SINGLE CELL MULTIOMIC PROFILING OF DISRUPTED REGULATORY NETWORKS IN COVID19 ASSOCIATED MULTISYSTEM INFLAMMATORY SYNDROME IN CHILDREN

BackgroundChildren infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) usually present minimal symptoms or are asymptomatic. Nevertheless, a subset of children 2-6 weeks after the initial SARS-CoV-2 infection develops a postinfectious SARS-CoV-2-related multisystem inflammatory syndrome in (MIS-C). Recently, transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation, however, the underlying pathophysiology of MIS-C is not fully understood [1].ObjectivesThe purpose of our project is to characterize the complexity of cell populations and capture cellular heterogeneity to uncover the regulatory networks and interactions that are disrupted during MIS-C flare with simultaneous profiling of gene expression and open chromatin regions from the same nuclei.MethodsSamples of peripheral blood mononuclear cells from patients with MIS-C diagnosed at the University Children's Hospital, University Medical Center Ljubljana, were collected during the initial presentation before any treatment and at 6-12 months in remission. The primary aim is to identify which regulatory networks are driving inflammation in MIS-C flare, for which we are performing single cell Multiome ATAC + Gene Expression Sequencing. To enable simultaneous profiling of epigenomic landscape and gene expression from the same nuclei, we are using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit from 10X Genomics.ResultsWe included 32 patients with MIS-C from whom we collected paired blood samples during the initial presentation before treatment and at 6-12 months in remission. In single cell multiomic experiment we included 10 patients with paired samples, with the most viable cell count prior cryopreservation. All samples that are included into multiomic single cell analysis have 75% - 99% viability prior cryopreservation. In the protocol the key is to remove remaining granulocytes causing high mitochondrial RNA burden and extensively optimize the dilution factor of lysis buffer and the length of cell lysis step in order to get intact nuclei with no significant blebbing. Afterward, the single cell ATAC libraries as well as single-cell gene expression libraries are constructed and sequenced. Data are undergoing pairwise analysis to compare the cell population heterogeneity, expression profile and open chromatin landscape in the time of the initial presentation of MIS-C and in the remission, with Cell ranger software as well as with R package scREG [2], and custom scripting. In the second step we will inspect if the resulting altered transcriptomic signature from single-cell experiment is present on larger cohort. In that regard, we will perform bulk transcriptomic profiling on all paired collected samples during the initial presentation of MIS-C before treatment and at 6-12 months in remission.ConclusionThe results of this project are expected to enlighten the underlying pathophysiology of MIS-C flare and thus support clinical decision on more targeted treatment. The identified disrupted networks during MIS-C flare could lead the way to establish an early diagnosis and improve long-term outcome, including prevention of myocardial and neuropsychological impairment. Moreover, a better understanding of the disrupted regulatory networks that are driving inflammation in MIS-C, could lead to new insights into diseases with similar clinical presentations as is Kawasaki Disease.References[1]Sacco, K., Castagnoli, R., Vakkilainen, S. et al. Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19. Nat Med 28, 1050–1062 (2022).[2]Duren, Z., Chang, F., Naqing, F. et al. Regulatory analysis of single cell multiome gene expression and chromatin accessibility data with scREG. Genome Biol 23, 114 (2022).AcknowledgementsThis research was supported by Slovenian research agency grant J3-3061 and Interreg ITA-SLO project Cattedra.Disclosure of InterestsNone Declared.

Whole exome sequencing of MIS-C patients: Analysis of genes involved in inborn errors of type I IFN immunity, hemophagocytic lymphohistiocytosis (HLH), Kawasaki disease (KD) and TLR7 gene

