User-Centered Design to Develop Automated Short Message Service Messaging for Patient Support of Rheumatic Heart Disease Secondary Antibiotic Prophylaxis.

BACKGROUND: The Active Community Case Management Platform is a cloud-based technology developed to facilitate rheumatic heart disease case management by health care providers. This study aimed to design and pilot an automated short message service (SMS) intervention to support secondary prophylaxis adherence. METHODS AND RESULTS: We developed a concise library of messages to support secondary antibiotic prophylaxis. The SMS intervention used TextIT, an interface that enables users to send out interactive SMS messages at scale. The message bank was piloted in a cohort of 50 patients with rheumatic heart disease randomized into 2 groups. Group 1 received standard support (nurse-led/Active Community Case Management Platform). Group 2 received standard support plus SMS intervention for 2 months in the Lira and Gulu districts of Northern Uganda. We collected qualitative data on participants' experiences and assessed treatment adherence. Using a sequential user-centered process consisting of 4 phases (phases 1-4), we developed a message bank (n=43) deployed during our pilot study. There were no between-group differences in treatment adherence or acceptance. Interviews of participants indicated that the intervention was viewed positively. A total of 75% of SMS recipients responded to the messages, and 25% called the study staff to acknowledge receipt of text messages. CONCLUSIONS: This study has successfully developed a bank of SMS messages to support secondary antibiotic prophylaxis adherence. We have demonstrated the feasibility and acceptability of SMS technology in rheumatic heart disease care management. Future work will include integrating automated SMS into the Active Community Case Management Platform and a larger study of integrated SMS to reduce health care worker burden for patient support and improve adherence to secondary antibiotic prophylaxis.

Development and User Testing of a Dynamic Tool for Rheumatic Heart Disease Management.

OBJECTIVE: Most rheumatic heart disease (RHD) registries are static and centralized, collecting epidemiological and clinical data without providing tools to improve care. We developed a dynamic cloud-based RHD case management application with the goal of improving care for patients with RHD in Uganda. METHODS: The Active Community Case Management Tool (ACT) was designed to improve community-based case management for chronic disease, with RHD as the first test case. Global and local partner consultation informed selection of critical data fields and prioritization of application functionality. Multiple stages of review and revision culminated in user testing of the application at the Uganda Heart Institute. RESULTS: Global and local partners provided feedback of the application via survey and interview. The application was well received, and top considerations included avenues to import existing patient data, considering a minimum data entry form, and performing a situation assessment to tailor ACT to the health system setup for each new country. Test users completed a postuse survey. Responses were favorable regarding ease of use, desire to use the application in regular practice, and ability of the application to improve RHD care in Uganda. Concerns included appropriate technical skills and supports and potential disruption of workflow. CONCLUSION: Creating the ACT application was a dynamic process, incorporating iterative feedback from local and global partners. Results of the user testing will help refine and optimize the application. The ACT application showed potential for utility and integration into existing care models in Uganda.

Evaluating the implementation of a dynamic digital application to enable community-based decentralisation of rheumatic heart disease case management in Uganda: protocol for a hybrid type III effectiveness-implementation study.

INTRODUCTION: Rheumatic heart disease (RHD) affects over 39 million people worldwide, the majority in low-income and middle-income countries. Secondary antibiotic prophylaxis (SAP), given every 3-4 weeks can improve outcomes, provided more than 80% of doses are received. Poor adherence is strongly correlated with the distance travelled to receive prophylaxis. Decentralising RHD care has the potential to bridge these gaps and at least maintain or potentially increase RHD prophylaxis uptake. A package of implementation strategies was developed with the aim of reducing barriers to optimum SAP uptake. METHODS AND ANALYSIS: A hybrid implementation-effectiveness study type III was designed to evaluate the effectiveness of a package of implementation strategies including a digital, cloud-based application to support decentralised RHD care, integrated into the public healthcare system in Uganda. Our overarching hypothesis is that secondary prophylaxis adherence can be maintained or improved via a decentralisation strategy, compared with the centralised delivery strategy, by increasing retention in care. To evaluate this, eligible patients with RHD irrespective of their age enrolled at Lira and Gulu hospital registry sites will be consented for decentralised care at their nearest participating health centre. We estimated a sample size of 150-200 registrants. The primary outcome will be adherence to secondary prophylaxis while detailed implementation measures will be collected to understand barriers and facilitators to decentralisation, digital application tool adoption and ultimately its use and scale-up in the public healthcare system. ETHICS AND DISSEMINATION: This study was approved by the Institutional Review Board (IRB) at Cincinnati Children's Hospital Medical Center (IRB 2021-0160) and Makerere University School of Medicine Research Ethics Committee (Mak-SOMREC-2021-61). Participation will be voluntary and informed consent or assent (>8 but <18) will be obtained prior to participation. At completion, study findings will be communicated to the public, key stakeholders and submitted for publication.

