and clinical diagnosis (Stage 3). While these stages may, in fact, not be discrete and vary by individual, the format suggests important applications of precision medicine to diagnosis, prevention, prognosis, treatment and monitoring. In this paper, applications of precision medicine in type 1 diabetes are discussed, with both opportunities and barriers to global implementation highlighted. Several groups have implemented components of precision medicine, yet the integration of the necessary steps to achieve both short- and long-term solutions will need to involve researchers, patients, families, and healthcare providers to fully impact and reduce the burden of type 1 diabetes.

Genome-wide association studies (GWAS) have identified hundreds of genetic risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWAS and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotype information to identify quantitative trait loci (QTL) for gene expression and splicing in coronary arteries obtained from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary arteries and 19% exhibited cell-type-specific expression. Colocalization analysis with GWAS identified subgroups of eGenes unique to CAD and blood pressure. Fine-mapping highlighted additional eGenes of interest, includingTBX20andIL5. Splicing (s)QTLs for 1,690 genes were also identified, among whichTOR1AIP1andULK3sQTLs demonstrated the importance of evaluating splicing events to accurately identify disease-relevant gene expression. Our work provides the first human coronary artery eQTL resource from a patient sample and exemplifies the necessity of diverse study populations and multi-omic approaches to characterize gene regulation in critical disease processes.Study Design Overview

Translating genetic discoveries for type 1 diabetes (T1D) into mechanistic insight can reveal novel biology and therapeutic targets but remains a major challenge. We developed the T1D Knowledge Portal (T1DKP), a disease-specific resource of genetic and functional annotation data that enables users to develop hypotheses for T1D-based research and target discovery. The T1DKP can be used to query genes and genomic regions for genetic associations, identify epigenomic features, access results of bioinformatic analyses, and obtain expert-curated resources. The T1DKP is available athttp://t1d.hugeamp.org.

Identifying genetic determinants for longitudinal changes in albumin excretion in individuals with type 1 diabetes may help identify those that are predisposed to renal, retinal and cardiovascular complications. Most studies have focussed on genetic predisposition to diabetic kidney disease and used cross-sectional measurements of urinary albumin excretion, but with limited success. Here, we utilise the wealth of longitudinal data and bio-samples collected from cohorts of childhood-onset type 1 diabetes followed over the last 30 years to describe a novel trajectory phenotype quantifying urinary albumin excretion changes during childhood and adolescence. We conducted a genome-wide association study and fine-mapping analysis for albumin excretion in 1584 individuals, finding one signal for cross-sectional albumin excretion close toGALNTL6(rs150766792), which validated in a previous independent study, and a novel genome-wide significant signal for albumin excretion trajectory in theCDH18gene region (rs145715205). Our trajectory phenotype quantifies albumin progression and offers a complementary measure to an albumin excretion phenotype based on a single measurement (i.e. most recent data collection) or an average of repeated measurements in longitudinal studies. It can be used to identify genetic or other risk factors which predict better or worse prognosis, thus facilitating the development of new preventive and therapeutic approaches.

Genome-wide association studies (GWASs) have revolutionized our understanding of the genetics of complex diseases, such as osteoporosis; however, the challenge has been converting associations to causal genes. Studies have demonstrated the utility of transcriptomics data in linking disease-associated variants to genes; though for osteoporosis, few population transcriptomics datasets have been generated on bone or bone cells, and an even smaller number have profiled individual cell-types. To begin to evaluate approaches to address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions, a popular model of osteoblast differentiation and activity, from five Diversity Outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model for the generation of cell-type specific transcriptomic profiles of mesenchymal lineage cells derived from large populations of mice to inform genetic studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were nearly identical from a transcriptomic perspective to cells isolated directly from bone. We also demonstrated the ability to multiplex single cells and subsequently assign cells to their “mouse-of-origin” using demultiplexing approaches based on genotypes inferred from coding SNPs. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell-types. SCENIC was used to reconstruct gene regulatory networks (GRNs) and we showed that identified cell-types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of BMD heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell-type specific transcriptomic profiles of mesenchymal lineage cells in large mouse, and potentially human, populations.

Genome-wide association studies have identified numerous loci with allelic associations to Type 1 Diabetes (T1D) risk. Most disease-associated variants are enriched in regulatory sequences active in lymphoid cell types, suggesting that lymphocyte gene expression is altered in T1D. We assayed gene expression between T1D cases and healthy controls in two autoimmunity-relevant lymphocyte cell types, memory CD4+/CD25+ T-regulatory cells (Treg) and memory CD4+/CD25- T-cells, using a splicing event-based approach to characterize tissue-specific transcriptomes. Limited differences in isoform usage between T1D cases and controls were observed in memory CD4+/CD25- T-cells. In Tregs, 553 genes demonstrated differences in isoform usage between cases and controls, particularly RNA recognition and splicing factor genes. Many of these genes are regulated by the variable inclusion of exons that can trigger nonsense mediated decay. Our results suggest that dysregulation of gene expression, through shifts in alternative splicing in Tregs, contributes to T1D etiology.

Alzheimer’s disease (AD) is the most prevalent cause of dementia1. Although there is no effective treatment for AD, passive immunotherapy with monoclonal antibodies against amyloid beta (Aβ) is a promising therapeutic strategy2,3. Meningeal lymphatic drainage has an important role in the accumulation of Aβ in the brain4, but it is not known whether modulation of meningeal lymphatic function can influence the outcome of immunotherapy in AD. Here we show that ablation of meningeal lymphatic vessels in 5xFAD mice (a mouse model of amyloid deposition that expresses five mutations found in familial AD) worsened the outcome of mice treated with anti-Aβ passive immunotherapy by exacerbating the deposition of Aβ, microgliosis, neurovascular dysfunction, and behavioural deficits. By contrast, therapeutic delivery of vascular endothelial growth factor C improved clearance of Aβ by monoclonal antibodies. Notably, there was a substantial overlap between the gene signature of microglia from 5xFAD mice with impaired meningeal lymphatic function and the transcriptional profile of activated microglia from the brains of individuals with AD. Overall, our data demonstrate that impaired meningeal lymphatic drainage exacerbates the microglial inflammatory response in AD and that enhancement of meningeal lymphatic function combined with immunotherapies could lead to better clinical outcomes. Meningeal lymphatic drainage can affect the microglial inflammatory response and anti-amyloid-β immunotherapy in mouse models of Alzheimer’s disease.

Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher’s exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch’s t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Type 1 diabetes (T1D) is a complex disease and more than 100 genetic loci influencing T1D risk have been identified to date. Discovering the genes and biological mechanisms through which these variants impact T1D requires the integration of multiple data types. We have created the T1D Knowledge Portal (T1DKP; type1diabetesgenetics.org) to help researchers utilize genetic and functional genomic data to generate hypotheses about the genes involved in T1D and its complications. The T1DKP aggregates genetic association data from the T1D community along with relevant functional genomics (e.g., accessible chromatin, 3D conformation, gene expression, gene perturbation) data, which are loaded via a sister resource, the Common Metabolic Diseases Genome Atlas (cmdga.org) . Using the HuGeAMP software platform developed for the Common Metabolic Diseases Knowledge Portal (CMDKP; cmdkp.org) within the Accelerating Medicines Partnership for Common Metabolic Diseases, the T1DKP integrates data by applying bioinformatic methods such as meta-analysis of genetic association results to generate “bottom-line” p-values, enrichment of genetic association within tissue-specific genomic annotations, and more. Interactive visualizations allow the user to explore results and to perform custom, on-the-fly analyses. The T1DKP also includes expert-curated lists of predicted effector genes of T1D loci, displaying the supporting evidence behind each prediction. Since the T1DKP is nested within and built upon the same framework as the CMDKP, users may navigate seamlessly to see results relevant to other metabolic disorders. In total the T1DKP project aims to accelerate our understanding and treatment of T1D by providing decision support for researchers as they prioritize loci, variants, and genes for experimental study. Disclosure M.C.Costanzo: Other Relationship; Pfizer Inc. P.V.Kudtarkar: None. U.Nayak: None. S.Onengut-gumuscu: None. Y.Sun: None. S.S.Rich: None. J.Flannick: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. N.Burtt: n/a. Funding National Institutes of Health (5UM1DK105554-07)

DNAme is associated with past HbA1c in DCCT/EDIC but these associations were examined when there was separation in HbA1c distribution by trial intervention arm. Thus, we performed an epigenome-wide association study (EWAS) of DNAme and HbA1c in a T1D cohort without intervention or risk factor exclusion to identify loci with DNAme associated with concurrent and future HbA1c over 28 years. Whole blood DNAme was assayed via Illumina EPIC beadchip in the Pittsburgh Epidemiology of Diabetes Complications (EDC) study of childhood onset ( Disclosure R.G.Miller: Stock/Shareholder; Becton, Dickinson and Company. J.Mychaleckyj: None. S.Onengut-gumuscu: None. T.J.Orchard: None. T.Costacou: None. Funding American Diabetes Association (1-19-JDF-109) ; NIH/NIDDK (R01-DK034818) , Rossi Memorial Fund

DNA methylation (DNAme) has been associated with risk of future CAD in the general population and is a potential target for pharmacological intervention. In T1D, DNAme has been associated with microvascular complications but its association with CAD has not been examined in this high risk population. Thus, we performed an epigenome-wide association study (EWAS) of DNAme and 28-year major CAD (CAD death, nonfatal myocardial infarction) incidence in an observational T1D cohort. Whole blood DNAme was assayed via Illumina EPIC beadchip in the Pittsburgh Epidemiology of Diabetes Complications (EDC) study of childhood onset ( Disclosure R.G.Miller: Stock/Shareholder; Becton, Dickinson and Company. J.Mychaleckyj: None. S.Onengut-gumuscu: None. T.J.Orchard: None. T.Costacou: None. Funding American Diabetes Association (1-19-JDF-109) ; NIH/NIDDK R01-DK034818, Rossi Memorial Fund

Modeling the course of C-peptide decline in autoantibody (Ab) positive individuals is important for type 1 diabetes (T1D) prediction and implementation of T1D prevention trials. Single nucleotide polymorphisms in TCF7L2, JAZF1, SLC30A8, INS, PTPRK, G6CP2, CLEC16, PTPN22 and HLA genes are associated with persistent C-peptide at or after T1D diagnosis. We sought to determine whether 12 SNPs in these genes and the T1D genetic risk score-2 (GRS2) can predict C-peptide trajectory before diagnosis of T1D. We studied Ab positive at-risk participants in the TrialNet Pathway to Prevention Study who had ImmunoChip data (N=1217, age at initial screen (mean±SD) 16.1±12.7 years, 51.5% female, 81.0% non-Hispanic white) . Over a mean follow up of 3.4±2. years, 255 (21.0%) developed multiple Ab and 336 (27.6%) developed clinical T1D. We analyzed the influence of these 12 SNPs and the T1D GRS2 on C-peptide AUC during progression from single to multiple Abs, from single Ab to clinical T1D, and from multiple Abs to clinical T1D. Analyses were adjusted for baseline C-peptide AUC, age, glucose AUC, BMI Z-score and HbA1c; the presence of high-risk HLA haplotypes (DR3 and DR4-DQ8) ; and the first 3 principal components. The type 2 diabetes (T2D) -associated TCF7L2 rs7901695 and rs4506565 SNPs were significantly associated with higher C-peptide AUC during progression from multiple Abs to T1D (p In conclusion, T2D-associated SNPs in the TCF7L2 gene and lower T1D GRS2 predict higher C-peptide particularly in progression from multiple Abs to clinical T1D. Disclosure T.M.Triolo: None. M.J.Redondo: Advisory Panel; Provention Bio, Inc. H.M.Parikh: None. M.Tosur: Advisory Panel; Provention Bio, Inc. P.Gottlieb: Advisory Panel; Janssen Research & Development, LLC, ViaCyte, Inc., Other Relationship; IM Therapeutics, Research Support; Caladrius Biosciences, Inc., Immune Tolerance Network, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk, Precigen, Inc., Tolerion, Inc. R.A.Oram: Consultant; Janssen Research & Development, LLC, Research Support; Randox R & D. S.Onengut-gumuscu: None. J.Krischer: None. S.S.Rich: None. A.Steck: None. Funding NIH K12 DK094712NIH RDK121843NIH RDK124395

