The most common genetic cause of intellectual and developmental disability is trisomy of human chromosome 21 (trisomy 21) or Down syndrome. Relative to the general population, individuals with Down syndrome heterogeneously experience atypical morphogenesis, a distinct neurocognitive profile, and a unique spectrum of diverse medical conditions that impact every major organ system. How trisomy 21 results in the highly variable manifestations of Down syndrome remains largely unknown and an active area of heavy investigation with therapeutic implications. For example, common inflammatory and metabolic signatures have begun to emerge across various co-occurring conditions in Down syndrome with assorted impacts on diverse yet intertwined organ systems that could directly or indirectly impact brain health. Here, we review current progress, resources, knowledge gaps, and bottlenecks for precision medicine approaches to promote brain health across the lifespan among individuals with Down syndrome within the larger context of research efforts geared towards our other distinct yet intertwined organ systems. Within this framework, we advocate for interdisciplinary pursuit of systems-level biomarkers to facilitate holistic intervention strategies that precisely benefit individuals with trisomy 21 each experiencing Down syndrome in their own unique way. To this end, we quantitatively assess clinical studies that are actively recruiting participants with Down syndrome and provide historical context through summary figures sourced to user-friendly tables that have been curated from federal websites to empower efficient exploration of research opportunities for interdisciplinary collaborations.

Mitochondrial dysfunction and Aβ accumulation are hallmarks of Alzheimer’s disease (AD). However, the role of these pathologies in Down Syndrome associated Alzheimer’s Disease (DSAD) is unknown. Decades of research describe a relationship between mitochondrial function and Aβ production. Amyloid precursor protein (APP), from which Aβ is generated, is found in mitochondria. APP and Aβ alter mitochondrial function, while mitochondrial function alters Aβ production from APP. How these interactions contribute to DSAD pathology and progression are unknown. Here we interrogated the association of full-length APP with mitochondria, mitochondrial function, and AD pathological hallmarks. ND (n = 10, without DS) and DS associated Alzheimer’s Disease (DSAD, n = 10) postmortem brain tissue was obtained from the University of California Irvine. A human iPSC line was purchased from WiCell with Trisomy 21 and a isogenic control line which underwent Crispr/Cas9 genome editing to remove the extra chromosome 21 copy. iPSC models were differentiated into neurons, astrocytes, and cerebral organoids using StemCell Technologies reagents and protocols. We examined mitochondrial function using a Seahorse XF analyzer. We measured full-length APP protein levels in whole cell extracts and mitochondrial fractions via Western Blotting. We measured Aβ levels with ELISA kits from ThermoFisher. DSAD postmortem brain tissue had reduced mitochondrial function regardless of sex. Full-length APP levels were significantly higher in mitochondrial fractions in DSAD brain tissue. Full-length APP levels in mitochondrial fractions correlated with mitochondrial function. Higher mitochondrial APP (full-length) levels associated with lower mitochondrial function. iPSC derived models showed similar phenotypes to postmortem brain tissues, including increased mitochondrial APP levels, and decreased mitochondrial function. We describe a relationship between mitochondrial APP accumulation, and mitochondrial function. These data support a centralized role for mitochondrial function in APP physiology and APP may play a role in modulating mitochondrial function. Further, DSAD postmortem tissue and iPSC models show significant mitochondrial dysfunction.

Individuals with Down syndrome, the genetic condition caused by trisomy 21, exhibit strong inter-individual variability in terms of developmental phenotypes and diagnosis of co-occurring conditions. The mechanisms underlying this variable developmental and clinical presentation await elucidation. We report an investigation of human chromosome 21 gene overexpression in hundreds of research participants with Down syndrome, which led to the identification of two major subsets of co-expressed genes. Using clustering analyses, we identified three main molecular subtypes of trisomy 21, based on differential overexpression patterns of chromosome 21 genes. We subsequently performed multiomics comparative analyses among subtypes using whole blood transcriptomes, plasma proteomes and metabolomes, and immune cell profiles. These efforts revealed strong heterogeneity in dysregulation of key pathophysiological processes across the three subtypes, underscored by differential multiomics signatures related to inflammation, immunity, cell growth and proliferation, and metabolism. We also observed distinct patterns of immune cell changes across subtypes. These findings provide insights into the molecular heterogeneity of trisomy 21 and lay the foundation for the development of personalized medicine approaches for the clinical management of Down syndrome.

Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), is characterized by delayed neurodevelopment, accelerated aging, and increased risk of many co-occurring conditions. Hypoxemia and dysregulated hematopoiesis have been documented in DS, but the underlying mechanisms and clinical consequences remain ill defined. We report an integrative multi-omic analysis of ∼400 research participants showing that people with DS display transcriptomic signatures indicative of elevated heme metabolism and increased hypoxic signaling across the lifespan, along with chronic overproduction of erythropoietin, elevated biomarkers of tissue-specific hypoxia, and hallmarks of stress erythropoiesis. Elevated heme metabolism, transcriptional signatures of hypoxia, and stress erythropoiesis are conserved in a mouse model of DS and associated with overexpression of select triplicated genes. These alterations are independent of the hyperactive interferon signaling characteristic of DS. These results reveal lifelong dysregulation of key oxygen-related processes that could contribute to the developmental and clinical hallmarks of DS.

Background: Individuals with Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), display clear signs of immune dysregulation, including high rates of autoimmunity and severe complications from infections. Although it is well established that T21 causes increased interferon responses and JAK/STAT signaling, elevated autoantibodies, global immune remodeling, and hypercytokinemia, the interplay between these processes, the clinical manifestations of DS, and potential therapeutic interventions remain ill defined. Methods: We report a comprehensive analysis of immune dysregulation at the clinical, cellular, and molecular level in hundreds of individuals with DS, including autoantibody profiling, cytokine analysis, and deep immune mapping. We also report the interim analysis of a Phase II clinical trial investigating the safety and efficacy of the JAK inhibitor tofacitinib through multiple clinical and molecular endpoints. Results: We demonstrate multi-organ autoimmunity of pediatric onset concurrent with unexpected autoantibody-phenotype associations in DS. Importantly, constitutive immune remodeling and hypercytokinemia occur from an early age prior to autoimmune diagnoses or autoantibody production. Analysis of the first 10 participants to complete 16 weeks of tofacitinib treatment shows a good safety profile and no serious adverse events. Treatment reduced skin pathology in alopecia areata, psoriasis, and atopic dermatitis, while decreasing interferon scores, cytokine scores, and levels of pathogenic autoantibodies without overt immune suppression. Conclusions: JAK inhibition is a valid strategy to treat autoimmune conditions in DS. Additional research is needed to define the effects of JAK inhibition on the broader developmental and clinical hallmarks of DS. Funding: NIAMS, Global Down Syndrome Foundation. Clinical trial number: NCT04246372 .

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.

Individuals with Down syndrome (DS) display chronic hyperactivation of interferon signaling. However, the clinical impacts of interferon hyperactivity in DS are ill-defined. Here, we describe a multiomics investigation of interferon signaling in hundreds of individuals with DS. Using interferon scores derived from the whole blood transcriptome, we defined the proteomic, immune, metabolic, and clinical features associated with interferon hyperactivity in DS. Interferon hyperactivity associates with a distinct proinflammatory phenotype and dysregulation of major growth signaling and morphogenic pathways. Individuals with the highest interferon activity display the strongest remodeling of the peripheral immune system, including increased cytotoxic T cells, B cell depletion, and monocyte activation. Interferon hyperactivity accompanies key metabolic changes, most prominently dysregulated tryptophan catabolism. High interferon signaling stratifies a subpopulation with elevated rates of congenital heart disease and autoimmunity. Last, a longitudinal case study demonstrated that JAK inhibition normalizes interferon signatures with therapeutic benefit in DS. Together, these results justify the testing of immune-modulatory therapies in DS.