Comparison of approaches to determine echocardiographic outcomes for children with latent rheumatic heart disease.

BACKGROUND: Screening programmes using echocardiography offer opportunity for intervention through identification and treatment of early (latent) rheumatic heart disease (RHD). We aimed to compare two methods for classifying progression or regression of latent RHD: serial review method and blinded, side-by-side review. METHODS: A four-member expert panel reviewed 799 enrolment (in 2018) and completion (in 2020) echocardiograms from the GOAL Trial of latent RHD in Uganda to make consensus determination of normal, borderline RHD or definite RHD. Serial interpretations (enrolment and completion echocardiograms read at two different time points, 2 years apart, not beside one another) were compared with blinded side-by-side comparisons (enrolment and completion echocardiograms displayed beside one another in random order on same screen) to determine outcomes according to prespecified definitions of disease progression (worsening), regression (improving) or no change. We calculated inter-rater agreement using Cohen's kappa. RESULTS: There were 799 pairs of echocardiogram assessments included. A higher number, 54 vs 38 (6.8% vs 4.5%), were deemed as progression by serial interpretation compared with side-by-side comparison. There was good inter-rater agreement between the serial interpretation and side-by-side comparison methods (kappa 0.89). Disagreement was most often a result of the difference in classification between borderline RHD and mild definite RHD. Most discrepancies between interpretation methods (46 of 47, 98%) resulted from differences in valvular morphological evaluation, with valves judged to be morphologically similar between enrolment and final echocardiograms when compared side by side but classified differently on serial interpretation. CONCLUSIONS: There was good agreement between the methods of serial and side-by-side interpretation of echocardiograms for change over time, using the World Heart Federation criteria. Side-by-side interpretation has higher specificity for change, with fewer differences in the interpretation of valvular morphology, as compared with serial interpretation.

Diagnoses during the First Three Years of Life for Children with Prenatal Opioid Exposure and Neonatal Abstinence Syndrome Using a Large Maternal Infant Data Hub.

PURPOSE: The purpose of this study was to examine patterns of health and developmental outcomes in children with prenatal opioid exposure (POE) and neonatal abstinence syndrome (NAS) compared to children without exposure during the first three years of life. DESIGN AND METHODS: This was a secondary data analysis of the Maternal and Infant Data Hub (MIDH), a de-identified dataset originating from the Midwest region of the United States, consisting of newborn billing records and corresponding maternal and child electronic medical records. For these analyses, the repository included data on more than 20,000 children born between 2013 and 2019. Diagnoses were identified with International Classification of Diseases, ninth and tenth Revision, Clinical Modification codes (ICD-9/10-CM). Firth logistic regression was used to assess whether incidence of each diagnosis code differed by exposure group. RESULTS: Among 20,389 children in the dataset, 13,173 were unexposed; 455 were POE, and 199 were POE + NAS. There were significant differences in frequency of diagnoses between groups, specifically regarding growth and development, infection, mental health, musculoskeletal, neonatal, sensory, and social issues. When comparing exposed groups, children with POE + NAS experienced more negative health outcomes than children with only POE across all years. CONCLUSIONS: This study implicates POE as a significant variable associated with many health and developmental outcomes of children during the first three years of life. PRACTICE IMPLICATIONS: It is crucial to understand and identify health risks observed more frequently in exposed children during such a critical period of growth and brain development.

