ABSTRACT
Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.
ABSTRACT
Background: Vacuoles, E1 enzyme, X-linked, autoinfammatory, somatic (VEXAS) syndrome is a recently identifed disorder caused by somatic mutations in the UBA1 gene of myeloid cells. Various manifestations of pulmonary involvement have been reported, but a detailed description of lung involvement and radiologic fndings is lacking. Objectives: To describe lung involvement in VEXAS syndrome. Methods: A retrospective cohort study was conducted of all patients iden-tifed at the Mayo Clinic with VEXAS syndrome since October 2020. Clinical records and chest high resolution computed tomography (HRCT) scans were reviewed. Results: Our cohort comprised 22 white men with a median age of 69 years (IQR 62-74, range 57-84). Hematologic disorders including multiple myeloma, myelodysplastic syndrome and pancytopenia were present in 10 patients (45%), rheumatologic diseases including granulomatosis with poly-angiitis, IgG4-related disease, polyarteritis nodosa, relapsing polychondritis, and rheumatoid arthritis were found in 10 patients (45%), and 4 patients had dermatologic presentations including Sweet syndrome, Schnitzer-like syndrome or drug rash with eosinophilia skin syndrome (DRESS). VEXAS syndrome-related features included fever (18, 82%), skin lesions (20, 91%), lung infiltrates (12, 55%), chondritis (10, 45%), venous thromboembolism (12, 55%), macrocytic anemia (21, 96%), and bone marrow vacuoles (21, 96%). Other manifestations observed were arthritis, scleritis, hoarseness and hearing loss. Median erythrocyte sedimentation rate (ESR) was 69 mm/1st hour (IQR 34.3-118.8) and median C-reactive protein (CRP) of 55.5 mg/dL (IQR 11.4-98.8). The somatic mutations affecting methionine-41 (p.Met41) in UBA1 gene were: 11 (50%) p.Met41Thr, 7 (32%) p.Met41Val, 2 (9%) p.Met41Leu, and 2 (9%) in the splice site. All patients received glu-cocorticoids (GC) (median duration of treatment was 2.6 years);21 (96%) received conventional immunosuppressive agents (methotrexate, aza-thioprine, mycophenolate, leflunomide, cyclosporin, hydroxychloroquine, tofacitinib, ruxolitinib) and 9 (41%) received biologic agents (rituximab, tocilizumab, infliximab, etanercept, adalimumab, golimumab, abatacept). Respiratory symptoms included dyspnea and cough present in 21 (95%) and 12 (55%), respectively, and were documented prior to VEXAS diagnosis. Most of the patients were non-smokers (14, 64%) and obstructive sleep apnea (OSA) was present in 11 patients (50%). Seven patients (32%) used non-invasive ventilation, 6 used C-PAP, and 1 used Bi-PAP. Bronchoalveolar lavage (BAL) was available in 4 patients, and the findings were compatible with neutrophilic alveolitis in 3. Two patients had lung biopsies (2 transbronchial and 1 surgical) that showed ATTR amyloidosis and organizing pneumonia with lymphoid interstitial pneumonia, respectively. Pulmonary function tests were available in 9 (41%) patients and showed normal results in 5;3 patients had isolated reduction in DLCO and 1 with mild restriction. On chest HRCT, 16 patients (73%) had parenchymal changes including ground-glass opacities in 9, septal thickening in 4, and nodules in 3;pleural effusions were present in 3 patients, air-trapping in 3 patients and tracheomalacia in 1 patient. Follow-up chest HRCT was available for 8 patients (36%), the ground-glass opacities resolved in 5 patients, 3 patients manifested new or increased ground-glass opacities, and 1 patient had increased interlobular septal thickening. After 1 year of follow-up, 4 patients (17%) had died;3 due to pneumonia (2 COVID-19,1 bacterial) and 1 due to heart failure. VEXAS flares occurred in 18 patients (82%), the maximum number of relapses was 7, and they were mainly managed with GC and with changes in the immuno-suppressive regimen. Conclusion: Pulmonary involvement was documented by chest HRCT in most patients with VEXAS syndrome. Respiratory symptoms occurred in over one half of patients and about 20% had PFT abnormalities. The pulmonary manifestations of VEXAS are nonspecifc and characterized predominantly by infamma-tory parenchymal involvement.