Background: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines—cytokine storm. Objective: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). Methods: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. Results: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10−4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10−5, OR = 0.82. Conclusion: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

Oxylipins, pro-inflammatory and pro-resolving lipid mediators, are associated with the risk of type 1 diabetes (T1D) and may be influenced by diet. This study aimed to develop a nutrient pattern related to oxylipin profiles and test their associations with the risk of T1D among youth. The nutrient patterns were developed with a reduced rank regression in a nested case-control study (n = 335) within the Diabetes Autoimmunity Study in the Young (DAISY), a longitudinal cohort of children at risk of T1D. The oxylipin profiles (adjusted for genetic predictors) were the response variables. The nutrient patterns were tested in the case-control study (n = 69 T1D cases, 69 controls), then validated in the DAISY cohort using a joint Cox proportional hazards model (n = 1933, including 81 T1D cases). The first nutrient pattern (NP1) was characterized by low beta cryptoxanthin, flavanone, vitamin C, total sugars and iron, and high lycopene, anthocyanidins, linoleic acid and sodium. After adjusting for T1D family history, the HLA genotype, sex and race/ethnicity, NP1 was associated with a lower risk of T1D in the nested case-control study (OR: 0.44, p = 0.0126). NP1 was not associated with the risk of T1D (HR: 0.54, p-value = 0.1829) in the full DAISY cohort. Future studies are needed to confirm the nested case-control findings and investigate the modifiable factors for oxylipins.

Background Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of end stage kidney disease (ESKD) in individuals with type 1 diabetes at advanced kidney disease stage. Methods Gene-based exome array analysis of 15,449 genes in 5 large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time-to-ESKD, testing the top gene in a 6th cohort (N=2,372/1,115 events all cohorts) and replicating in two retrospective case-control studies (N=1,072 cases, 752 controls). Deep resequencing of the top associated gene in 5 cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. Results Protein coding variants in the hydroxysteroid 17-beta dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (N=4,196; p-value=3.3x10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other chronic kidney disease-associated renal pathologies. Conclusions HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across several cell types. Genome-wide association studies (GWAS) have identified over 170 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis -regulatory elements (CREs). Here, we applied single-cell ATAC-seq to profile 28,316 cells across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped over 320,000 accessible sites across all cells, identified cell type-specific elements, transcription factors, and prioritized functional CAD risk variants via quantitative trait locus and sequence-based predictive modeling. Using differential peak analyses we identified a number of candidate mechanisms for smooth muscle cell transition states (e.g. fibromyocytes). By integrating these profiles with GWAS meta-analysis summary data we resolved cell type-specific putative binding sites for the majority of CAD risk variants. In particular, we prioritized functional variants predicted to alter MEF2 binding in smooth muscle cells at the MRAS locus. We also identify variants predicted to alter macrophage-specific regulation of LIPA. We further employed DNA to gene linkage to nominate disease-associated key driver transcription factors such as PRDM16 and TBX2. Together, this single cell atlas provides a critical step towards interpreting cis -regulatory mechanisms in the vessel wall across the continuum of CAD risk.

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.

Multiple cross-sectional and longitudinal studies have shown that serum levels of the chemokine ligand 2 (CCL-2) are associated with type 1 diabetes (T1D), although the direction of effect differs. We assessed CCL-2 serum levels in a longitudinal cohort to clarify this association, combined with genetic data to elucidate the regulatory role of CCL-2 in T1D pathogenesis. The Diabetes Autoimmunity Study in the Young (DAISY) followed 310 subjects with high risk of developing T1D. Of these, 42 became persistently seropositive for islet autoantibodies but did not develop T1D (non-progressors); 48 did develop T1D (progressors). CCL-2 serum levels among the three study groups were compared using linear mixed models adjusting for age, sex, HLA genotype, and family history of T1D. Summary statistics were obtained from the CCL-2 protein quantitative trait loci (pQTL) and CCR2 expression QTL (eQTL) studies. The T1D fine mapping association data were provided by the Type 1 Diabetes Genetics Consortium (T1DGC). Serum CCL-2 levels were significantly lower in both progressors (p = 0.004) and non-progressors (p = 0.005), compared to controls. Two SNPs (rs1799988 and rs746492) in the 3p21.31 genetic locus, which includes the CCL-2 receptor, CCR2, were associated with increased CCR2 expression (p = 8.2e-5 and 5.2e-5, respectively), decreased CCL-2 serum level (p = 2.41e-9 and 6.21e-9, respectively), and increased risk of T1D (p = 7.9e-5 and 7.9e-5, respectively). The 3p21.31 genetic region is associated with developing T1D through regulatory control of the CCR2/CCL2 immune pathway., Highlights • Serum CCL-2 levels are lower in individuals with islet autoantibodies and type 1 diabetes compared to controls. • Serum CCL-2 levels are associated with the 3p21.31 genetic locus. • The 3p21.31 genetic locus is associated with type 1 diabetes. • The 3p21.31 genetic locus is associated with gene expression of the CCL-2 receptor, CCR2.