Monocyte and bone marrow macrophage transcriptional phenotypes in systemic juvenile idiopathic arthritis reveal TRIM8 as a mediator of IFN-γ hyper-responsiveness and risk for macrophage activation syndrome.

OBJECTIVES: Systemic juvenile idiopathic arthritis (SJIA) confers high risk for macrophage activation syndrome (MAS), a life-threatening cytokine storm driven by interferon (IFN)-γ. SJIA monocytes display IFN-γ hyper-responsiveness, but the molecular basis of this remains unclear. The objective of this study is to identify circulating monocyte and bone marrow macrophage (BMM) polarisation phenotypes in SJIA including molecular features contributing to IFN response. METHODS: Bulk RNA-seq was performed on peripheral blood monocytes (n=26 SJIA patients) and single cell (sc) RNA-seq was performed on BMM (n=1). Cultured macrophages were used to define consequences of tripartite motif containing 8 (TRIM8) knockdown on IFN-γ signalling. RESULTS: Bulk RNA-seq of SJIA monocytes revealed marked transcriptional changes in patients with elevated ferritin levels. We identified substantial overlap with multiple polarisation states but little evidence of IFN-induced signature. Interestingly, among the most highly upregulated genes was TRIM8, a positive regulator of IFN-γ signalling. In contrast to PBMC from SJIA patients without MAS, scRNA-seq of BMM from a patient with SJIA and MAS identified distinct subpopulations of BMM with altered transcriptomes, including upregulated IFN-γ response pathways. These BMM also showed significantly increased expression of TRIM8. In vitro knockdown of TRIM8 in macrophages significantly reduced IFN-γ responsiveness. CONCLUSIONS: Macrophages with an 'IFN-γ response' phenotype and TRIM8 overexpression were expanded in the bone marrow from an MAS patient. TRIM8 is also upregulated in SJIA monocytes, and augments macrophage IFN-γ response in vitro, providing both a candidate molecular mechanism and potential therapeutic target for monocyte hyper-responsiveness to IFNγ in cytokine storms including MAS.

Definition and validation of serum biomarkers for optimal differentiation of hyperferritinaemic cytokine storm conditions in children: a retrospective cohort study.

BACKGROUND: Cytokine storm syndromes are life-threatening complications that can occur in children with rheumatic conditions (macrophage activation syndrome [MAS]), inherited cytotoxicity defects (ie, primary haemophagocytic lymphohistiocytosis [HLH]), or as a result of infection or malignancies (ie, secondary HLH). To adequately steer treatment, an early and clear discrimination of these entities is essential. We aimed to define and validate serum biomarker profiles that can differentiate between primary HLH, secondary HLH (predominantly infection-associated), and MAS associated with systemic juvenile idiopathic arthritis (systemic JIA-MAS). METHODS: In this multicentre, retrospective, cohort study, serum samples from patients (0-18 years) with a clinical diagnosis of primary HLH, secondary HLH, or systemic JIA-MAS were analysed by immunoassays for 55 cytokines and chemokines. Serum samples were collected from patients treated at seven clinical centres in Europe and North America. 15 serum biomarkers were validated using an independent commercial assay, and the diagnostic accuracy of the best performing biomarkers was tested in an independent validation cohort. FINDINGS: Serum samples were collected between Dec 7, 2010, and Jan 26, 2018. In the discovery cohort of 43 patients (24 girls and 19 boys) multi-marker analyses revealed distinct serum biomarker profiles associated with primary or secondary HLH versus systemic JIA-MAS. Ten biomarkers were identified that were differentially elevated in either HLH or systemic JIA-MAS and distinguished between these clinical entities, six of which were tested in an independent validation cohort of 79 patients (34 girls and 45 boys). Serum concentrations of S100A12 and interleukin-18, as well as ratios of both S100A12 and IL-18 with chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 were identified as the most promising candidates for differential diagnostics. INTERPRETATION: At initial presentation, when it is unclear whether a patient with excessive hyperferritinaemic inflammation has primary HLH, infection-associated secondary HLH, or MAS, high serum concentrations of S100A12 indicate an initial differential diagnosis of systemic JIA-MAS, thus helping to guide subsequent treatment decisions. We therefore suggest the inclusion of serum S100A12 and IL-18 in the diagnostic investigations for hyperferritinaemic syndromes; however, the definition and introduction of universially applicable cutoff values are still required. FUNDING: German Research Foundation, the Center for Interdisciplinary Clinical Research at University Hospital Muenster, the EU's Horizon 2020 research and innovation programme, and the Deutsche Kinderkrebsstiftung.