ABSTRACT
Background: SARS-Cov-2 infections are associated with increased mortality and morbidity, largely due to inflammatory cascades and cytokine release syndrome (CRS). Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence and subsequent expansion of somatic, leukemia-associated driver mutations in apparently healthy individuals with normal blood counts. CHIP has been associated with increased inflammation, with cytokines such as IL1-b, IL6 and TNF-a being elevated at baseline in affected individuals. We hypothesized that the presence of CHIP in patients with COVID-19 would result in excessive inflammation-related mortality and morbidity. Methods: We used the Mayo Clinic COVID-19 database to identify patients with COVID-19 on whom peripheral blood mononuclear cells (PBMC) were available for research use (IRB approved). We carried out target-capture next generation sequencing for 220 CH related genes, by previously described methods (1000 x coverage, variant allele fraction/VAF detection limit >0.5%;Kusne Y et al AJH 2021). CHIP was defined by the presence of a CH mutation with a VAF>1% in an individual with normal baseline blood counts. Demographics, blood counts, and inflammatory markers (CRP and cytokine levels- ELISA assay) at COVID-19 diagnosis and during follow-up (as clinically indicated) were collected. COVID-19 disease severity was classified based on the presence and severity of CRS, graded using the Penn Grading Scale (Porter et. al., 2018), and the WHO ordinal scale (WHO Blueprint, 2020). We used Fisher's exact test and the Wilcoxon rank sum test to compare categorical and continuous variables. Survival analysis was performed using the Kaplan-Meier method. We accounted for differences in age and sex using multivariable-adjusted proportional hazards regression models. Results: Seventy-two CHIP mutations were detected in 56 (25%) of the 227 patients with COVID-19 that had PBMC available;median age 69 years (range;42-99 years), 61% male. Fifteen (26%) patients had 2 CHIP mutations, while 1 patient had 3 CHIP mutations. Common mutations encountered included DNMT3A (32%), TET2 (19%), SF3B1 (8%), ASXL1 (6%), MPL (5%), and TP53 (5%;Figure 1A). COVID-19 patients with CHIP were older in age (median 69 vs 57 years;p<0.0001) and had higher baseline MCP-1 (p=0.04) levels. However, there were no differences in sex, comorbidities, blood counts, IL1-b, IL6 and TNF-a levels between the two groups. The median follow-up for the entire cohort was 9 months. The relative change from baseline in blood counts and inflammatory markers (CRP and cytokines) during follow-up was similar in CHIP and non-CHIP patients, with the exception that COVID-19-onset neutropenia was more common in CHIP patients (8% vs 1%;p=0.017) compared to those without CHIP. At last follow up neutropenia had resolved in all patients. Both groups had comparable number of patients with CRS (61% CHIP vs 53% non-CHIP patients, p=0.354, Figure 1B), however, CHIP patients had more severe CRS (median Penn Grade 3 versus 2 in non-CHIP, p=0.018, Figure 1C). Based on the WHO ordinal scale, CHIP patients were more likely to experience hospitalization with severe disease and death (61% versus 45% in non-CHIP, p = 0.049). Moreover, COVID-19 CHIP patients experienced worse overall survival in comparison to patients without CHIP (median 13.1 months vs not reached, p<0.001, Figure 1D). This association remained consistent after adjusting for age and sex at the time of COVID-19 diagnosis (HR 3.15, 95% 1.32 - 7.55, p = 0.010). At last follow-up, 22 deaths were documented: 13 (23%) in patients with CHIP and 9 (5%) in the non-CHIP group (p=0.02), with the primary cause for mortality being hypoxic respiratory failure (62% in CHIP vs 44% non-CHIP, p=0.04). Conclusions: In this study, we observe an age-independent impact of CHIP on COVID-19 associated inflammatory morbidity (CRS) and mortality (hypoxemic respiratory failure). We are currently carrying out detailed single cell (ssDNA, RNA and ATAC-seq) and proteomic studies (O-link PEA assays) to better elucidate this pathophysiology. [Formula presented] Disclosures: Patnaik: Kura Oncology: Research Funding;StemLine: Research Funding.