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important ‘signaling node’ in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the ‘ELMO1 interactome’ in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis., Osteoporosis and bone fractures affect millions of patients worldwide and are often due to increased bone resorption. Here the authors identify the cytoplasmic protein ELMO1 as an important ‘signaling node’ promoting the bone resorption function of osteoclasts.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across several cell types. Genome-wide association studies (GWAS) have identified over 170 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis-regulatory elements (CREs). Here, we applied single-cell ATAC-seq to profile 28,316 cells across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped ~320,000 accessible sites across all cells, identified cell type-specific elements, transcription factors, and prioritized functional CAD risk variants via quantitative trait locus and sequence-based predictive modeling. We identified a number of candidate mechanisms for smooth muscle cell transition states and identified putative binding sites for risk variants. We further employed CRE to gene linkage to nominate disease-associated key driver transcription factors such as PRDM16 and TBX2. This single cell atlas provides a critical step towards interpreting cis-regulatory mechanisms in the vessel wall across the continuum of CAD risk.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across cell types. Genome-wide association studies have identified over 200 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis-regulatory elements. Here, we applied single-nucleus assay for transposase-accessible chromatin with sequencing to profile 28,316 nuclei across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped ~320,000 accessible sites across all cells, identified cell-type-specific elements and transcription factors, and prioritized functional CAD risk variants. We identified elements in smooth muscle cell transition states (for example, fibromyocytes) and functional variants predicted to alter smooth muscle cell- and macrophage-specific regulation of MRAS (3q22) and LIPA (10q23), respectively. We further nominated key driver transcription factors such as PRDM16 and TBX2. Together, this single-nucleus atlas provides a critical step towards interpreting regulatory mechanisms across the continuum of CAD risk.

Development of islet autoimmunity (IA) and type 1 diabetes (T1D) are associated with high-risk HLA class II loci as well as non-HLA genes. Monozygotic (MZ) twins have a high rate of concordance to T1D progression after the first twin develops T1D, especially for those diagnosed at a young age. We analyzed 52 T1D-associated non-HLA SNPs from ImmunoChip data in 159 MZ twins from the Twin Family Study: 79 twin probands with T1D and their 80 unaffected cotwins/triplets (including one set of triplets). Subjects are enrolled into the Twin Study when the proband is diagnosed with T1D and cotwins are followed longitudinally for the development of IA and/or T1D. In the cotwins, we analyzed the association between each of the non-HLA SNPs and IA after adjusting for HLA DR3/4*0302. In the twin probands with T1D, we used a linear regression model to evaluate the association of non-HLA SNPs with diabetes age at onset after adjusting for HLA DR3/4*0302. Median (IQR) age of diagnosis of the twin probands was 9.9 (5.4-13.9) years and age of last visit for the cotwins was 17.5 (11.2-26.7) years. Of the 80 cotwins, 41 (51.3%) developed IA and 15 (18.8%) were diagnosed with T1D. After adjusting for HLA DR3/4*0302, SNPs in CTLA4 (rs3087243), IL2 (rs4505848), IKZF1 (rs62447205), and INS (rs7111341) were found to be associated with the development of IA in cotwin subjects (all p Disclosure T. M. Triolo: None. F. Dong: None. H. C. Broncucia: None. S. Onengut-gumuscu: None. A. Steck: None. S. S. Rich: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K12DK094712)

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics., Osteoporosis GWAS faces two challenges, causal gene discovery and a lack of phenotypic diversity. Here, the authors use the Diversity Outbred mouse population to inform human GWAS using networks and map genetic loci for 55 bone traits, identifying new potential bone strength genes.

Background: Type 1 diabetes (T1D) genetic risk score (GRS) and islet autoantibody (AAb) specificity and titers influence T1D risk; however, these factors collected at baseline in cross sectional studies have not been effectively incorporated in the current models for prediction of progression along pre-clinical stages of T1D. Our aim was to develop an updated model of T1D prediction for research and clinical practice. Methods: AAb-positive relatives of individuals with T1D (n=1,233, 52% female, 88% European ancestry, age mean 15 yrs [SD 12.2]) from the TrialNet Pathway to Prevention were followed for a median of 3.1 years (IQR 1.1 -7.1). T1D developed in 498/1,233 (40.4%). T1D GRS, AAb number, AAb combination, AAb titer, age and sex were evaluated on the combined T1D prediction model, estimating its performance using 3-fold cross-validation (10x repeated). Results: Machine learning (survival random forest) and traditional methods (Cox models, extended Cox model) performed equivalently as measured by 5 yr horizon time-dependent AUC ROC (respectively 0.74, 0.74, 0.72), on calibration by integrated Brier score (respectively 0.15, 0.15, 0.16), and by calibration plots. AAb combinations with specific variables predicted T1D. In multivariate analysis, T1D GRS was a significant predictor of T1D in individuals positive for GADA, IAA or both. For any autoantibody combination including IA2A, the IA2A titer was predictive while the T1D GRS was not. Variable importance analyses showed that T1D was predicted by IA2A titer level, followed by T1D GRS and combinations of specific AAbs. Conclusion: We modeled individual estimates of T1D risk with T1D GRS, AAb characteristics, age, and sex. T1D prediction improved by adding T1D GRS and AAb characteristics to other variables, and was similar using machine learning or traditional models. T1D GRS was a significant predictor only in the absence of IA2A. In the presence of IA2A, IA2A titer was the most influential factor. Disclosure L. A. Ferrat: None. M. J. Redondo: Advisory Panel; Self; Provention Bio, Inc. A. Steck: None. H. M. Parikh: None. L. You: None. S. Onengut-gumuscu: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. S. S. Rich: None. J. Krischer: None. R. A. Oram: Consultant; Self; Janssen Research & Development, LLC. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK121843-01); JDRF (3-SRA-2019-827-S-B)

Significant progress has been made in elucidating genetic risk factors influencing Type 1 diabetes (T1D); however, features other than genetic variants that initiate and/or accelerate islet autoimmunity that lead to the development of clinical T1D remain largely unknown. We hypothesized that genetic and environmental risk factors can both contribute to T1D through dynamic alterations of molecular interactions in physiologic networks. To test this hypothesis, we utilized longitudinal blood transcriptomic profiles in The Environmental Determinants of Diabetes in the Young (TEDDY) study to generate gene co-expression networks. In network modules that contain immune response genes associated with T1D, we observed highly dynamic differences in module connectivity in the 600 days (~ 2 years) preceding clinical diagnosis of T1D. Our results suggest that gene co-expression is highly plastic and that connectivity differences in T1D-associated immune system genes influence the timing and development of clinical disease.