IL-18 as a biomarker linking systemic juvenile idiopathic arthritis and macrophage activation syndrome.

OBJECTIVES: Systemic juvenile idiopathic arthritis (sJIA) is a childhood arthritis with features of autoinflammation and high risk of macrophage activation syndrome (MAS). IL-18 has been shown to have key roles in sJIA and MAS. We aimed to examine IL-18 levels in sJIA in relation to disease activity and history of MAS and other disease biomarkers namely S100 proteins and CXCL9. METHODS: Total IL-18, CXCL9 and S100 proteins levels were determined in 40 sJIA patients, and IL-18 levels were compared between patients with regards to disease activity, history of MAS, and other biomarkers. RESULTS: Total IL-18 levels were significantly higher in patients with active sJIA (median 16 499 pg/ml; interquartile range (IQR) 4816-61 839), and remained persistently elevated even in the majority of patients with inactive disease (1164 pg/ml; IQR 587-3444). Patients with history of MAS had significantly higher IL-18 levels (13 380 pg/ml; IQR 4212-62 628) as compared with those without MAS history (956.5 pg/ml; IQR 276.3-4262.5). Total IL-18 performed well with area under the curve of 0.8145 and 0.84 in predicting disease activity and history of MAS, respectively. We observed moderate correlation between IL-18 and CXCL9 (R = 0.56), S100A8/A9 (R = 0.47) and S100A12 (R = 0.46). The correlation was stronger for ferritin (R = 0.74) and overall for those with active disease. CONCLUSION: Total IL-18 levels were elevated in the majority of sJIA patients regardless of clinical features, but were higher in patients with active disease and history of MAS. Change in IL-18 may reflect increased disease activity or development of MAS.

Discovery of tear biomarkers in children with chronic non-infectious anterior uveitis: a pilot study.

BACKGROUND: Biomarkers in easily obtained specimens that accurately predict uveitis in children with juvenile idiopathic arthritis (JIA) are needed. Aqueous humor has been studied for biomarkers, but is not routinely available. We evaluated tears from children with chronic anterior uveitis (CAU) for biomarkers reported in aqueous humor. In this pilot study, we used Schirmer strips to collect tears from seven children (nine eyes); three children had JIA- associated uveitis (JIA-U) and four had idiopathic disease (I-CAU). Liquid chromatography-tandem mass spectrometry was used to identify and quantify tear proteins. The Mann-Whitney U test identified differential tear protein expression between children with JIA-U and those with I-CAU. RESULTS: S100A9, LAP3, TTR, MIF, sCD14, S100A8, and SAA1 were detected in tears of all children; the same cytokines have been reported in aqueous humor of children with JIA-U. Tears from children with JIA-U had higher expression of proteins associated with inflammatory arthritis (SEMA3G, TIMP1, HEXB, ERN1, and SAA1) than tears from those with I-CAU. In addition, we found higher expression of sCD14, S100A8, and SAA1, but lower expression of S100A9, LAP3, TTR, and MIF, in tears from children with JIA-U compared to tears from those with I-CAU. CONCLUSIONS: Tears contain similar cytokine profiles to aqueous humor in children with CAU and may be a clinically useful source of disease biomarkers. Tears from children with JIA-U also contain cytokines associated with inflammatory arthritis; furthermore, differential expression of other tear proteins as well may provide clues to intrinsic differences between JIA-U and I-CAU, despite their similar clinical phenotypes.

