Snyder, Melissa R.

Abstract Background Celiac disease (CeD) is a chronic autoimmune disorder that can affect people of any age. Both ingestion of gluten and the presence of human leukocyte antigens (HLA)-DQ2 or DQ8 are required for disease development. In adults, correlation between HLA-DQ2/DQ8 allele combinations and tissue transglutaminase (tTG) antibody positivity has been well described. In comparison, this association in pediatric patients is less defined. In this study, we evaluated the roles of HLA-DQ2 and HLA-DQ8 as predictors of tTG-IgA positivity in a pediatric cohort, aiming to enhance understanding of CeD risk assessment in this diagnostic group. Methods Retrospective data were collected from individuals who underwent CeD diagnostic testing at Mayo Clinic Laboratories between 2011 and 2024; all patients were tested for total IgA, HLA-DQ typing, and tTG-IgA. Total IgA was measured using quantitative nephelometry on the BN™ II System (Siemens); HLA-DQ typing was performed using reverse sequence-specific oligonucleotide (SSO) DNA typing (LABType SSO kits, One Lambda, Thermo). tTG-IgA was tested using an enzyme-linked immunosorbent assay (QUANTA Lite® h-tTG IgA, Werfen Diagnostics). Those with total IgA below the age-specific reference interval were excluded from this analysis. Logistic regression was used to evaluate the association between HLA-DQ haplotypes and tTG-IgA positivity to assess the risk gradient across different HLA-DQ genotypes in children. Results The cohort identified in this study consisted of 115,499 individuals for whom total IgA, HLA-DQ typing and tTG-IgA testing was performed. Within this cohort, 20,676 (17.9%) were pediatric patients (age<=18 at the time of testing). Pediatric patients tested for CeD exhibited a slightly higher prevalence of at least one copy of HLA-DQ2 or DQ8 compared to adults (57.6% vs 55.4%, p<0.001). Additionally, these patients showed significantly higher rates of tTG-IgA positive across all genotypes, particularly those carrying HLA-DQ2.5 or DQ8, with positivity rates 1.2 to 2.3 times higher than in adults (p<=0.01). However, in non-permissive genotypes, the positivity rates were 0.3% (p=0.95), for both populations, indicating comparable specificity. Individuals with homozygous HLA-DQ2.5/DQ2.5 and HLA-DQ2.5/DQ2.2 showed the highest risks for tTG-IgA positivity with ORs of 85.7 (95% CI: 55.1-137.7) and 78.5 (95% CI: 51.3-124.3), respectively. Moderate risk was observed for HLA-DQ2.5/DQ8 with an OR of 49.0 (95% CI: 32.1-77.3), followed by single copy HLA-DQ2.5 (OR=35.0, 95% CI: 24.0-53.4) and homozygous HLA-DQ8/DQ8 (OR=21.7, 95% CI: 11.7-39.4). Although single copies of HLA-DQ2.2 and HLA-DQ8 showed lower risks with ORs of 5.4 and 8.9 (95% CI: 3.2-9.2 and 5.7–13.9), their combination increased the OR to 22.6 (95% CI: 13.0-39.2), similar to homozygous HLA-DQ8. Conclusion This large cohort study demonstrates the predictive value of HLA-DQ genotypes for pediatric CeD seropositivity. Notably, pediatric patients showed slightly higher prevalence and positivity rates compared to adults, particularly among those with homozygous HLA-DQ2.5/DQ2.5 and HLA-DQ2.5/DQ2.2 genotypes, which exhibited the highest risks for tTG-IgA positivity. The findings confirm that HLA-DQ2.5 is the main driver of serology positivity and suggest that HLA-DQ8 also influences serological positivity in pediatric patients, which can enhance targeted screening and potentially inform decisions regarding the necessity of duodenal biopsy.

