Genome-wide association study identifies multiple HLA loci for sarcoidosis susceptibility.

Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.

Standardization of flow cytometry and cell sorting to enable a transcriptomic analysis in a multi-site sarcoidosis study.

The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.

Cognitive Difficulties and Health-Related Quality of Life in Sarcoidosis: An Analysis of the GRADS Cohort.

RATIONALE: Subjective cognitive difficulties are common among sarcoidosis patients; however, previous studies have not modeled the link between cognitive difficulties and health-related quality of life (HRQOL). OBJECTIVES: To determine whether cognitive difficulties are associated with HRQOL in sarcoidosis patients after adjusting for demographics, fatigue, and physical disease severity measures. METHODS: We performed a secondary analysis of the Genomic Research in Alpha-1 antitrypsin Deficiency and Sarcoidosis (GRADS) study data. We examined the association between self-reported cognitive difficulties (Cognitive Failures Questionnaire (CFQ)) and HRQOL (SF12v2 mental and physical component scores) while adjusting for the demographics, fatigue, and physical disease severity measures (i.e., organ involvement, forced vital capacity). RESULTS: Approximately one-fourth of the patients with sarcoidosis endorsed cognitive difficulties. More frequent cognitive difficulties and more severe fatigue were significantly associated with worse mental HRQOL in the fully adjusted model, while older age was associated with better mental HRQOL. The association between cognitive difficulties and physical HRQOL was not significant in the final model. More severe fatigue, joint involvement, and reduced forced vital capacity (FVC) were associated with worse physical HRQOL, while higher income and higher education were associated with better physical HRQOL. CONCLUSIONS: Perceived cognitive difficulties are associated with diminished HRQOL after adjusting for demographics, organ involvement, pulmonary function, and fatigue. The association between cognitive difficulties and reduced HRQOL primarily occurs through the impact on mental components of HRQOL.

Quantitative Chest CT Assessment of Small Airways Disease in Post-Acute SARS-CoV-2 Infection.

Background The long-term effects of SARS-CoV-2 infection on pulmonary structure and function remain incompletely characterized. Purpose To test whether SARS-CoV-2 infection leads to small airways disease in patients with persistent symptoms. Materials and Methods In this single-center study at a university teaching hospital, adults with confirmed COVID-19 who remained symptomatic more than 30 days following diagnosis were prospectively enrolled from June to December 2020 and compared with healthy participants (controls) prospectively enrolled from March to August 2018. Participants with post-acute sequelae of COVID-19 (PASC) were classified as ambulatory, hospitalized, or having required the intensive care unit (ICU) based on the highest level of care received during acute infection. Symptoms, pulmonary function tests, and chest CT images were collected. Quantitative CT analysis was performed using supervised machine learning to measure regional ground-glass opacity (GGO) and using inspiratory and expiratory image-matching to measure regional air trapping. Univariable analyses and multivariable linear regression were used to compare groups. Results Overall, 100 participants with PASC (median age, 48 years; 66 women) were evaluated and compared with 106 matched healthy controls; 67% (67 of 100) of the participants with PASC were classified as ambulatory, 17% (17 of 100) were hospitalized, and 16% (16 of 100) required the ICU. In the hospitalized and ICU groups, the mean percentage of total lung classified as GGO was 13.2% and 28.7%, respectively, and was higher than that in the ambulatory group (3.7%, P < .001 for both comparisons). The mean percentage of total lung affected by air trapping was 25.4%, 34.6%, and 27.3% in the ambulatory, hospitalized, and ICU groups, respectively, and 7.2% in healthy controls (P < .001). Air trapping correlated with the residual volume-to-total lung capacity ratio (ρ = 0.6, P < .001). Conclusion In survivors of COVID-19, small airways disease occurred independently of initial infection severity. The long-term consequences are unknown. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Elicker in this issue.

Defining CD4 T helper and T regulatory cell endotypes of progressive and remitting pulmonary sarcoidosis (BRITE): protocol for a US-based, multicentre, longitudinal observational bronchoscopy study.