Brief Report: Novel UNC13D Intronic Variant Disrupting an NF-κB Enhancer in a Patient With Recurrent Macrophage Activation Syndrome and Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (JIA) and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH type 3 (FHLH3), but the role of noncoding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. METHODS: A custom enrichment library was constructed to sequence a genomic region of ~1 Mb flanking UNC13D in 24 patients with systemic JIA, recurrent MAS, and negative results of prior genetic (exon/coding) testing. The functional consequences of intronic variants were assessed using quantitative polymerase chain reaction in patient-derived peripheral blood mononuclear cells (PBMCs), electromobility shift assay, in vitro transcriptional enhancer assays, and natural killer (NK) cell degranulation assays. RESULTS: We evaluated a patient with systemic JIA and recurrent MAS in whom a novel functional intronic variant in UNC13D, c.117+143A>G, was observed. This variant occurred in a proposed regulatory region that drives lymphocyte-specific UNC13D expression and is associated with reduced transcript levels in patient PBMCs. This variant also disrupted NF-κB binding to a functional transcriptional enhancer, leading to reduced enhancer activity in vitro. Partial knockdown of UNC13D expression also led to impaired NK cell degranulation. An additional patient was identified with a previously described UNC13D intronic variant, for a total noncoding variant hit rate of 8.3% (2 of 24). CONCLUSION: These findings highlight the notion that intronic variants in key regulatory regions may be associated with MAS in patients with systemic JIA and support deep sequencing approaches when causative coding variants are not identified.

Urine S100 proteins as potential biomarkers of lupus nephritis activity.

BACKGROUND: Improved, noninvasive biomarkers are needed to accurately detect lupus nephritis (LN) activity. The purpose of this study was to evaluate five S100 proteins (S100A4, S100A6, S100A8/9, and S100A12) in both serum and urine as potential biomarkers of global and renal system-specific disease activity in childhood-onset systemic lupus erythematosus (cSLE). METHODS: In this multicenter study, S100 proteins were measured in the serum and urine of four cSLE cohorts and healthy control subjects using commercial enzyme-linked immunosorbent assays. Patients were divided into cohorts on the basis of biospecimen availability: (1) longitudinal serum, (2) longitudinal urine, (3) cross-sectional serum, and (4) cross-sectional urine. Global and renal disease activity were defined using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and the SLEDAI-2K renal domain score. Nonparametric testing was used for statistical analysis, including the Wilcoxon signed-rank test, Kruskal-Wallis test, Mann-Whitney U test, and Spearman's rank correlation coefficient. RESULTS: All urine S100 proteins were elevated in patients with active LN compared with patients with active extrarenal disease and healthy control subjects. All urine S100 protein levels decreased with LN improvement, with S100A4 demonstrating the most significant decrease. Urine S100A4 levels were also higher with proliferative LN than with membranous LN. S100A4 staining in the kidney localized to mononuclear cells, podocytes, and distal tubular epithelial cells. Regardless of the S100 protein tested, serum levels did not change with cSLE improvement. CONCLUSIONS: Higher urine S100 levels are associated with increased LN activity in cSLE, whereas serum S100 levels do not correlate with disease activity. Urine S100A4 shows the most promise as an LN activity biomarker, given its pronounced decrease with LN improvement, isolated elevation in urine, and positive staining in resident renal cells.