Abstract Background: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for hematological neoplasms. In this study, we make use of a natural inflammatory response occurring during COVID-19 to define the impact of these mutations on inflammation and outcomes. Methods: A cohort of 243 community-based patients (Olmsted County, MN) with COVID-19 were identified. CH status was determined on PBMC using error corrected sequencing (lower detection limit 0.5% VAF). In a subset of patients, we use multi-omics interrogation of the genome, transcriptome, and epigenome at single cell (sc) resolution along with DNA methylation, in PBMC, from patients with COVID-19 and CH. Results: We detected 97 CH mutations in 72 (29.6%) of 243 patients with COVID-19. The most frequent being DNMT3A (n=30, 30%) and TET2 (n=26, 28%). The presence of CH negatively impacted overall survival when restricted to TET2 and DNMT3A mutations. However, only DNMT3Amt CH retained age and comorbidity independent prognostication (HR 2.84, p = 0.022). We then assessed for changes in DNA methylation using Illumina MethylationEPIC array on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=4) CH. Site specific DNA methylation changes revealed significant loss of methylation in DNMT3Amt CH in comparison to TET2mt patients, with 10,944 hypomethylated and 1,160 hypermethylated sites. We found that actively transcribed states (Tx, TxWk) were more commonly hypomethylated in DNMT3Amt CH while enhancers (Enh) and promoters (TssA, TssAFlnk) were more commonly hypomethylated in TET2mt CH. We then conducted scDNA-seq in DNMT3Amt (n=3) and TET2mt (n=1) CH in COVID-19 and observed that while in TET2mt CH, mutations were largely restricted to classical and intermediate monocytes, in DNMT3Amt CH, mutations were seen in myeloid and lymphoid lineage cells. We performed scRNA-seq on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=6) CH. Differential gene expression analysis identified 1,569 upregulated genes in DNMT3Amt patients and 205 downregulated. Pathway analysis demonstrated an upregulation of genes involved in T cell survival, function, and inflammation in DNMT3Amt patients compared to TET2mt patients with a significant overexpression of GIMAP1, GIMAP4, IRF2, IL32 in multiple cell types. We correlated transcriptomic changes with chromatin accessibility using the 10X Genomics Multiome platform to profile both gene expression and open chromatin from the same samples. Analysis of global distribution of cut sites and differentially accessible peaks showed increased chromatin accessibility in DNMT3Amt CH, especially CD4+ T lymphocytes and NK cells. Conclusion: We demonstrate an age, sex, and comorbidity-independent adverse impact on mortality associated with DNMT3Amt CH in the context of COVID-19 and highlight transcriptomic and epigenetic differences between DNMT3A and TET2mt CH, with single cell resolution. Citation Format: Jenna A Fernandez, Wazim Mohammed Ismail, Moritz Binder, Terra Lasho, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek Mangaonkar, Jeong-Heon Lee, Liguo Wang, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Jonathan J. Harrington, Melissa R. Snyder, Keith D. Robertson, Alexandre Gaspar-Maia, Mrinal Patnaik. Divergent impacts of DNMT3A and TET2 mutant clonal hematopoiesis on COVID-19 related outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A012.

Abstract DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19–related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19–related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.

Abstract Background DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposing epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematological neoplasms. While individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Results Here we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant CH on COVID-19-related cytokine release severity and mortality. Using single cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid cells. Using single cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of IL32, a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Conclusions We demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19 related inflammatory morbidity and mortality. DNMT3Amt CH involves myeloid and lymphoid cells and in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can serve as a potential biomarker for adverse inflammatory outcomes.

Background: Over 30% of patients with COVID-19 have persistent symptoms that last beyond 30 days and referred to as Long COVID. Long COVID has been associated with a persistent elevation in peripheral cytokines including interleukin-6, interleukin-1β, and tumor necrosis factor-α. This study reports cytokine profiles of patients in our clinic across SARS-COV-2 variant epochs. Methods: The clinical cytokine panel was analyzed in patients with Long COVID during periods that were stratified according to variant epoch. The 4 variant epochs were defined as: (1) wild-type through alpha, (2) alpha/beta/gamma, (3) delta, and (4) omicron variants. Results: A total of 390 patients had the clinical cytokine panel performed; the median age was 48 years (IQR 38-59) and 62% were female. Distribution by variant was wild-type and alpha, 50% (n = 196); alpha/beta/gamma, 7.9% (n = 31); delta, 18% (n = 72); and omicron, 23% (n = 91). Time to cytokine panel testing was significantly longer for the earlier epochs. Tumor necrosis factor-α ( P < .001) and interleukin 1β ( P < .001) were significantly more elevated in the earlier epochs (median of 558 days in wild-type through Alpha epoch vs 263 days in omicron epoch, P < .001)). Nucleocapsid antibodies were consistently detected across epochs. Discussion: When stratified by variant epoch, patients with early epoch Long COVID had persistently elevated peripheral pro-inflammatory cytokine levels when compared to later epoch Long COVID. Patients with Long COVID have similar clusters of symptoms across epochs, suggesting that the underlying pathology is independent of the peripheral cytokine signature.

