Longitudinal Phenotypes Research Articles

THE EFFECTS OF BIOLOGIC AND SMALL MOLECULE INHIBITOR THERAPY ON THE MUCOSAL IMMUNE SYSTEM IN ULCERATIVE COLITIS

Abstract Ulcerative colitis (UC) is a subtype of inflammatory bowel disease (IBD) that causes relapsing and remitting inflammation in the colon and rectum. Numerous treatment options are available for patients with UC including biologics and small molecule inhibitors that target the immune system. Although these therapies have dramatically improved options for patients with IBD, there remains a modest ceiling for their overall efficacy. Currently there are no biomarkers that can guide treatment decisions in patients with UC. Some studies have reported certain molecules or immune populations that are associated with treatment resistance or as predictors of treatment response. However, their clinical utility have yet to be fully determined because there is a paucity of longitudinal studies examining the direct effects of current biologics and small molecule inhibitors approved for UC on the immune biology of the colon mucosa. In this study, we perform longitudinal immune phenotyping using flow cytometry in patients with UC before and after therapy. Colon biopsy tissue was taken prior to treatment from 10 UC patients who were then followed and repeat biopsies were obtained from the same patients after 6-12 months of treatment. Tissue was analyzed using high dimensional flow cytometry with a panel of 24 markers of both lymphoid and myeloid lineages. Pre-treatment samples with inflammation correlated with higher populations of CD4+ T cells and CD4+ FOXP3 regulatory T cells also positive for TIGIT and GITR. Inflammation also corresponded with the presence of a CD33+ HLA-DR- population classically known as myeloid-derived suppressor cells (MDSC). Overall, therapy significantly decreased immune cell infiltration in the colon, most notably in B and T cell populations. However, certain treatments increased the MDSC population along with the stromal compartment of non-immune cells. A comprehensive analysis of the populations present during active inflammation and the treatment-induced changes in mucosal immunity will help identify biomarkers to predict response to therapy. In addition, examining the interplay between immune cells and the surrounding stroma can may lead to important findings that improve our understanding of each drug’s mechanism and efficacy. Future experiments with single cell RNA and T cell receptor sequencing on the same patients will identify effects of biologic therapy on the T cell repertoire and interactions between immune, epithelial, and stromal cells in the colonic mucosa. With the advent of novel sequencing and immune profiling assays, we now have the ability to harness the power of precision medicine to make more informed therapeutic decisions for IBD patients that improve clinical outcomes.

Novel loci for hyperglycemia identified by QTL mapping of longitudinal phenotypes and congenic analysis

We previously reported that four hyperglycemia loci are located on three chromosomes in the Nagoya-Shibata-Yasuda (NSY) mouse model, commonly used to study type 2 diabetes. However, we did not search for hyperglycemia loci across all chromosomes. In this study, we performed quantitative trait loci (QTLs) mapping of longitudinal phenotypes from crosses between NSY (hyperglycemic) and C3H (normoglycemic) mice. We identified four new QTLs for hyperglycemia, namely Nidd5nsy, Nidd6nsy, Nidd1c3h, and Nidd2c3h, on Chromosome 1, 4, 10, and 13, respectively. These QTLs were associated with hyperglycemia in young mice and had attenuated effects in older mice. Nidd5nsy and Nidd6nsy were hyperglycemic with NSY alleles, and Nidd1c3h and Nidd2c3h were hyperglycemic with C3H alleles. We further bred Nidd5nsy congenic mice and demonstrated that Nidd5nsy has a strong effect on hyperglycemia when young, accompanied by insulin resistance and visceral fat accumulation. These results showed that the effects of individual QTLs strengthened or weakened with age, and that the sum of the effects of QTLs captured the age-related deterioration of glucose tolerance in individuals. Our results support the importance of longitudinal phenotypes in the genetic analysis of polygenic traits and have implications for the genetic basis and pathogenesis of type 2 diabetes in humans.

Longitudinally tracking personal physiomes for precision management of childhood epilepsy.