Proinflammatory Cytokine Environments Can Drive Interleukin-17 Overexpression by γ/δ T Cells in Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Systemic-onset juvenile idiopathic arthritis (JIA) is speculated to follow a biphasic course, with an initial systemic disease phase driven by innate immune mechanisms and interleukin-1ß (IL-1ß) as a key cytokine and a second chronic arthritic phase that may be dominated by adaptive immunity and cytokines such as IL-17A. Although a recent mouse model points to a critical role of IL-17-expressing γ/δ T cells in disease pathology, in humans, both the prevalence of IL-17 and the role of IL-17-producing cells are still unclear. METHODS: Serum samples from systemic JIA patients and healthy pediatric controls were analyzed for the levels of IL-17A and related cytokines. Whole blood samples were studied for cellular expression of IL-17 and interferon-γ (IFNγ). CD4+ and γ/δ T cells isolated from the patients and controls were assayed for cytokine secretion in different culture systems. RESULTS: IL-17A was prevalent in sera from patients with active systemic JIA, while both ex vivo and in vitro experiments revealed that γ/δ T cells overexpressed this cytokine. This was not seen with CD4+ T cells, which expressed strikingly low levels of IFNγ. Therapeutic IL-1 blockade was associated with partial normalization of both cytokine expression phenotypes. Furthermore, culturing healthy donor γ/δ T cells in serum from systemic JIA patients or in medium spiked with IL-1ß, IL-18, and S100A12 induced IL-17 overexpression at levels similar to those observed in the patients' cells. CONCLUSION: A systemic JIA cytokine environment may prime γ/δ T cells in particular for IL-17A overexpression. Thus, our observations in systemic JIA patients strongly support a pathophysiologic role of these cells, as proposed by the recent murine model.

Monocyte MicroRNA Expression in Active Systemic Juvenile Idiopathic Arthritis Implicates MicroRNA-125a-5p in Polarized Monocyte Phenotypes.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is an inflammatory disease of childhood in which cells of the monomyelocytoid lineage are thought to be key effector cells. Monocytes from patients with systemic JIA have a distinct phenotype, with features of both M1 and M2 alternative activation. MicroRNAs are critical regulators of monocyte polarization and function, but cellular microRNAs in systemic JIA have not been examined systematically. METHODS: MicroRNA TaqMan arrays were used to determine the expression profiles of monocytes from children with systemic JIA. Expression of microRNA-125a-5p (miR-125a-5p) and its contribution to monocyte polarization were examined using in vitro-polarized THP-1 cells and primary human monocytes. RESULTS: A total of 110 microRNAs were found to be differentially expressed in monocytes from patients with active systemic JIA, including molecules implicated in rheumatoid arthritis pathogenesis, cytokine production, and monocyte polarization. MicroRNA-125a-5p was identified as being highly up-regulated in monocytes from children with active systemic JIA, as compared to those from children with clinically inactive JIA or those with active polyarticular JIA, and correlated with systemic features of the disease. In vitro, monocyte miR125a-5p expression was increased after polarization under M2b or M2c conditions. Inhibition of miR-125a-5p showed that this microRNA contributed to full polarization of M2b regulatory macrophages. In contrast, miR-125a-5p overexpression enhanced M2b polarization and altered other polarized populations, including increasing the production of M2 markers. Indeed, in vitro overexpression of this microRNA altered the macrophage phenotype toward that observed in systemic JIA. CONCLUSION: Children with active systemic JIA have profound alterations in the expression of microRNAs that are implicated in monocyte function and polarization. One of these microRNAs, miR-125a-5p, is also a regulator of immunoregulatory M2b macrophages.

Relationship of cell-free urine MicroRNA with lupus nephritis in children.

BACKGROUND: MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of genes. The objective of this study was to investigate whether select urinary cell-free microRNA's may serve as biomarkers in children with active lupus nephritis (LN) and to assess their relationship to the recently identified combinatorial urine biomarkers, a.k.a. the LN-Panel (neutrophil gelatinase associated lipocalin, monocyte chemotactic protein 1, transferrin, and beta-trace protein). METHODS: miRNAs (125a, 127, 146a, 150 and 155) were measured using real-time polymerase chain reaction in the urine pellet (PEL) and supernatant (SUP) in 14 patients with active LN, 10 patients with active extra-renal lupus, and 10 controls. The concentrations of the LN-Panel biomarkers (neutrophil gelatinase associated lipocalin, monocyte chemotactic protein-1, transferrin, beta-trace protein) was assayed. Traditional laboratory and clinical measures of LN and lupus (complements, protein to creatinine ratio; Systemic Lupus Erythematosus Disease Activity Index) were also measured. RESULTS: All tested miRNAs in the SUP, but not the PEL, were associated with the LN-Panel biomarkers (0.3 < |r Pearson| < 0.73; p < 0.05), miRNA125a, miRNA127,miRNA146a also with C3 and dsDNA antibody levels (|r Pearson| > 0.24; p < 0.05), and miRNA146a with the renal domain of the SLEDAI (|r Pearson| = 0.32; p < 0.05). Mean miRNA levels of patients with active LN did not statistically (P > 0.05) differ from those of SLE patients without LN or controls. CONCLUSION: Levels of cell-free miR-125a, miR-150, and miR-155 in the urine supernatant are associated with the expression of LN-Panel biomarkers and some LN measures. These miRNA's may complement, but are unlikely superior to the LN-Panel for estimating concurrent LN activity.

Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.

BACKGROUND: Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. METHODS: Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. RESULTS: Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. CONCLUSIONS: This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection.

Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis.

OBJECTIVE: Macrophage activation syndrome (MAS), a life-threatening complication of systemic juvenile idiopathic arthritis (JIA), resembles familial hemophagocytic lymphohistiocytosis (HLH), a constellation of autosomal-recessive immune disorders resulting from deficiency in cytolytic pathway proteins. We undertook this study to test our hypothesis that MAS predisposition in systemic JIA could be attributed to rare gene sequence variants affecting the cytotolytic pathway. METHODS: Whole-exome sequencing was used in 14 patients with systemic JIA and MAS and in their parents to identify protein-altering single-nucleotide polymorphisms/indels in known HLH-associated genes. To discover new candidate genes, the entire whole-exome sequencing data were filtered to identify protein-altering, rare recessive homozygous, compound heterozygous, and de novo variants with the potential to affect the cytolytic pathway. RESULTS: Heterozygous protein-altering rare variants in the known genes (LYST,MUNC13-4, and STXBP2) were found in 5 of 14 patients with systemic JIA and MAS (35.7%). This was in contrast to only 4 variants in 4 of 29 patients with systemic JIA without MAS (13.8%). Homozygosity and compound heterozygosity analysis applied to the entire whole-exome sequencing data in systemic JIA/MAS revealed 3 recessive pairs in 3 genes and compound heterozygotes in 73 genes. We also identified 20 heterozygous rare protein-altering variants that occurred in at least 2 patients. Many of the identified genes encoded proteins with a role in actin and microtubule reorganization and vesicle-mediated transport. "Cellular assembly and organization" was the top cellular function category based on Ingenuity Pathways Analysis (P < 3.10 × 10(-5) ). CONCLUSION: Whole-exome sequencing performed in patients with systemic JIA and MAS identified rare protein-altering variants in known HLH-associated genes as well as in new candidate genes.

Follistatin-like protein 1 and the ferritin/erythrocyte sedimentation rate ratio are potential biomarkers for dysregulated gene expression and macrophage activation syndrome in systemic juvenile idiopathic arthritis.

OBJECTIVE: Follistatin-like protein 1 (FSTL-1) is a secreted glycoprotein overexpressed in certain inflammatory diseases. Our objective was to correlate FSTL-1 levels with gene expression, known biomarkers, and measures of disease activity in systemic juvenile idiopathic arthritis (sJIA), including macrophage activation syndrome (MAS). METHODS: FSTL-1 serum levels were measured by ELISA in 28 patients with sJIA, including 7 patients who developed MAS, and 30 healthy controls. Levels were correlated with erythrocyte sedimentation rate (ESR), ferritin, and soluble interleukin-2 receptor-α (sIL-2Rα). Gene expression based on FSTL-1 levels was analyzed in peripheral blood mononuclear cells (PBMC). RESULTS: Serum levels of FSTL-1 were elevated at time of presentation of sJIA (mean 200.7 ng/ml) and decreased to normal (mean 133.7 ng/ml) over 24 months (p < 0.01). FSTL-1 levels were markedly elevated during acute MAS (mean 279.8 ng/ml) and decreased to normal following treatment (p < 0.001). FSTL-1 levels correlated with serum markers of inflammation, including sIL-2Rα and ferritin. Ferritin/ESR ratio was superior to ferritin, sIL-2Rα, and FSTL-1 in discriminating MAS from new-onset sJIA. PBMC from patients with FSTL-1 levels > 200 ng/ml showed altered expression of genes related to innate immunity, erythropoiesis, and natural killer cell dysfunction. Two patients with the highest FSTL-1 levels at disease onset (> 300 ng/ml) ultimately developed MAS. CONCLUSION: Elevated pretreatment serum FSTL-1 levels in sJIA are associated with dysregulated gene expression suggestive of occult MAS, and may have utility in predicting progression to overt MAS. Ferritin/ESR ratio may be superior to ferritin alone in discriminating overt MAS from new-onset sJIA.