Down syndrome (DS), the genetic condition caused by trisomy 21, is characterized by variable cognitive impairment, immune dysregulation, dysmorphogenesis and increased prevalence of diverse co-occurring conditions. The mechanisms by which trisomy 21 causes these effects remain largely unknown. We demonstrate that triplication of the interferon receptor (IFNR) gene cluster on chromosome 21 is necessary for multiple phenotypes in a mouse model of DS. Whole-blood transcriptome analysis demonstrated that IFNR overexpression associates with chronic interferon hyperactivity and inflammation in people with DS. To define the contribution of this locus to DS phenotypes, we used genome editing to correct its copy number in a mouse model of DS, which normalized antiviral responses, prevented heart malformations, ameliorated developmental delays, improved cognition and attenuated craniofacial anomalies. Triplication of the Ifnr locus modulates hallmarks of DS in mice, suggesting that trisomy 21 elicits an interferonopathy potentially amenable to therapeutic intervention.

Background and AimsThe lack of easily measurable biomarkers remains a challenge in executing clinical trials for diabetic neuropathy (DN). Plasma Neurofilament light chain (NFL) concentration is a promising biomarker in immune-mediated neuropathies. Longitudinal studies evaluating NFL in DN have not been performed. MethodsA nested case-control study was performed on participants with youth-onset type 2 diabetes enrolled in the prospective Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study. Plasma NFL concentrations were measured at 4-year intervals from 2008 to 2020 in 50 participants who developed DN and 50 participants with type 2 diabetes who did not develop DN. ResultsNFL concentrations were similar in the DN and no DN groups at the first assessment. Concentrations were higher in DN participants at all subsequent assessment periods (all p < .01). NFL concentrations increased over time in both groups, with higher degrees of change in DN participants (interaction p = .045). A doubling of the NFL value at Assessment 2 in those without DN increased the odds of ultimate DN outcome by an estimated ratio of 2.86 (95% CI: [1.30, 6.33], p = .0046). At the final study visit, positive Spearman correlations (controlled for age, sex, diabetes duration, and BMI) were observed between NFL and HbA1c (0.48, p < .0001), total cholesterol (0.25, p = .018), and low-density lipoprotein (LDL (0.30, p = .0037)). Negative correlations were observed with measures of heart rate variability (-0.42 to -0.46, p = <.0001). InterpretationThe findings that NFL concentrations are elevated in individuals with youth-onset type 2 diabetes, and increase more rapidly in those who develop DN, suggest that NFL could be a valuable biomarker for DN.

Trisomy 21 causes Down syndrome, a condition characterized by cognitive impairments, immune dysregulation, and atypical morphogenesis. Using whole blood transcriptome analysis, we demonstrate that specific overexpression of four interferon receptors encoded on chromosome 21 associates with chronic interferon hyperactivity and systemic inflammation in Down syndrome. To define the contribution of interferon receptor overexpression to Down syndrome phenotypes, we used genome editing to correct interferon receptor gene dosage in mice carrying triplication of a large genomic region orthologous to human chromosome 21. Normalization of interferon receptor copy number attenuated lethal antiviral responses, prevented heart malformations, decreased developmental delays, improved cognition and normalized craniofacial anomalies. Therefore, interferon receptor gene dosage determines major hallmarks of Down syndrome, indicating that trisomy 21 elicits an interferonopathy amenable to therapeutic intervention. One-Sentence Summary Correction of interferon receptor gene dosage rescues multiple key phenotypes in a mouse model of trisomy 21.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), is characterized by stunted growth, cognitive impairment, and increased risk of diverse neurological conditions. Although signs of lifelong neurodegeneration are well documented in DS, the mechanisms underlying this phenotype await elucidation. Here we report a multi-omics analysis of neurodegeneration and neuroinflammation biomarkers, plasma proteomics, and immune profiling in a diverse cohort of more than 400 research participants. We identified depletion of insulin growth factor 1 (IGF1), a master regulator of growth and brain development, as the top biosignature associated with neurodegeneration in DS. Individuals with T21 display chronic IGF1 deficiency downstream of growth hormone production, associated with a specific inflammatory profile involving elevated tumor necrosis factor alpha (TNF-α). Shorter children with DS show stronger IGF1 deficiency, elevated biomarkers of neurodegeneration, and increased prevalence of autism and other conditions. These results point to disruption of IGF1 signaling as a potential contributor to stunted growth and neurodegeneration in DS.