Our current understanding of human physiology and activities is largely derived from sparse and discrete individual clinical measurements. To achieve precise, proactive, and effective health management of an individual, longitudinal, and dense tracking of personal physiomes and activities is required, which is only feasible by utilizing wearable biosensors. As a pilot study, we implemented a cloud computing infrastructure to integrate wearable sensors, mobile computing, digital signal processing, and machine learning to improve early detection of seizure onsets in children. We recruited 99 children diagnosed with epilepsy and longitudinally tracked them at single-second resolution using a wearable wristband, and prospectively acquired more than one billion data points. This unique dataset offered us an opportunity to quantify physiological dynamics (e.g., heart rate, stress response) across age groups and to identify physiological irregularities upon epilepsy onset. The high-dimensional personal physiome and activity profiles displayed a clustering pattern anchored by patient age groups. These signatory patterns included strong age and sex-specific effects on varying circadian rhythms and stress responses across major childhood developmental stages. For each patient, we further compared the physiological and activity profiles associated with seizure onsets with the personal baseline and developed a machine learning framework to accurately capture these onset moments. The performance of this framework was further replicated in another independent patient cohort. We next referenced our predictions with the electroencephalogram (EEG) signals on selected patients and demonstrated that our approach could detect subtle seizures not recognized by humans and could detect seizures prior to clinical onset. Our work demonstrated the feasibility of a real-time mobile infrastructure in a clinical setting, which has the potential to be valuable in caring for epileptic patients. Extension of such a system has the potential to be leveraged as a health management device or longitudinal phenotyping tool in clinical cohort studies.

Abstract IA001: Intercepting pancreatic cancer development with oncogene targeted immunotherapy

Abstract Pancreatic adenocarcinoma (PDAC) progression is triggered by a complex interaction of genetic mutations, stromal cell interactions and tumor microenvironmental (TME) signals. Diagnosis usually occurs late in disease progression, making treatment challenging and survival rates extremely poor. PDACs are also considered non-immunogenic, therefore newly emerging immunotherapies that have been successful in other cancers have not significantly progressed PDAC treatment options. Resistance to immunotherapies progresses as normal cells undergo the earliest genetic changes that transform them into early pre-malignant lesions. The two most common premalignant lesions are pancreatic intra-epithelial neoplasms (PanINs) and intraductal papillary mucinous neoplasm (IPMN). Both types of premalignant lesions eventually accumulate additional genetic changes that lead to early-stage invasive cancer and eventually, to late stage PDAC. This transformation process is influenced by both tumor-intrinsic and extrinsic forces within the developing TME. Accumulating data suggests that immune resistance mechanisms also evolve with this progression, which has led to the hypothesis that early immune intervention may be the best time to intervene to slow or even halt disease progression and improve treatment outcomes. We know that tumor initiation to metastases takes years to decades, providing a unique window of opportunity for the prevention of premalignant progression. KRAS mutations are an early oncogenic event present in over 90% of PanINs and 75% of IPMNs. Initial studies examining cancer vaccines targeted to early oncogenic mutations are showing promise in animal models. A listeria-based vaccine engineered to express oncogenic KrasG12D combined with and without regulatory T cell depletion by an anti-CD25 antibody (PC61) and cyclophosphamide showed increased T cell infiltration, decreased disease progression, and increased survival in Kras-driven and p53 mutated genetically engineered mice (KrasG12D/+Trp53R172H/+;Pdx-1-Cre (KPC) mice) with early PanIN lesions but not those with later stage PanINs (Keenan et. al. 2014. Gastroenterology 146: 1784). Other early oncogenic mutations in PDACs may prove to be therapeutic targets for vaccine development. We recently initiated a pilot study to examine the feasibility and safety of targeting mKRAS in patients with resected PDAC. For this, we developed a clinical–grade pooled peptide vaccine targeting 6 common KRAS mutations. This vaccine has been tested in combination with checkpoint blockade in 10 PDAC patients who had undergone surgery plus peri–operative chemotherapy and had remained disease–free (NCT04117087). Longitudinal immune phenotyping showed a robust peripheral mKRAS–specific T cell response against the most vaccinated epitopes. Flow cytometry revealed that these CD4 and CD8 T cells were activated, polyfunctional and displayed a memory phenotype. Based on the favorable immunogenicity and safety profile of our peptide vaccine in patients with resected PDAC, we have initiated a Prevention Study testing our mKRAS vaccine in individuals at high risk for PDAC. This study is currently enrolling and early results will be described. Citation Format: Elizabeth M. Jaffee. Intercepting pancreatic cancer development with oncogene targeted immunotherapy [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA001.