The limited role of interferon-γ in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is an autoinflammatory syndrome in which the myelomonocytic lineage appears to play a pivotal role. Inflammatory macrophages are driven by interferon-γ (IFNγ), but studies have failed to demonstrate an IFN- induced gene signature in active systemic JIA. This study sought to characterize the status of an IFN-induced signature within affected tissue and to gauge the integrity of IFN signaling pathways within peripheral monocytes from patients with systemic JIA. METHODS: Synovial tissue from 12 patients with active systemic JIA and 9 with active extended oligoarticular JIA was assessed by real-time polymerase chain reaction to quantify IFN-induced chemokine gene expression. Peripheral monocytes from 3 patients with inactive systemic JIA receiving anti-interleukin-1ß (anti-IL-1ß) therapy, 5 patients with active systemic JIA, and 8 healthy controls were incubated with or without IFNγ to gauge changes in gene expression and to measure phosphorylated STAT-1 (pSTAT-1) levels. RESULTS: IFN-induced chemokine gene expression in synovium was constrained in active systemic JIA compared to the known IFN-mediated extended oligoarticular subtype. In unstimulated peripheral monocytes, IFN-induced gene expression was similar between the groups, except that lower levels of STAT1, MIG, and PIAS were observed in patients with active disease, while higher levels of PIAS1 were observed in patients with inactive disease. Basal pSTAT-1 levels in monocytes tended to be higher in systemic JIA patients compared to healthy controls, with the highest levels seen in those with inactive disease. Upon stimulation of monocytes, the fold increase in gene expression was roughly equal between groups, except for a greater increase in STAT1 in patients with inactive systemic JIA compared to controls, and a greater increase in IRF1 in those with active compared to inactive disease. Upon stimulation, the fold increase in pSTAT-1 was highest in monocytes from patients with inactive systemic JIA. CONCLUSION: Monocytes in patients with active systemic JIA retain the ability to respond to IFNγ, suggesting that the lack of an IFN-induced gene signature in patients with active disease reflects a limited in vivo exposure to IFNγ. In patients with inactive systemic JIA who received treatment with anti-IL-1ß, hyperresponsiveness to IFNγ was observed.

Short-term stability study of RNA at room temperature.

The quality of RNA preserved in different stabilization matrices was investigated after 2 weeks of storage at room temperature. RNA samples in RNAstable (Biomatrica), GenTegra (IntegenX), and RNAshell (Imagene) were compared to RNA stored at -80°C (the current gold standard for RNA preservation) and with liquid or dried RNA stored at room temperature without additives in this multi-center study. One center prepared all of the RNA samples, and five participating laboratories applied the samples to the matrices and stored them for 2 weeks at room temperature. Samples were shipped to three testing laboratories, where the 336 RNA samples were rehydrated and then analyzed for RNA recovery, purity, and integrity. Parallel RNA quality analyses and real-time PCR analyses were performed at each of the three testing laboratories. Each of the RNA matrices tested was shown to be fit-for-purpose for short-term room temperature storage in terms of total RNA recovery and rRNA integrity. All but one of the matrices was judged to be fit-for-purpose for mRNA integrity when assessed by real-time PCR analysis. In a follow-up study, RNase-contaminated samples were shown to provide accurate real-time PCR results when stored for up to 3.5 months in either RNAshell or RNAstable.