AIMS: To examine candidate insulin resistance single nucleotide polymorphisms (SNPs) for associations with glycemic control, insulin resistance, BMI, and complications in an observational type 1 diabetes (T1D) cohort: the Pittsburgh Epidemiology of Diabetes Complications (EDC) study.METHODS: In 422 European-ancestry participants, we assessed associations using additive models between 15 candidate SNPs and 25-year mortality, cardiovascular disease, microalbuminuria, overt nephropathy and proliferative retinopathy, and 25-year mean HbA1c, estimated glucose disposal rate (eGDR, inverse measure of insulin resistance), and BMI.RESULTS: The A allele of rs12970134 was associated with higher mean HbA1c (β = +0.34 ± 0.09, p = 0.00009) and nominally associated with worse eGDR (p = 0.02). Further analyses suggest the HbA1c association may be modified by diabetes therapy regimen: rs12970134 AA genotype was associated with higher HbA1c under non-intensive therapy conditions (CONCLUSIONS: rs12970134, near MC4R, is strongly associated with HbA1c in this cohort. Further exploration of this genomic region is warranted, as it may hold promise for discovering new therapeutic targets to improve glycemic control in T1D.

AIMS We aimed to identify long-term HbA1c trajectories and examine associated characteristics in an observational, childhood-onset (

For polygenic traits, associations with genetic variants can be detected over many chromosome regions, owing to the availability oflarge sample sizes. Most variants, however, have small effects on disease risk and, therefore, unravelling the causal variants, target genes, and biology of these variants is challenging. Here, we define the Bigger or False Discovery Rate (BFDR) as the probability that either a variant is a false-positive or a randomly drawn, true-positive association exceeds it in effect size. Using the BFDR, we identified 302 previously unreported signals with larger effect associations with type 1 diabetes and autoimmune thyroid disease. Out of 239 genome-wide significant signals in both diseases, only 66 (28%) show evidence for having a large effect using the BFDR, further demonstrating how using a combination of effect size and significance, rather than significance alone, is important in identifying SNPs and candidate genes for further investigation.

We report the largest and most ancestrally diverse genetic study of type 1 diabetes (T1D) to date (61,427 participants), yielding 152 regions associated to false discovery rate < 0.01, including 36 regions associated to genome-wide significance for the first time. Credible sets of disease-associated variants are specifically enriched in immune cell accessible chromatin, particularly in CD4+ effector T cells. Colocalization with chromatin accessibility quantitative trait loci (QTL) in CD4+ T cells identified five regions where differences in T1D risk and chromatin accessibility are potentially driven by the same causal variant. Allele-specific chromatin accessibility further refined the set of putative causal variants with functional relevance in CD4+ T cells and integration of whole blood expression QTLs identified candidate T1D genes, providing high-yield targets for mechanistic follow-up. We highlight rs72938038 in BACH2 as a candidate causal T1D variant, where the T1D risk allele leads to decreased enhancer accessibility and BACH2 expression in T cells. Finally, we prioritise potential drug targets by integrating genetic evidence, functional genomic maps, and immune protein-protein interactions, identifying 12 genes implicated in T1D that have been targeted in clinical trials for autoimmune diseases. These findings provide an expanded genomic landscape for T1D, including proposed genetic regulatory mechanisms of T1D-associated variants and genetic support for therapeutic targets for immune intervention.

Type 1 diabetes arises from the autoimmune destruction of insulin-producing beta-cells of the pancreas, resulting in dependence on exogenously administered insulin to maintain glucose homeostasis. In this study, our aim was to identify genetic risk factors that contribute to progression from islet autoimmunity to clinical type 1 diabetes. We analyzed 6.8 million variants derived from whole genome sequencing of 160 islet autoantibody positive subjects, including 87 who had progressed to type 1 diabetes. The Cox proportional-hazard model for survival analysis was used to identify genetic variants associated with progression. We identified one novel region, 20p12.1 (TASP1; genome-wide P –8) and three regions, 1q21.3 (MRPS21–PRPF3), 2p25.2 (NRIR), 3q22.1 (COL6A6), with suggestive evidence of association (P –8) with progression from islet autoimmunity to type 1 diabetes. Once islet autoimmunity is initiated, functional mapping identified two critical pathways, response to viral infections and interferon signaling, as contributing to disease progression. These results provide evidence that genetic pathways involved in progression from islet autoimmunity differ from those pathways identified once disease has been established. These results support the need for further investigation of genetic risk factors that modulate initiation and progression of subclinical disease to inform efforts in development of novel strategies for prediction and intervention of type 1 diabetes.

Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage.Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (Protein coding variants in the hydroxysteroid 17

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. Methods and Results: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we used Diversity Outbred (DO) mice to directly address these limitations by performing the first systems genetics analysis of 55 complex skeletal phenotypes. We applied a network approach to cortical bone RNA-seq data to discover 72 genes likely to be causal for human BMD GWAS associations, including the novel genes SERTAD4 and GLT8D2. We also performed GWAS in the DO for a wide-range of bone traits and identified Qsox1 as a novel gene influencing cortical bone accrual and bone strength. Our results provide a new perspective on the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

Background: Type 1 diabetes (T1D) is determined by unknown environmental factors in genetically susceptible individuals. The design of most genome wide association studies of T1D involves comparison of cases (with varying duration of disease) with controls; hence, the genetic variants associated with T1D reflect current disease, ignoring variants that play a key role in the initiation of islet autoimmunity and the progression of subclinical disease. In this study, we focus on the genetic contribution to progression of islet autoimmunity using whole-genome sequencing in a collection of participants of the Diabetes AutoImmunity Study in the Young (DAISY). Methods: A total of 160 persistent islet autoantibody positive DAISY subjects were sequenced, including 87 subjects who progressed to T1D and 73 “non-progressors” over a mean of 11 years since seroconversion. Following whole genome sequence alignment, single nucleotide polymorphisms (SNPs) and small insertion/deletions (indels) were identified (∼6.8 million variants), and statistical analyses performed. Results: Genes with multiple SNPs associated with progression to T1D in subjects with islet autoimmunity (P < 8.5x10-8) did not overlap with known T1D risk loci; instead, four novel regions were identified - 1q21.3 (MRPS21-PRPF3), 2p25.2 (NRIR), 3q22.1 (COL6A6), and 20p12.1 (TASP1). Functional mapping of the associated SNPs within these risk loci indicates pathways critical for response to viral infections and response to interferon signaling contribute to progression to T1D once islet autoimmunity is initiated. Discussion: This study presents evidence that different genetic pathways may contribute to progression of islet autoimmunity from those identified once disease is established and support the need for follow-up studies to understand genetic risk factors that modulate progression of subclinical disease. Disclosure S. Onengut-Gumuscu: None. U. Paila: None. W. Chen: None. A. Ratan: None. Z. Zhu: None. A. Steck: None. B.I. Frohnert: None. K. Waugh: None. B. Webb-Robertson: None. J.M. Norris: None. L. Lange: None. M. Rewers: None. S.S. Rich: None.

Motivation: Twin studies estimate 50% of type 1 diabetes (T1D) risk is genetic. Genetic association has implicated T cells as central to the disease process. Thus, we generated chromatin accessibility profiles from CD4+ T cells isolated from 135 T1D cases and 144 controls and investigated whether (1) there are patterns of chromatin accessibility that differ from T1D patients compared to healthy controls; (2) established T1D risk variants influence chromatin accessibility. Methods: Frozen PBMCs were provided by the Type 1 Diabetes Genetics Consortium (T1DGC). CD4+ T cells were isolated using bead purification and Assay for Transposase Accessible Chromatin (ATAC-seq) was performed. ATAC-seq samples were processed using the PEPATAC pipeline and R packages edgeR and limm. Results: We tested for differences in accessibility between individuals with and without T1D adjusting for sex, age, and experimental variability. We identified 516 chromatin regions peaks with differential accessibility by T1D status (2 with increased and 514 with decreased accessibility at FDR Conclusion: These results provide hypotheses about molecular mechanisms of T1D in CD4+ T cells. Disclosure C.C. Robertson: None. W. Chen: None. S. Onengut-Gumuscu: None. P.J. Concannon: Stock/Shareholder; Self; Amgen. Other Relationship; Self; 10x Genomics. S.S. Rich: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK111906)

The risk for autoimmunity and subsequently type 1 diabetes is 10-fold higher in children with a first-degree family history of type 1 diabetes (FDR children) than in children in the general population (GP children). We analyzed children with high-risk HLA genotypes (n = 4,573) in the longitudinal TEDDY birth cohort to determine how much of the divergent risk is attributable to genetic enrichment in affected families. Enrichment for susceptible genotypes of multiple type 1 diabetes–associated genes and a novel risk gene, BTNL2, was identified in FDR children compared with GP children. After correction for genetic enrichment, the risks in the FDR and GP children converged but were not identical for multiple islet autoantibodies (hazard ratio [HR] 2.26 [95% CI 1.6–3.02]) and for diabetes (HR 2.92 [95% CI 2.05–4.16]). Convergence varied depending upon the degree of genetic susceptibility. Risks were similar in the highest genetic susceptibility group for multiple islet autoantibodies (14.3% vs .12.7%) and diabetes (4.8% vs. 4.1%) and were up to 5.8-fold divergent for children in the lowest genetic susceptibility group, decreasing incrementally in GP children but not in FDR children. These findings suggest that additional factors enriched within affected families preferentially increase the risk of autoimmunity and type 1 diabetes in lower genetic susceptibility strata.

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2—that is, ‘smell’ (‘find-me’ signals or sensing factors released by apoptotic cells), ‘taste’ (phagocyte–apoptotic cell contact) and ‘ingestion’ (corpse internalization)—activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells. Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk−/−Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk−/− mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk+/+ and Mertk−/− mice. Prior to the onset of photoreceptor degeneration, Mertk−/− mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexedin situhybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker,Neuromedin BmRNA (Nmb). Most (∼75%) RTN neurons express low-to-moderate levels ofNmband display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. TheseNmb-low RTN neurons also express varying levels of transcripts forGal,Penk, andAdcyap1, and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20–25%), typically larger than average, express very high levels ofNmbmRNA. TheseNmb-high RTN neurons do not expressFosafter hypercapnia and have low-to-undetectable levels ofKcnk5orGpr4transcripts; they also expressAdcyap1, but are essentially devoid ofPenkandGaltranscripts. In male rats,Nmbis also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum,Nmbis a selective marker of the RTN in rodents;Nmb-low neurons, the vast majority, are central respiratory chemoreceptors, whereasNmb-high neurons likely have other functions.SIGNIFICANCE STATEMENTCentral respiratory chemoreceptors regulate arterial PCO2by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexedin situhybridization, we show here that a single marker,Neuromedin BmRNA (Nmb), identifies RTN neurons in rodents. We also suggest that >75% of theseNmbneurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (Kcnk5andGpr4). The other RTN neurons express very high levels ofNmb, but low levels ofKcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin, and do not respond to hypercapnia. Their function is unknown.