Early childhood wheezing phenotypes and determinants in a South African birth cohort: longitudinal analysis of the Drakenstein Child Health Study

Developmental trajectories of childhood wheezing in low-income and middle-income countries (LMICs) have not been well described. We aimed to derive longitudinal wheeze phenotypes from birth to 5 years in a South African birth cohort and compare those with phenotypes derived from a UK cohort. We used data from the Drakenstein Child Health Study (DCHS), a longitudinal birth cohort study in a peri-urban area outside Cape Town, South Africa. Pregnant women (aged ≥18 years) were enrolled during their second trimester at two public health clinics. We followed up children from birth to 5 years to derive six multidimensional indicators of wheezing (including duration, temporal sequencing, persistence, and recurrence) and applied Partition Around Medoids clustering to derive wheeze phenotypes. We compared phenotypes with a UK cohort (the Avon Longitudinal Study of Parents and Children [ALSPAC]). We investigated associations of phenotypes with early-life exposures, including all-cause lower respiratory tract infection (LRTI) and virus-specific LRTI (respiratory syncytial virus, rhinovirus, adenovirus, influenza, and parainfluenza virus) up to age 5 years. We investigated the association of phenotypes with lung function at 6 weeks and 5 years. Between March 5, 2012, and March 31, 2015, we enrolled 1137 mothers and there were 1143 livebirths. Four wheeze phenotypes were identified among 950 children with complete data: never (480 children [50%]), early transient (215 children [23%]), late onset (104 children [11%]), and recurrent (151 children [16%]). Multivariate adjusted analysis indicated that LRTI and respiratory syncytial virus-LRTI, but not other respiratory viruses, were associated with increased risk of recurrent wheeze (odds ratio [OR] 2·79 [95% CI 2·05-3·81] for all LTRIs; OR 2·59 [1·30-5·15] for respiratory syncytial virus-LRTIs). Maternal smoking (1·88 [1·12-3·02]), higher socioeconomic status (2·46 [1·23-4·91]), intimate partner violence (2·01 [1·23-3·29]), and male sex (2·47 [1·50-4·04]) were also associated with recurrent wheeze. LRTI and respiratory syncytial virus-LRTI were also associated with early transient and late onset clusters. Wheezing illness architecture differed between DCHS and ALSPAC; children included in ALSPAC in the early transient cluster wheezed for a longer period before remission and late-onset wheezing started at an older age, and no persistent phenotype was identified in DCHS. At 5 years, airway resistance was higher in children with early or recurrent wheeze compared with children who had never wheezed. Airway resistance increased from 6 weeks to 5 years among children with recurrent wheeze. Effective strategies to reduce maternal smoking and psychosocial stressors and new preventive interventions for respiratory syncytial virus are urgently needed to optimise child health in LMICs. UK Medical Research Council; The Bill & Melinda Gates Foundation; National Institutes of Health Human Heredity and Health in Africa; South African Medical Research Council; Wellcome Trust.

The longitudinal triglyceride phenotype in heterozygotes with LPL pathogenic variants

Biallelic pathogenic variants in lipoprotein lipase (LPL) cause familial chylomicronemia syndrome with severe hypertriglyceridemia (HTG), defined as plasma triglycerides (TG) > 10 mmol/L (> 885 mg/dL). TG levels in individuals with one copy of a pathogenic LPL gene variant is less familiar; some assume that the phenotype is intermediate between homozygotes and controls. We undertook an evaluation of the longitudinal TG phenotype of individuals heterozygous for pathogenic LPL variants. Medically stable outpatients were evaluated based on having: (1) a single copy of a rare pathogenic LPL variant; and (2) serial fasting TG measurements obtained over > 1.5 years of follow-up. Fifteen patients with a single pathogenic LPL variant were followed for a mean of 10.3 years (range 1.5 to 30.3 years). TG levels varied widely both within and between patients. One patient had normal TG levels < 2.0 mmol/L (< 175 mg/dL) continuously, while four patients had at least one normal TG level. Most patients fluctuated between mild-to-moderate and severe HTG: five patients had only mild-to-moderate HTG, with TG levels ranging from 2.0 to 9.9 mmol/L (175 to 885 mg/dL), while 6 patients had at least one instance of severe HTG. Of the 203 total TG measurements from these patients, 14.8%, 67.0% and 18.2% were in the normal, mild-to-moderate and severe HTG ranges, respectively. The heterozygous LPL deficient phenotype is highly variable both within and between patients. Heterozygosity confers susceptibility to a wide range of TG phenotypes, with severity likely depending on secondary factors.