Individuals with Down syndrome (DS, trisomy 21) display consistent activation of the interferon (IFN) response, hyperactive JAK/STAT signaling, and chronic dysregulation of the immune system, which could be explained by the fact that four IFN receptors are encoded on chromosome 21. IFN hyperactivity may explain the high prevalence of immune skin conditions in this population, including alopecia areata (AA), hidradenitis suppurativa (HS), psoriasis, atopic dermatitis, and vitiligo. We investigated the use of the JAK inhibitor Tofacitinib in an open label clinical trial enrolling individuals with DS ages 12-50 with moderate-to-severe AA, HS, psoriasis, atopic dermatitis, and/or vitiligo. All participants received 5mg of Tofacitinib (Xeljanz, Pfizer) BID. Transcriptional IFN scores, plasma cytokine scores, and skin pathology were assessed at baseline and 16 weeks. Results from the first 10 participants indicate that Tofacitinib is well tolerated in people with DS. Both IFN scores and cytokine scores were significantly decreased at 16-weeks (p<0.05). Overall skin pathology improved in 7 of 10 participants (IGA, p<0.05). For AA, 5 of 6 participants responded to Tofacitinib treatment (SALT, p<0.05), as did the two participants with atopic dermatitis, and the single participant with psoriatic arthritis. Two of five participants with HS showed improvement. Tofacitinib appears to be a safe treatment for patients with DS. For individuals with DS and AA, treatment is efficacious in stimulating hair re-growth, and on-label use for psoriatic arthritis can be appropriate in DS. Additional research is required in individuals with DS and HS. Tofacitinib represents a viable treatment option for people with DS and immune skin conditions when clinically indicated.

Down syndrome (DS) is the result of trisomy in chromosome 21 (T21) and is associated with an increased risk of diseases, particularly acute myeloid leukemia (AML). However, people with DS have variable disease risks and severities that have yet to be explained. We hypothesize that differing characteristics of clonal hematopoiesis (CH) may coincide with and/or contribute to poor health outcomes seen in people with DS and can help explain differences in disease outcome. To characterize CH, we use a rare-mutation detection technique called Duplex Seq, only requiring a blood draw to obtain the DNA from leukocytes. This targeted-sequencing tool uses a double-stranded tag to incorporate mutational data from both strands of DNA, enabling high sensitivity through the elimination of DNA processing errors. Using bioinformatics and COSMIC’s Cancer Gene Census, we have established differences in mutational patterns that enable us to distinguish people with DS from people without DS and characterize the variability among people with DS. Notably, protein-altering mutations in genes responsible for the differentiation of hematopoietic stem and progenitor cells are more prevalent among people with T21 than their counterparts who are disomic in chromosome 21 (D21). Coupling our genomic findings with other -omics approaches, we have drawn associations between the observed mutations and phenotypes of these individuals. Using mouse models of DS (Dp16, Dp17, and Dp10), we plan to investigate the susceptibility of CH and leukemogenesis with the use of a Moloney Murine Leukemia virus, expecting the mice modeling DS to have more prevalent CH and more leukemias. Furthermore, based on our analyses of leukocytic gene expression differences between people with DS with or without CH, we believe that people with DS have a varying reduction in autophagy that enables the presence of harmful CH and increased disease risk in some individuals. Using a GFP-LC3-RFP construct, we plan to quantify and compare basal autophagic flux between the mouse models of DS and their control littermates. This will enable us to draw the links between DS, CH, and autophagy. Future work will be centered around manipulating autophagy using transgenic mice and non-invasive interventions to see if deleterious phenotypes and risks to AML can be reversed. Overall, these studies offer a means by which disease risk in people with DS can be monitored through CH and a potential mechanism that can be altered by non-intrusive means to improve longevity and quality of life. Citation Format: Edward James Evans, Ross Granrath, Keith Smith, Joaquin Espinosa, Andrew Thorburn, James DeGregori. Linking Down syndrome, clonal hematopoiesis, and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 763.