Genome-wide association studies (GWAS) have identified multiple, shared allelic associations with many autoimmune diseases. However, the pathogenic contributions of variants residing in risk loci remain unresolved. The location of the majority of shared disease-associated variants in noncoding regions suggests they contribute to risk of autoimmunity through effects on gene expression in the immune system. In the current study, we test this hypothesis by applying RNA sequencing to CD4+, CD8+, and CD19+ lymphocyte populations isolated from 81 subjects with type 1 diabetes (T1D). We characterize and compare the expression patterns across these cell types for three gene sets: all genes, the set of genes implicated in autoimmune disease risk by GWAS, and the subset of these genes specifically implicated in T1D. We performed RNA sequencing and aligned the reads to both the human reference genome and a catalog of all possible splicing events developed from the genome, thereby providing a comprehensive evaluation of the roles of gene expression and alternative splicing (AS) in autoimmunity. Autoimmune candidate genes displayed greater expression specificity in the three lymphocyte populations relative to other genes, with significantly increased levels of splicing events, particularly those predicted to have substantial effects on protein isoform structure and function (e.g., intron retention, exon skipping). The majority of single-nucleotide polymorphisms within T1D-associated loci were also associated with one or more cis-expression quantitative trait loci (cis-eQTLs) and/or splicing eQTLs. Our findings highlight a substantial, and previously underrecognized, role for AS in the pathogenesis of autoimmune disorders and particularly for T1D.

We examined the association between plasma 25-hydroxyvitamin D [25(OH)D] concentration and islet autoimmunity (IA) and whether vitamin D gene polymorphisms modify the effect of 25(OH)D on IA risk. We followed 8,676 children at increased genetic risk of type 1 diabetes at six sites in the U.S. and Europe. We defined IA as positivity for at least one autoantibody (GADA, IAA, or IA-2A) on two or more visits. We conducted a risk set sampled nested case-control study of 376 IA case subjects and up to 3 control subjects per case subject. 25(OH)D concentration was measured on all samples prior to, and including, the first IA positive visit. Nine polymorphisms in VDR, CYP24A, CYP27B1, GC, and RXRA were analyzed as effect modifiers of 25(OH)D. Adjusting for HLA-DR-DQ and ancestry, higher childhood 25(OH)D was associated with lower IA risk (odds ratio = 0.93 for a 5 nmol/L difference; 95% CI 0.89, 0.97). Moreover, this association was modified by VDR rs7975232 (interaction P = 0.0072), where increased childhood 25(OH)D was associated with a decreasing IA risk based upon number of minor alleles: 0 (1.00; 0.93, 1.07), 1 (0.92; 0.89, 0.96), and 2 (0.86; 0.80, 0.92). Vitamin D and VDR may have a combined role in IA development in children at increased genetic risk for type 1 diabetes.

Context Genome-wide association studies identified >50 type 1 diabetes (T1D) associated non-human leukocyte antigens (non-HLA) loci. Objective The purpose of this study was to assess the contribution of non-HLA single nucleotide polymorphisms (SNPs) to risk of disease progression. Design and Setting The TrialNet Pathway to Prevention Study follows relatives of T1D patients for development of autoantibodies (Abs) and T1D. Participants Using the Immunochip, we analyzed 53 diabetes-associated, non-HLA SNPs in 1016 Ab-positive, at-risk non-Hispanic white relatives. Main Outcome Measure Effect of SNPs on the development of multiple Abs and T1D. Results Cox proportional analyses included all substantial non-HLA SNPs, HLA genotypes, relationship to proband, sex, age at initial screening, initial Ab type, and number. Factors involved in progression from single to multiple Abs included age at screening, relationship to proband, HLA genotypes, and rs3087243 (cytotoxic T lymphocyte antigen-4). Significant factors for diabetes progression included age at screening, Ab number, HLA genotypes, rs6476839 [GLIS family zinc finger 3 (GLIS3)], and rs3184504 [SH2B adaptor protein 3 (SH2B3)]. When glucose area under the curve (AUC) was included, factors involved in disease progression included glucose AUC, age at screening, Ab number, relationship to proband, HLA genotypes, rs6476839 (GLIS3), and rs7221109 (CCR7). In stratified analyses by age, glucose AUC, age at screening, sibling, HLA genotypes, rs6476839 (GLIS3), and rs4900384 (C14orf64) were significantly associated with progression to diabetes in participants Conclusions In conclusion, we identified five non-HLA SNPs associated with increased risk of progression from Ab positivity to disease that may improve risk stratification for prevention trials.

The receptor for advanced glycation end products (RAGE) is underexpressed in idiopathic pulmonary fibrosis (IPF) lung, but the role of RAGE in human lung fibrosis remains uncertain.To examine (1) the association between IPF risk and variation at rs2070600, a functional missense variant in AGER (the gene that codes for RAGE), and (2) the associations between plasma-soluble RAGE (sRAGE) levels with disease severity and time to death or lung transplant in IPF.We genotyped the rs2070600 single-nucleotide polymorphism in 108 adults with IPF and 324 race-/ethnicity-matched control subjects. We measured plasma sRAGE by ELISA in 103 adults with IPF. We used generalized linear and additive models as well as Cox models to control for potential confounders. We repeated our analyses in 168 (genetic analyses) and 177 (sRAGE analyses) adults with other forms of interstitial lung disease (ILD).There was no association between rs2070600 variation among adults with IPF (P = 0.31). Plasma sRAGE levels were lower among adults with IPF and other forms of ILD than in control subjects (P 0.001). The rs2070600 allele A was associated with a 49% lower sRAGE level (95% confidence interval [CI], 11 to 71%; P = 0.02) among adults with IPF. In adjusted analyses, lower sRAGE levels were associated with greater disease severity (14% sRAGE decrement per 10% FVC decrement; 95% CI, 5 to 22%) and a higher rate of death or lung transplant at 1 year (adjusted hazard ratio, 1.9 per logarithmic unit of sRAGE decrement; 95% CI, 1.2-3.3) in IPF. Similar findings were observed in a heterogeneous group of adults with other forms of ILD.Lower plasma sRAGE levels may be a biological measure of disease severity in IPF. Variation at the rs2070600 single-nucleotide polymorphism was not associated with IPF risk.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