Longitudinal phenotypes in patients with acute respiratory distress syndrome: a multi-database study

BackgroundPreviously identified phenotypes of acute respiratory distress syndrome (ARDS) have been limited by a disregard for temporal dynamics. We aimed to identify longitudinal phenotypes in ARDS to test the prognostic and predictive enrichment of longitudinal phenotypes, and to develop simplified models for phenotype identification.MethodsWe conducted a multi-database study based on the Chinese Database in Intensive Care (CDIC) and four ARDS randomized clinical trials (RCTs). We employed latent class analysis (LCA) to identify longitudinal phenotypes using 24-hourly data from the first four days of invasive ventilation. We used the Cox regression model to explore the association between time-varying respiratory parameters and 28-day mortality across phenotypes. Phenotypes were validated in four RCTs, and the heterogeneity of treatment effect (HTE) was investigated. We also constructed two multinomial logistical regression analyses to develop the probabilistic models.FindingsA total of 605 ARDS patients in CDIC were enrolled. The three-class LCA model was identified and had the optimal fit, as follows: Class 1 (n = 400, 66.1% of the cohort) was the largest phenotype over all study days, and had fewer abnormal values, less organ dysfunction and the lowest 28-day mortality rate (30.5%). Class 2 (n = 102, 16.9% of the cohort) was characterized by pulmonary mechanical dysfunction and had the highest proportion of poorly aerated lung volume, the 28-day mortality rate was 47.1%. Class 3 (n = 103, 17% of the cohort) was correlated with extra-pulmonary dysfunction and had the highest 28-day mortality rate (56.3%). Time-varying mechanical power was more significantly associated with 28-day mortality in Class 2 patients compared to other phenotypes. Similar phenotypes were identified in four RCTs. A significant HTE between phenotypes and treatment strategies was observed in the ALVEOLI (high PEEP vs. low PEEP) and the FACTT trials (conservative vs. liberal fluid management). Two parsimonious probabilistic models were constructed to identify longitudinal phenotypes.InterpretationWe identified and validated three novel longitudinal phenotypes for ARDS patients, with both prognostic and predictive enrichment. The phenotypes of ARDS can be accurately identified with simple classifier models, except for Class 3.

Systematic dissection, preservation, and multiomics in whole human and bovine hearts

ObjectivesWe sought to develop a rigorous, systematic protocol for the dissection and preservation of human hearts for biobanking that expands previous success in postmortem transcriptomics to multiomics from paired tissue. BackgroundExisting cardiac biobanks consist largely of biopsy tissue or explanted hearts in select diseases and are insufficient for correlating whole organ phenotype with clinical data. MethodsWe demonstrate optimal conditions for multiomics interrogation (ribonucleic acid (RNA) sequencing, untargeted metabolomics) in hearts by evaluating the effect of technical variables (storage solution, temperature) and simulated postmortem interval (PMI) on RNA and metabolite stability. We used bovine (n=3) and human (n=2) hearts fixed in PAXgene or snap-frozen with liquid nitrogen. ResultsUsing a paired Wald test, only two of the genes assessed were differentially expressed between left ventricular samples from bovine hearts stored in PAXgene at 0 and 12 hours PMI (FDR q<0.05). We obtained similar findings in human left ventricular samples, suggesting stability of RNA transcripts at PMIs up to 12 hours. Different library preparation methods (mRNA poly-A capture vs. rRNA depletion) resulted in similar quality metrics with both library preparations achieving >95% of reads properly aligning to the reference genomes across all PMIs for bovine and human hearts. PMI had no effect on RNA Integrity Number or quantity of RNA recovered at the time points evaluated. Of the metabolites identified (855 total) using untargeted metabolomics of human left ventricular tissue, 503 metabolites remained stable across PMIs (0, 4, 8, 12 hours). Most metabolic pathways retained several stable metabolites. ConclusionsOur data demonstrate a technically rigorous, reproducible protocol that will enhance cardiac biobanking practices and facilitate novel insights into human CVD. Condensed abstractCardiovascular disease (CVD) is the leading cause of mortality worldwide. Current biobanking practices insufficiently capture both the diverse array of phenotypes present in CVDs and the spatial heterogeneity across cardiac tissue sites. We have developed a rigorous and systematic protocol for the dissection and preservation of human cardiac biospecimens to enhance the availability of whole organ tissue for multiple applications. When combined with longitudinal clinical phenotyping, our protocol will enable multiomics in hearts to deepen our understanding of CVDs.