INTRODUCTION: Sarcoidosis is a multiorgan granulomatous disorder thought to be triggered and influenced by gene-environment interactions. Sarcoidosis affects 45-300/100 000 individuals in the USA and has an increasing mortality rate. The greatest gap in knowledge about sarcoidosis pathobiology is a lack of understanding about the underlying immunological mechanisms driving progressive pulmonary disease. The objective of this study is to define the lung-specific and blood-specific longitudinal changes in the adaptive immune response and their relationship to progressive and non-progressive pulmonary outcomes in patients with recently diagnosed sarcoidosis. METHODS AND ANALYSIS: The BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints study is a US-based, NIH-sponsored longitudinal blood and bronchoscopy study. Enrolment will occur over four centres with a target sample size of 80 eligible participants within 18 months of tissue diagnosis. Participants will undergo six study visits over 18 months. In addition to serial measurement of lung function, symptom surveys and chest X-rays, participants will undergo collection of blood and two bronchoscopies with bronchoalveolar lavage separated by 6 months. Freshly processed samples will be stained and flow-sorted for isolation of CD4 +T helper (Th1, Th17.0 and Th17.1) and T regulatory cell immune populations, followed by next-generation RNA sequencing. We will construct bioinformatic tools using this gene expression to define sarcoidosis endotypes that associate with progressive and non-progressive pulmonary disease outcomes and validate the tools using an independent cohort. ETHICS AND DISSEMINATION: The study protocol has been approved by the Institutional Review Boards at National Jewish Hospital (IRB# HS-3118), University of Iowa (IRB# 201801750), Johns Hopkins University (IRB# 00149513) and University of California, San Francisco (IRB# 17-23432). All participants will be required to provide written informed consent. Findings will be disseminated via journal publications, scientific conferences, patient advocacy group online content and social media platforms.

Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia.

OBJECTIVE: Cardiac sarcoidosis is difficult to diagnose, often requiring expensive and inconvenient advanced imaging techniques. Circulating exosomes contain genetic material, such as microRNA (miRNA), that are derived from diseased tissues and may serve as potential disease-specific biomarkers. We thus sought to determine whether circulating exosome-derived miRNA expression patterns would distinguish cardiac sarcoidosis (CS) from acute myocardial infarction (AMI). METHODS: Plasma and serum samples conforming to CS, AMI or disease-free controls were procured from the Biologic Specimen and Data Repository Information Coordinating Center repository and National Jewish Health. Next generation sequencing (NGS) was performed on exosome-derived total RNA (n = 10 for each group), and miRNA expression levels were compared after normalization using housekeeping miRNA. Quality assurance measures excluded poor quality RNA samples. Differentially expressed (DE) miRNA patterns, based upon >2-fold change (p < 0.01), were established in CS compared to controls, and in CS compared to AMI. Relative expression of several DE-miRNA were validated by qRT-PCR. RESULTS: Despite the advanced age of the stored samples (~5-30 years), the quality of the exosome-derived miRNA was intact in ~88% of samples. Comparing plasma exosomal miRNA in CS versus controls, NGS yielded 18 DE transcripts (12 up-regulated, 6 down-regulated), including miRNA previously implicated in mechanisms of myocardial injury (miR-92, miR-21) and immune responses (miR-618, miR-27a). NGS further yielded 52 DE miRNA in serum exosomes from CS versus AMI: 5 up-regulated in CS; 47 up-regulated in AMI, including transcripts previously detected in AMI patients (miR-1-1, miR-133a, miR-208b, miR-423, miR-499). Five miRNAs with increased DE in CS included two isoforms of miR-624 and miR-144, previously reported as markers of cardiomyopathy. CONCLUSIONS: MiRNA patterns of exosomes derived from CS and AMI patients are distinct, suggesting that circulating exosomal miRNA patterns could serve as disease biomarkers. Further studies are required to establish their specificity relative to other cardiac disorders.

Delphi consensus recommendations for a treatment algorithm in pulmonary sarcoidosis.

Pulmonary sarcoidosis presents substantial management challenges, with limited evidence on effective therapies and phenotypes. In the absence of definitive evidence, expert consensus can supply clinically useful guidance in medicine. An international panel of 26 experts participated in a Delphi process to identify consensus on pharmacological management in sarcoidosis with the development of preliminary recommendations.The modified Delphi process used three rounds. The first round focused on qualitative data collection with open-ended questions to ensure comprehensive inclusion of expert concepts. Rounds 2 and 3 applied quantitative assessments using an 11-point Likert scale to identify consensus.Key consensus points included glucocorticoids as initial therapy for most patients, with non-biologics (immunomodulators), usually methotrexate, considered in severe or extrapulmonary disease requiring prolonged treatment, or as a steroid-sparing intervention in cases with high risk of steroid toxicity. Biologic therapies might be considered as additive therapy if non-biologics are insufficiently effective or are not tolerated with initial biologic therapy, usually with a tumour necrosis factor-α inhibitor, typically infliximab.The Delphi methodology provided a platform to gain potentially valuable insight and interim guidance while awaiting evidenced-based contributions.