Objectives Individuals with Down syndrome (DS) display high risk of celiac disease (CD), but the mechanisms underlying this increased susceptibility await elucidation. Here, we examined the prevalence of HLA genotypes associated with CD risk in the general population and tested a previously developed genetic risk score (GRS) for CD in people with DS. Methods HLA genotypes were obtained for 204 individuals with DS in the Human Trisome Project cohort study, of whom 9% had CD. We compared HLA genotype frequencies in those with and without CD against frequencies observed in the general population. CD permissive HLA haplotypes explored were DQ2.5, DQ2.2, DQ8.1, and DQ7.5. We also analyzed 38 non-HLA-DQ alleles used to generate the CD GRS. Results Frequencies of risk genotypes were different for CD in DS versus CD in the general population. For example, we observed lower frequency of DQ2.5/DQ2.5 and higher prevalence of DQ7.5/X and X/X in CD in DS. Although GRS values were significantly increased in those with CD and DS, their predictive power was decreased relative to the general population. Transcriptome analysis revealed dysregulated expression of many genes composing the GRS in DS. Proteomics analysis showed that GRS values correlate with elevation of specific immune factors in DS. Conclusions The genetic risk profile of CD in DS is different relative to the general population, which is likely due to dysregulation of immune pathways in DS. Larger studies are needed to elucidate pathogenic mechanisms and to develop a validated GRS for CD in DS. What is Known What is New ### Competing Interest Statement R.J.S. serves on the advisory committee for Mirum Pharma and Abireo Pharma and is a consultant for Astellas. ### Funding Statement This study was funded by the NIH Office of the Director via the NIH INCLUDE Project through NIAID grant R01AI150305, and NCATS grants 5UL1TR002535 and KL2-TR002534. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional funding was provided by the Linda Crnic Institute for Down Syndrome, the GLOBAL Down Syndrome Foundation, the Anna and John J. Sie Foundation, the GI & Liver Innate Immune Program, the Human Immunology and Immunotherapy Initiative, and the University of Colorado School of Medicine. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Colorado Multiple Institutional Review Board (COMIRB) gave ethical approval of this work under protocol numbers COMIRB# 15-2170 and COMIRB# 20-1977. See also [clinicaltrials.gov][1] entry [NCT02864108][2]. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * (CD) : Celiac Disease (D21) : Disomy 21 (DS) : Down Syndrome (GRS) : Genetic Risk Score (HLA) : Human Leukocyte Antigen (IFN) : Interferon (T21) : Trisomy 21 [1]: http://clinicaltrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02864108&atom=%2Fmedrxiv%2Fearly%2F2022%2F09%2F29%2F2022.09.27.22280436.atom

COVID19 is a heterogeneous medical condition involving diverse underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Low antibody titers associate with hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, and depletion of lymphocytes, neutrophils, and platelets. Upon seroconversion, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased D-dimer, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.

ABSTRACTThe impacts of IFN signaling on COVID19 pathology are multiple, with protective and harmful effects being documented. We report here a multi-omics investigation of IFN signaling in hospitalized COVID19 patients, defining the biosignatures associated with varying levels of 12 different IFN ligands. Previously we showed that seroconversion associates with decreased production of select IFN ligands (Galbraith et al, 2021). We show now that the antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of ligands, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage associate with levels of IFNB and IFNA6. Differential IFN ligand production is linked to distinct constellations of circulating immune cells. Lastly, IFN ligands associate differentially with activation of the kynurenine pathway, dysregulated fatty acid metabolism, and altered central carbon metabolism. Altogether, these results reveal specialized IFN ligand action in COVID19, with potential diagnostic and therapeutic implications.IMPACT STATEMENTAnalysis of multi-omics signatures associated with 12 different IFN ligands reveals their specialized action in COVID19.