DUPILUMAB REBALANCES IMMUNE RESPONSE IN PEDIATRIC TYPE-2 DISORDERS: IMPACTS ON T-CELL CLONES AND FUNCTIONALITY

<h3>Introduction</h3> Dupilumab demonstrates efficacy in treating a variety of Type-2 disorders refractory to conventional therapies. Improvement in clinical outcomes establishes a need to investigate mechanistic pathways of potential immunomodulation. <h3>Methods</h3> 11 patients, two with moderate-to-severe asthma and 9 with moderate-top-severe AD were recruited to give blood samples pre and three months post-Dupilumab. Longitudinal phenotyping of leukocytes using mass cytometry (CyTOF) and lineage tracing of single-cells using mitochondrial single-cell ATAC-seq (mt-scATAC-seq) was conducted in PBMC and purified T-cells. Clones were inferred using a community-based detection algorithm to cluster cells based on mitochondrial variant allele-frequency (VAF). Gene scores were computed based on open-chromatin regions in the gene and promoter. Differential expression pre- and post-dupilumab was done for each cell cluster population. <h3>Results</h3> CyTOF immunophenotyping showed increased Th1 and Th17 cells following dupilumab, consistent with increased activity of IL-6, TNF, IFNa, and IFNg genes in lymphocytes by mt-scATAC-Seq. Rebalancing of adaptive immunity was accompanied by upregulation of Myc, E2F, MTORC1, PI3K-AKT, and p53 signaling intermediates, and was associated with upregulation of genes controlling apoptosis, unfolded protein response, and oxidative phosphorylation. Changes in frequency of T-cell clonal lineages <i>in vivo</i> were detected following dupilumab. <h3>Conclusion</h3> Data supports clone-specific effects of dupilumab in pediatric Type-2 disorders. This approach enables analysis of changes in gene networks in T-cell clones and populations following treatment. Upregulation of non-Type-2 signaling and changes at the clonal level suggest Dupilumab restores balance to the immune response.

Effect of perfluoroalkyl exposure in pregnancy and infancy on intrauterine and childhood growth and anthropometry. Sub study from COPSAC2010 birth cohort.

Perfluoroalkyl substances PFOS and PFOA are persistent and bioaccumulative exogenous chemicals in the human body with a range of suspected negative health effects. It is hypothesised that exposure during prenatal and early postnatal life might have particularly detrimental effects on intrauterine and childhood growth. In a Danish longitudinal mother-child cohort we investigate effect of PFOS and PFOA in pregnancy and infancy on intrauterine and childhood growth and anthropometry. COPSAC2010 is an ongoing population based mother-child cohort of 738 pregnant women and their children followed from 24 week gestation with longitudinal deep clinical phenotyping until age 10 years. In this observational cohort sub study plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics in the mothers at week 24 and 1 week postpartum and in the children at ages 6 and 18 months and calibrated using a targeted pipeline. We examined associations to intrauterine and childhood growth and anthropometry, including interactions with child sex. Untargeted and targeted blood metabolomics profiles were integrated to investigate underlying mechanisms. Pregnancy plasma PFOA concentrations were associated with lower birth size -0.19 [-0.33; -0.05] BMI z-score per 1-ng/mL and increased childhood height (z-scored) at age 6: 0.18 [0.05; 0.31], but there was no association between childs' own infancy plasma PFOA concentration and height. Pregnancy plasma PFOS concentrations were also associated with lower birth BMI (-0.04 [-0.08; -0.01]), but in childhood pregnancy plasma PFOS concentration interacted with child sex on BMI and fat percentage at 6 years with negative associations in girls and positive in boys. The effect of maternal plasma PFOS concentration on lower girl BMI was borderline mediated through increasing child plasma lactosyl-ceramide levels (p-mediation=0.08). Similarly the effect of maternal plasma PFOS concentration on higher boy fat percentage was borderline mediated through increasing child plasma lactosyl-ceramide levels (p-mediation=0.07). Infancy concentrations of plasma PFOS associated with lower height in childhood, -0.06 z-score at age 6 [-0.19; -0.03]. Higher PFOS and PFOA plasma concentrations during pregnancy had detrimental effects on fetal growth. The effects on childhood growth were not similar as PFOA increased child height, opposite of PFOS in multipollutant models suggesting a differing fetal programming effect. Sex specific growth effects were borderline mediated through an altered lactosyl-ceramide metabolism, proposing a possible mechanism of PFOS that has long-lasting health consequences in this observational study. All funding received by COPSAC are listed on www.copsac.com. The Lundbeck Foundation (Grant no R16-A1694); The Novo Nordic Foundation (Grant nos NNF20OC0061029, NNF170C0025014, NNF180C0031764) The Ministry of Health (Grant no 903516); Danish Council for Strategic Research (Grant no 0603-00280B) and The Capital Region Research Foundation have provided core support to the COPSAC research center. Effort from JALS is supported by R01HL123915, R01HL141826, and R01HL155742 from NIH/NHLBI. CEW was supported by the Swedish Heart Lung Foundation (HLF 20180290, HLF 20200693). BC has received funding for this project from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 946228). The funding agencies did not have any role in design and conduct of the study; collection, management, and interpretation of the data; or preparation, review, or approval of the manuscript.