Oligodendrocyte progenitor cells (OPCs) account for about 5% of total brain and spinal cord cells, giving rise to myelinating oligodendrocytes that provide electrical insulation to neurons of the CNS. OPCs have also recently been shown to regulate inflammatory responses and glial scar formation, suggesting functions that extend beyond myelination. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted phagocytic receptor that is highly expressed in several CNS cell types, including OPCs. Here, we have generated an oligodendroglia-specific knockout of LRP1, which presents with normal myelin development, but is associated with better outcomes in two animal models of demyelination (EAE and cuprizone). At a mechanistic level, LRP1 did not directly affect OPC differentiation into mature oligodendrocytes. Instead, animals lacking LRP1 in OPCs in the demyelinating CNS were characterized by a robust dampening of inflammation. In particular, LRP1-deficient OPCs presented with impaired antigen cross-presentation machinery, suggesting a failure to propagate the inflammatory response and thus promoting faster myelin repair and neuroprotection. Our study places OPCs as major regulators of neuroinflammation in an LRP1-dependent fashion.

Background In recent years, research has consistently proven the occurrence of genetic overlap across autoimmune diseases, which supports the existence of common pathogenic mechanisms in autoimmunity. Objectives The objective of this study was to further investigate this shared genetic component. Methods We performed a cross-disease meta-analysis of Immunochip data from 37,159 patients diagnosed with a seropositive autoimmune disease (11,489 celiac disease (CeD), 15,523 rheumatoid arthritis (RA), 3,477 systemic sclerosis (SSc), and 6,670 type 1 diabetes (T1D)) and 22,308 healthy controls of European origin using the R package ASSET. Results We identified 38 risk variants shared by at least two of the conditions analyzed, five of which represent new pleiotropic loci in autoimmunity. We also identified six novel genome-wide associations for the diseases studied. Cell-specific functional annotations and biological pathway enrichment analyses suggested that pleiotropic variants may deregulate gene expression in different subsets of T cells, especially Th17 and regulatory T cells. Finally, drug repositioning analysis evidenced several drugs that could represent promising candidates for CeD, RA, SSc and T1D treatment. Conclusion We have been able to advance in the knowledge of the genetic overlap existing in autoimmunity, thus shedding light on common molecular mechanisms of disease and suggesting novel drug targets that could be explored for the treatment of the autoimmune diseases studied. References [1] Richard-Miceli C, Criswell LA. Emerging patterns of genetic overlap across autoimmune disorders. Genome Med. 2012;4(1):6. [2] Bhattacharjee S, Rajaraman P, Jacobs KB, et al. A subset-based approach improves power and interpretation for the combined analysis of genetic association studies of heterogeneous traits. Am J Hum Genet. 2012;90(5):821-35 Disclosure of Interests Ana Marquez: None declared, Martin Kerick: None declared, Alexandra Zhernakova: None declared, Javier Gutierrez-Achury: None declared, Wei-Min Chen: None declared, Suna Onengut-Gumuscu: None declared, Isidoro Gonzalez-Alvaro: None declared, Luis Rodriguez-Rodriguez: None declared, Miguel A Gonzalez-Gay Grant/research support from: Prof. MA Gonzalez-Gay received grants/research supports from Abbvie, MSD, Jansen and Roche., Speakers bureau: Consultation fees/participation in company sponsored speaker’s bureau from Pfizer, Lilly, Sobi, Celgene, Novartis, Roche and Sanofi., Maureen Mayes Grant/research support from: Maureen Mayes is a clinical trial investigator for Boehringer-Ingelheim; Galapagos, Reata, Sanofi, Merck-Serono, Consultant for: Maureen Mayes is a member of scientific advisory boards for Galapagos NV (Pharma), Boehringer-Ingelheim, Mitsubishi-Tanabe, Astellas: Grant Review Board for Actelion., Speakers bureau: Maureen Mayes received personal fees for being a conference speaker on the use of autoantibodies in connective tissue diseases for Medtelligence, Soumya Raychaudhuri: None declared, Stephen S. Rich: None declared, Cisca Wijmenga: None declared, Javier Martin Ibanez: None declared

The MC4R gene (18q21.32) plays a role in regulating appetite and energy homeostasis and is consistently linked with obesity. A variant near MC4R (rs12970134) has also been associated with IR and type 2 diabetes, independent of BMI. We examined the association of rs12970134 with HbA1c, IR, and BMI in the Pittsburgh Epidemiology of Diabetes Complications study, a prospective T1D cohort (mean age 27, T1D duration 18 years at baseline, 1986-88). In 422 EDC participants of European ancestry with available DNA, we assessed associations between 16 published IR SNPs, including rs12970134, and longitudinal measures of HbA1c, estimated glucose disposal rate (eGDR, an inverse measure of IR), and BMI over 18 years of follow-up (1986-88 to 2004-06). General additive mixed models were adjusted for sex, T1D duration, and principal components of ancestry. The rs12970134 SNP was associated with longitudinal HbA1c (β=0.33, p=5x10-5, see Figure) and eGDR (β=-0.31, p=0.008) trajectories, but not BMI (β=0.21, p=0.38). The associations remained significant after adjusting for BMI and insulin dose. There was no evidence for a SNP x time interaction (p>0.25 for both); all groups showed a similar fall in HbA1c after publication of the DCCT results. Thus, the rs12970134 SNP near MC4R is associated with glycemic control and insulin resistance, despite no association with BMI, in this T1D cohort. Disclosure R.G. Miller: None. T. Costacou: None. S. Onengut-Gumuscu: None. W. Chen: None. S.S. Rich: None. T.J. Orchard: Consultant; Self; Boehringer Ingelheim International GmbH. Funding National Institutes of Health; Rossi Memorial Fund; JDRF