Introduction: After spring 2020, a series of reports from Europe and USA described clusters of children, presenting life-threatening multisystem inflammatory syndrome in children (MIS-C), associated with antecedent exposure to SARS-CoV-2 (1). In patients with life threatening COVID-19 3.5% were found to have inborn errors in type I IFN signalling pathway (2). A case series of 4 young patients with severe COVID-19 reported rare loss-of-function variants in the TLR7 gene associated with impaired type I IFN responses (3). Clinically, MIS-C shares features with secondary hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), which were also associated with possible infectious trigger and might share a common genetic cause (4). Objectives: We analysed whether MIS-C patients have an underlying presence of genetic variants in exomes associated with inborn errors of type I IFN immunity, HLH, KD and presence of variants in TLR7 gene. Methods: Blood was drawn from 17 MIS-C patients upon submission into the hospital, DNA from peripheral blood was isolated and whole exome sequencing was performed. Variants in the following genes were investigated: type I IFN immunity (TLR3, UNC93B1, TRAF3, TBK1, IRF3/9, IRF7, IFNAR1/2, STAT1/2, IKBKG, TRIF), HLH (AP3B1, CD27, FADD, FAS, FASLG, HPLH1, ITK, LYST, MAGT1, MYO5A, NLRC4, PRF1, RAB27A, RECQL4, SH2D1A, STX11, STXBP2, UNC13D, XIAP, TNFRSF9, CDC42), KD (ITPKC, CD40, FCGR2A, BLK, CASP3, TRX-CAT1-7, PGBD1, LTA, TSBP1, HLA-DQB1/2, HLA-DOB, IGHV1-69) and TLR7 genes. Analysis was focused on rare (GnomAD<0.01) exonic or splicing variants. Results: No common genetic denominators were found in analysed genes. Five rare variants were observed in four patients (4/17). According to ACMG classification variants of uncertain significance (VUS) were found in LYST (2), IKBKG (1), IRF3 (1) and NLRC4 (1) in heterozygous genotype. No clinical evidence was found in ClinVar database for any of the variants, except for one variant in LYST (c.3931A>G:p.M1311V) with uncertain significance for Chédiak-Higashi syndrome and medium prediction scores. Variants in LYST (c.5990C>G:p.A1997G), NLRC4 (c.772T>C:p.C258R) and IRF3 (c.325G>C: p.G109R) have high CADD, Mutation Taster, Polyphen and SIFT prediction scores. And IKBKG (c.325C>G:p.L109V) variant had medium prediction scores. Conclusion: Our findings suggest that MIS-C patients do not share a rare loss-of-function variant in type I IFN immunity genes, TLR7 gene or genes associated either with HLH or KD. Despite numerous clinical, immunological and genetic research of the MIS-C patients, the syndromes pathogenesis and etiologic cause remain elusive.

The effect of atmospheric pollutants on the grass pollen allergenicity

Background: Grass pollen is one of the most important allergen sources inducing respiratory allergies and Phl p 5 allergen of timothy pollen is considered one of the major parts of the allergenic activity of grass pollen. In this study, we evaluated seasonal variation in the concentration of both grass pollen and Phl p 5 allergens as well as the ratio allergen/pollen (pollen potency) in the air of Bratislava, Slovakia during two consecutive years, 2019-2020. These two years differed in terms of air pollution, as COVID-19 lockdown in 2020 improved air quality in a very emphatic manner in the study area. Therefore, the goal of this research was also to determine how environmental factors affect airborne pollen and aeroallergen levels and pollen potency. Method: Pollen sampling was performed using a Hirst-type sampler, while a cyclone sampler was used for the aeroallergen capturing. Allergenic molecules were quantified by ELISA assay. Results: In 2020, the year characterised by a less polluted atmosphere due to COVID-19 lockdown, we observed significantly higher Seasonal Poaceae Pollen Integral, the mean daily pollen value and even peak pollen value, while the mean daily pollen potency, the mean daily allergen concentration and peak allergen value were significantly lower than in 2019. Raised pollen concentrations were accompanied by increased ozone and carbon monoxide levels in 2020, whereas increased rainfall or relative humidity led to the reduction of pollen in the atmosphere. In 2020, the aeroallergen levels were associated mainly with pollen, but nitrogen dioxide in the air could increase the number of allergens per pollen. In contrast, the aeroallergen levels were associated with carbon monoxide in 2019. Conclusion: Based on our results it is evident that air pollutants can influence grass plants to produce pollen with altered allergenic content.