A longitudinal study of cytochrome P450 2D6 (CYP2D6) activity during adolescence.

CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7–15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4‐h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention‐deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model‐estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype‐based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.

Pulmonary Function Trajectories in People with HIV: Analysis of the Pittsburgh HIV Lung Cohort.

Rationale: Human immunodeficiency virus (HIV) infection is associated with chronic lung disease and impaired pulmonary function; however, longitudinal pulmonary function phenotypes in HIV are undefined. Objectives: To identify pulmonary function trajectories, their determinants, and outcomes. Methods: We used data from participants with HIV in the Pittsburgh HIV Lung Cohort with three or more pulmonary function tests between 2007 and 2020. We analyzed post-bronchodilator forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and FEV1/FVC, and diffusing capacity of the lung for carbon monoxide (DlCO) using group-based trajectory modeling to identify subgroups of individuals whose measurements followed a similar pattern over time. We examined the association between participant characteristics and trajectories using multivariable logistic regression. In exploratory adjusted analyses restricted to individuals with available plasma cytokine data, we investigated the association between 18 individual standardized cytokine concentrations and trajectories. We compared mortality, dyspnea prevalence, respiratory health status, and 6-minute-walk distance between phenotypes. Results: A total of 265 participants contributed 1,606 pulmonary function measurements over a median follow-up of 8.1 years. We identified two trajectories each for FEV1 and FVC: "low baseline, slow decline" and "high baseline, rapid decline." There were three trajectory groups for FEV1/FVC: "rapid decline," "moderate decline," and "slow decline." Finally, we identified two trajectories for DlCO: "baseline low" and "baseline high." The low baseline, slow decline FEV1 and FVC, rapid decline, and moderate decline FEV1/FVC, and baseline low DlCO phenotypes were associated with increased dyspnea prevalence, worse respiratory health status, and decreased 6-minute-walk distance. The baseline low DlCO phenotype was also associated with worse mortality. Current smoking and pack-years of smoking were associated with the adverse FEV1, FEV1/FVC, and DlCO phenotypes. Detectable viremia was the only HIV marker associated with the adverse DlCO phenotype. C-reactive protein and endothelin-1 were associated with the adverse FEV1 and FVC phenotypes, and endothelin-1 trended toward an association with the adverse DlCO phenotype. Conclusions: We identified novel, distinct longitudinal pulmonary function phenotypes with significant differences in characteristics and outcomes. These findings highlight the importance of lung dysfunction over time in people with HIV and should be validated in additional cohorts.

Phenotypes of disease severity in a cohort of hospitalized COVID-19 patients: Results from the IMPACC study.

SummaryBackgroundBetter understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management.MethodsImmunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed.FindingsThe median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28–3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13–2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63– 4.80) or troponin (OR 1.89; 95% 1.03–3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61–2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96–5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17–2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC.InterpretationIntegration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19.FundingNIH.

Longitudinal phenotyping of maternal antenatal depression in obese pregnant women supports multiple-hit hypothesis for fetal brain development, a secondary analysis of the UPBEAT study.