Genetic, Immunologic, and Environmental Basis of Sarcoidosis.

Sarcoidosis is a multisystem disease with tremendous heterogeneity in disease manifestations, severity, and clinical course that varies among different ethnic and racial groups. To better understand this disease and to improve the outcomes of patients, a National Heart, Lung, and Blood Institute workshop was convened to assess the current state of knowledge, gaps, and research needs across the clinical, genetic, environmental, and immunologic arenas. We also explored to what extent the interplay of the genetic, environmental, and immunologic factors could explain the different phenotypes and outcomes of patients with sarcoidosis, including the chronic phenotypes that have the greatest healthcare burden. The potential use of current genetic, epigenetic, and immunologic tools along with study approaches that integrate environmental exposures and precise clinical phenotyping were also explored. Finally, we made expert panel-based consensus recommendations for research approaches and priorities to improve our understanding of the effect of these factors on the health outcomes in sarcoidosis.

Computed Tomographic Scoring Systems in Sarcoidosis: Comparison With Cardiopulmonary Exercise Testing Parameters.

PURPOSE: The aim of the study was to correlate computed tomographic (CT) scoring systems for pulmonary sarcoidosis with cardiopulmonary exercise testing and evaluate which scoring system provides the most reliable information for assessing disease severity and predicting impairment of gas exchange during exercise. MATERIALS AND METHODS: The institutional review board approved this retrospective study. All 62 patients underwent thin-section CT, pulmonary function tests, and cardiopulmonary exercise test. Two observers scored CT images according to scoring systems published by Remy-Jardin and colleagues. Spearman rank correlation coefficients were calculated between CT patterns and pulmonary functional impairment parameters, and multiple regression analyses were performed to evaluate which CT abnormalities were significantly associated with pulmonary functional impairment parameters. RESULTS: Regardless of scoring system, PaO2max was significantly associated with the subscores of ground-glass opacity, linear opacity, and total CT scores. Multiple regression analyses showed that subscores of ground-glass and linear opacity in the Leung scoring system appeared to explain a significant amount of variance in functional parameters at rest and at maximal exercise. CONCLUSIONS: CT findings, particularly ground-glass opacity and linear abnormalities, can explain a significant amount of variance in cardiopulmonary exercise parameters. This suggests that CT-based scoring systems are valid measures of disease severity in sarcoidosis.

Safety and efficacy of ustekinumab or golimumab in patients with chronic sarcoidosis.

Sarcoidosis is characterised by non-caseating granulomas that secrete pro-inflammatory cytokines, including interleukin (IL)-12, IL-23, and tumour necrosis factor (TNF)-α. Ustekinumab and golimumab are monoclonal antibodies that specifically inhibit IL-12/IL-23 and TNF-α, respectively. Patients with chronic pulmonary sarcoidosis (lung group) and/or skin sarcoidosis (skin group) received either 180 mg ustekinumab at week 0 followed by 90 mg every 8 weeks, 200 mg golimumab at week 0 followed by 100 mg every 4 weeks, or placebo. Patients underwent corticosteroid tapering between weeks 16 and 28. The primary end-point was week 16 change in percentage predicted forced vital capacity (ΔFVC % pred) in the lung group. Major secondary end-points were: week 28 for ΔFVC % pred, 6-min walking distance, St George's Respiratory Questionnaire (lung group), and Skin Physician Global Assessment response (skin group). At week 16, no significant differences were observed in ΔFVC % pred with ustekinumab (-0.15, p = 0.13) or golimumab (1.15, p = 0.54) compared with placebo (2.02). At week 28, there were no significant improvements in the major secondary end-points, although a nonsignificant numerically greater Skin Physician Global Assessment response was observed following golimumab treatment (53%) when compared with the placebo (30%). Serious adverse events were similar in all treatment groups. Although treatment was well tolerated, neither ustekinumab nor golimumab demonstrated efficacy in pulmonary sarcoidosis. However, trends towards improvement were observed with golimumab in some dermatological end-points.

Predictors of cardiac sarcoidosis using commonly available cardiac studies.