SummaryBackgroundMaternal antenatal depression is associated with offspring psychological disorders, but obesity is also widely implicated in maternal depression and neurodevelopment. In pregnant women with obesity we explored interrelationships between antenatal depressive symptom trajectories and multiple exposures implicated in fetal neurodevelopment which could explain these associations, as a prelude to exploring associations with infant mental health.MethodsThe UK Pregnancies Better Eating and Activity Trial (UPBEAT) recruited multi-ethnic pregnant women with obesity (BMI >= 30kg/m2) between March 2009 and June 2014 from 8 UK sites and 1369 were included to model longitudinal antenatal depressive symptoms from Edinburgh Postnatal Depression Scale (EPDS) scores using Latent Class Growth Analysis. Classes were compared on maternal baseline demography, biomarkers of metabolism, inflammation and placental function, infection, diet and by pregnancy and birth outcomes. Odds ratios, mean differences and 95% Confidence Intervals were calculated using robust auxiliary modelling techniques.FindingsThe chosen model produced four classes: “Not Depressed” (n=575 [42%], “reference”), “Mild” (n=523 [37·5%]), “Moderate” (n=219 [16%]) and “Severe” (n=62 [4·5%]) symptom trajectories. Socio-economic deprivation and ethnic diversity were greater in Severe and Moderate classes. Dietary glycaemic load and saturated fat intake were higher in Severe and Moderate classes (at 17 and 27 weeks). Higher Interleukin-6, glycoprotein acetyls (17 weeks), glucose (34 weeks) and lower placental growth factor (PlGF, 17 and 27 weeks) was found in the Severe class. PlGF was lower in the Moderate class (27 weeks). Infection was least likely in the Not Depressed class across gestation. Risks of preterm birth were associated with Severe depressive symptoms (aOR 3·05[1·11 to 8·36]).InterpretationComprehensive phenotyping exposes important fetal exposures implicated in adverse neurodevelopment, differing by depression class. This study expands substantially on causal models of suboptimal fetal neurodevelopment and offers potential new targets for intervention in obese pregnant women.FundingJNS was funded by a PhD studentship from the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas’ NHS Foundation Trust and King's College London. UPBEAT was supported by the European Union's 7th Framework Programme (FP7/2007-2013), project EarlyNutrition; grant agreement no. 289346 and the National Institute for Health Research (NIHR) (UK) Programme Grants for Applied Research Programme (RP-0407-10452), Medical Research Council UK Project Grant (MR/L002477/1). Support was also provided by the Chief Scientist Office Scotland, Guy's and St Thomas’ Charity and Tommy's Charity (Registered charity no. 1060508). LP and SLW are funded by Tommy's Charity.

Monogenic disorders as mimics of juvenile idiopathic arthritis

BackgroundJuvenile idiopathic arthritis is the most common chronic rheumatic disease of childhood. The term JIA encompasses a heterogenous group of diseases. The variability in phenotype of patients affected by the disease means it is not uncommon for mimics of JIA to be misdiagnosed.Case presentationWe present four cases who were treated in single tertiary rheumatology centre for JIA who were subsequently diagnosed with a rare monogenic disease. All four patients shared the unifying features of presenting in early childhood and subsequently suffered with refractory disease, not amenable to usual standards of treatment. Multicentric Carpotarsal Osteolysis Syndrome and Camptodactyly-arthropathy-coxa vara-pericarditis syndrome are non-inflammatory conditions and patients typically present with arthropathy, normal inflammatory markers and atypical radiological features. Blau syndrome is an autosomal dominant condition and patients will typically have symmetrical joint involvement with a strong family history of arthritis, signifying the genetic aetiology.ConclusionsWe share our learning from these cases to add to the growing portfolio of JIA mimics and to highlight when to consider an alternative diagnosis. In cases of refractory disease and diagnostic uncertainty further imaging and genetic testing can play a crucial role in establishing the aetiology. In all of these cases the correct diagnosis was made due to careful, longitudinal clinical phenotyping and a close working relationship between rheumatology, radiology and clinical genetics; highlighting the importance of the multidisciplinary team in managing complex patients.

A Systematic Review of Artificial Intelligence and Machine Learning Applications to Inflammatory Bowel Disease, with Practical Guidelines for Interpretation.

BackgroundInflammatory bowel disease (IBD) is a gastrointestinal chronic disease with an unpredictable disease course. Computational methods such as machine learning (ML) have the potential to stratify IBD patients for the provision of individualized care. The use of ML methods for IBD was surveyed, with an additional focus on how the field has changed over time.MethodsOn May 6, 2021, a systematic review was conducted through a search of MEDLINE and Embase databases, with the search structure (“machine learning” OR “artificial intelligence”) AND (“Crohn* Disease” OR “Ulcerative Colitis” OR “Inflammatory Bowel Disease”). Exclusion criteria included studies not written in English, no human patient data, publication before 2001, studies that were not peer reviewed, nonautoimmune disease comorbidity research, and record types that were not primary research.ResultsSeventy-eight (of 409) records met the inclusion criteria. Random forest methods were most prevalent, and there was an increase in neural networks, mainly applied to imaging data sets. The main applications of ML to clinical tasks were diagnosis (18 of 78), disease course (22 of 78), and disease severity (16 of 78). The median sample size was 263. Clinical and microbiome-related data sets were most popular. Five percent of studies used an external data set after training and testing for additional model validation.DiscussionAvailability of longitudinal and deep phenotyping data could lead to better modeling. Machine learning pipelines that consider imbalanced data and that feature selection only on training data will generate more generalizable models. Machine learning models are increasingly being applied to more complex clinical tasks for specific phenotypes, indicating progress towards personalized medicine for IBD.

Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice

BackgroundThe zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity.MethodsHere, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied.ResultsMyt1l was necessary for survival beyond 24 h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact.LimitationsIn future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice.ConclusionsBehavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

OP-10 Gut microbiota dysbiosis as a driver of inflammation in Ocular Mucous Membrane Pemphigoid.

Gut microbiome profiles between OcMMP patients (n=49) and healthy controls (n=40) were compared by extracting DNA from faecal samples and amplified for the V4 region of the 16S rRNA gene followed by Illumina Miseq platform sequencing. Sequencing reads were processed using the bioinformatics pipeline available in the mothur v.1.44.1 software. Using multivariable model and adjustment for participant factors, OcMMP cohort was found to be associated with lower number of operational taxonomic units (OTUs) and Shannon Diversity Index when compared to healthy controls. OcMMP OTUs were found to be significantly correlated with both the bulbar conjunctival inflammation score (p=0.03) and the current use of systemic immunotherapy (p=0.02). Linear discriminant analysis effect size scores found Streptococcus and Lachnoclostridium enriched in OcMMP. By contrast, healthy controls were enriched with Oxalobacter, Clostridia uncultured genus-level group (UCG) 014, Christensenellaceae R-7 group and butyrate-producing bacteria such as Ruminococcus, Lachnospiraceae, Coprococcus, Roseburia, Oscillospiraceae UCG 003, 005, NK4A214 group (Log10 LDA score <2, FDR-adjusted p<0.05). In conclusion, OcMMP patients have gut dysbiosis that correlated with bulbar conjunctival inflammation and the use of systemic immunotherapies. This provides a framework for future longitudinal deep phenotyping studies on the role of the gut microbiome in the pathogenesis of OcMMP. Low L, Suleiman K, Shamdas M, Bassilious K, Poonit N, Rossiter AE, Acharjee A, Loman N, Murray PI, Wallace GR, Rauz S. Gut dysbiosis in ocular mucous membrane pemphigoid. Front. Cell. Infect. Microbiol. 2022;12:780354. https://doi.org/10.3389/fcimb.2022.780354.

EHypertension: A prospective longitudinal multi‐omics essential hypertension cohort

eHypertension: A prospective longitudinal multi‐omics essential hypertension cohort

Gut Dysbiosis in Ocular Mucous Membrane Pemphigoid.

Mucous Membrane Pemphigoid is an orphan multi-system autoimmune scarring disease involving mucosal sites, including the ocular surface (OcMMP) and gut. Loss of tolerance to epithelial basement membrane proteins and generation of autoreactive T cell and/or autoantibodies are central to the disease process. The gut microbiome plays a critical role in the development of the immune system. Alteration in the gut microbiome (gut dysbiosis) affects the generation of autoreactive T cells and B cell autoantibody repertoire in several autoimmune conditions. This study examines the relationship between gut microbiome diversity and ocular inflammation in patients with OcMMP by comparing OcMMP gut microbiome profiles with healthy controls. DNA was extracted from faecal samples (49 OcMMP patients, 40 healthy controls), amplified for the V4 region of the 16S rRNA gene and sequenced using Illumina Miseq platform. Sequencing reads were processed using the bioinformatics pipeline available in the mothur v.1.44.1 software. After adjusting for participant factors in the multivariable model (age, gender, BMI, diet, proton pump inhibitor use), OcMMP cohort was found to be associated with lower number of operational taxonomic units (OTUs) and Shannon Diversity Index when compared to healthy controls. Within the OcMMP cohort, the number of OTUs were found to be significantly correlated with both the bulbar conjunctival inflammation score (p=0.03) and the current use of systemic immunotherapy (p=0.02). The linear discriminant analysis effect size scores indicated that Streptococcus and Lachnoclostridium were enriched in OcMMP patients whilst Oxalobacter, Clostridia uncultured genus-level group (UCG) 014, Christensenellaceae R-7 group and butyrate-producing bacteria such as Ruminococcus, Lachnospiraceae, Coprococcus, Roseburia, Oscillospiraceae UCG 003, 005, NK4A214 group were enriched in healthy controls (Log10 LDA score < 2, FDR-adjusted p <0.05). In conclusion, OcMMP patients have gut dysbiosis correlating with bulbar conjunctival inflammation and the use of systemic immunotherapies. This provides a framework for future longitudinal deep phenotyping studies on the role of the gut microbiome in the pathogenesis of OcMMP.