Cardiac involvement affects ≤40% of patients with sarcoidosis and accounts for ≤25% of deaths. The diagnosis of cardiac sarcoidosis is challenging using the existing screening tests and often relies on expensive cardiac magnetic resonance imaging (cMRI) and cardiac 18-fluorodeoxuyglucose positron emission tomography (FDG-cPET). We developed a scoring system using common clinical tests to predict positive imaging findings using cMRI or FDG-cPET. A retrospective chart review of subjects undergoing cMRI or FDG-cPET was performed. The data were extracted and scored using a predetermined system. Our cohort was predominantly white, with a mean age of 55 years, and 60% were women. The scoring system was compared with the findings from cMRI and FDG-cPET to determine the ability to predict the imaging results that define cardiac sarcoidosis. The scoring system for the patients who had undergone both FDG-cPET and cMRI suggested predictability, but the differences were not statistically significant. However, the positive results from just 1 study were as predictive as having positive findings from both studies. A 1-point increase in the total score increased the probability of positive findings from cMRI or FDG-cPET by 14% (95% confidence interval 3% to 25% increase; p = 0.01). The scoring system seemed to be driven more by the findings from cMRI than by those from FDG-cPET. In patients who had undergone cMRI alone, for each 1-point increase in the total score, the probability of positive cMRI findings increased 11% (95% confidence interval 1% decrease to 25% increase, p = 0.08). All screening modalities were analyzed. No modality was sensitive or specific, although major findings (defined in our scoring system) were most predictive of positive imaging findings. In conclusion, commonly available cardiac screening tools used together in a composite score provide reasonable results to predict positive cardiac sarcoidosis findings on imaging, but the system needs refinement. Our data suggest that major findings from screening studies are more likely to correlate with cMRI findings than with findings from FDG-cPET.

Management of cardiac sarcoidosis in the United States: a Delphi study.

BACKGROUND: No formal guidelines exist to guide physicians caring for patients with sarcoidosis in their screening for management of patients with cardiac sarcoidosis. We conducted a modified Delphi study to investigate if a consensus could be reached on the best approaches for screening for and management of cardiac sarcoidosis. METHODS: A modified Delphi study design with two rounds of questionnaires was used to investigate if a consensus existed among sarcoid experts in the United States on the best management approaches for cardiac sarcoidosis. Experts were identified based on their national reputation as sarcoid experts and by being actively involved in sarcoidosis clinics at their institutions. RESULTS: Overall agreement was low to moderate. Agreement was reached on the role of history, physical examination, and 12-lead ECG in screening, echocardiogram, Holter monitor, myocardial fluorodeoxyglucose PET scan, and cardiac MRI in workup, and steroids in treatment. Agreement was not reached on the role of signal-averaged ECG in screening, optimum dose of prednisone, use of steroid-sparing agents, and duration of treatment. Several comments underscore the diverse approaches and uncertainty that exist in managing cardiac sarcoidosis. CONCLUSIONS: Our study highlights the dilemma that sarcoid experts face in their approach to cardiac sarcoidosis. It also highlights the lack of agreement among sarcoid experts on key aspects of diagnosis and management and stresses the importance of collaborative efforts to investigate the best strategies for screening for and management of cardiac sarcoidosis.

Role of repeated lung injury and genetic background in bleomycin-induced fibrosis.

Current hypotheses of the pathogenesis of many forms of pulmonary fibrosis suggest that (i) a stimulus results in repeated or prolonged episodes of lung injury, and (ii) genetic factors modulate the outcome of the injury. The commonly employed single-exposure bleomycin model results in only temporary fibrosis. Therefore, we evaluated whether repeated bleomycin exposures, in the setting of a genetic background more likely to develop a T helper 2 (Th2) response, would induce prolonged fibrosis. Lung fibrosis was induced by intratracheal bleomycin injection, either as a single exposure or as three consecutive exposures. We found that bleomycin induced a Th2-like environment in both Th1-biased C57BL/6J and Th2-biased DBA/2 mice. We also found histologic changes and collagen increases consistent with lung injury/fibrosis at early time points, but prolonged fibrosis only after multiple exposures in the Th2-biased DBA/2 mice. We also determined if impaired healing of bleomycin-induced injury would prolong fibrosis in the C57BL/6J mice. Endothelial nitric oxide (which protects endothelial cells from oxidant-induced injury) synthase knockout animals on a C57BL/6J background also had prolonged fibrosis, similar to DBA/2 mice, after multiple bleomycin exposures. This was specific to eNOS, as inducible nitric oxide synthase knockout animals cleared the fibrosis as effectively as wild-type C57BL/6J mice. This data indicate that healing of injury/fibrosis after bleomycin is complex and can be determined by a number of genetic and environmental factors.