AbstractBackgroundClot based assays used for lupus anticoagulant (LAC) detection are typically interpreted in a qualitative fashion and may not reflect LAC potency. In this cross‐sectional study, we describe a method for quantifying the LAC titer using serial (dependent) two‐fold dilutions in normal pooled plasma.MethodsSerial dilutions of 51 residual plasma samples from 50 patients were tested using the Russell's viper venom screening time (DRVVT) and activated partial thromboplastin screening time (APTT) methodologies. The measured clotting times and the corresponding dilution factors were then used to derive a four‐parameter logistic model. The LAC titer for each patient was interpolated as the sample dilution that corresponds to the upper reference interval limit of the corresponding assay.ResultsCalculated APTT and DRVVT LAC titers displayed a strong linear correlation (R2 = 0.84) between each other, but not with the degree of prolongation of the APTT/DRVVT screening time in the neat undiluted samples. Using data driven partitioning, patients could be grouped into low (<10) or high (≥10) DRVVT LAC titer. There were no significant differences in anticardiolipin (aCL) or anti‐beta 2 glycoprotein 1 (aB2GPI) antibody levels or prevalence of thromboembolic events between low and high LAC titer groups. In contrast, antiphosphatidylserine/prothrombin (aPS/PT) IgM antibody levels, but not IgG, were significantly higher in the high LAC titer group.ConclusionsThe degree of prolongation of the APTT/DRVVT screening time is not correlated with the LAC titer. Only aPS/PT IgM antibodies levels were strongly correlated with the LAC titers. Additional studies are warranted to determine clinical implications of high LAC titers.

Abstract Background Fecal calprotectin (FC) is a promising biomarker for assessing ulcerative colitis (UC) endoscopic activity. However, the optimal FC cutoff to identify each Mayo endoscopic subscore (MES) remains inconclusive. Methods The electronic medical records of 177 adult UC patients evaluated at Mayo Clinic Rochester from January 2017 to March 2023 were retrospectively reviewed, obtaining clinical data and US-based Werfen Diagnostics FC levels collected within 30 days before colonoscopy or flexible sigmoidoscopy. Three independent inflammatory bowel disease specialist endoscopists blindly reviewed the most severe endoscopic images for grading MES. Results The median interval between FC collection and endoscopy was 2 days. Fecal calprotectin showed strong positive correlations with MES (Spearman’s r = 0.709; P &lt; .01) and other clinical parameters. Fecal calprotectin cutoff of 60 mcg/g effectively distinguished MES 0 from MES 1-3 (sensitivity, 0.78; specificity, 0.97; area under the receiver operating characteristic curve [AUC], 0.901) and predicted clinical remission (Total Mayo Score ≤2 and no subscore &gt;1; sensitivity, 0.83; specificity, 0.98; AUC, 0.921). Fecal calprotectin cutoff of 110 mcg/g effectively differentiated MES 0-1 from MES 2-3 (sensitivity, 0.86; specificity, 0.87; AUC, 0.915), while a cutoff of 310 mcg/g distinguished MES 0-2 from MES 3 (sensitivity, 0.80; specificity, 0.76; AUC, 0.820). Conclusions This study supports the reliability and applicability of FC as a valuable marker of endoscopic inflammation, particularly in distinguishing MES 0 from MES 1-3 using the FC cutoff of 60 mcg/g. Sensitivity analysis demonstrated robust results.

Abstract Background The racial, ethnic and sex compositions of a study population may impact the clinical performance of a biomarker. In a prospective clinical study to evaluate biomarkers for asthmatic and allergic conditions in a large medical center in the upper Midwest, a largely White-alone patient population was encountered. Using several outreach strategies, potential eligible patients were contacted to enroll in a biomarker clinical study and to provide blood specimens. The patient demographics between the eligible candidates and the enrolled participants were compared. The analysis was carried out to understand the likelihood of enrollment in different race, ethnicity, and sex groups; and to identify factors that contribute to selection biases in enrolled cohorts. Methods Patients with eligible asthma and allergic conditions were selected based on relevant diagnostic codes in their electronic medical records, verified by clinical research coordinator. In accordance with Institutional Review Board approval, eligible patients were contacted via their electronic healthcare portal or with a phone call. The study information was also posted in an internal classified ad as an additional outreach strategy. Interested participants were then contacted a second time to complete consent. Upon consent, participants were considered as “enrolled”. Demographics information including sex, race, ethnicity, and age were de-identified for comparison analysis. Results Among all eligible patients, a disproportionally low representation of Blacks, Asians, and Hispanics (“non-White” groups) was observed, relative to regional demographics (22.2% non-White groups, 2021 census) and higher asthma and allergy prevalence in the non-White groups. In eligible adult patients (&gt;18 years old) with asthma, only 13.4% were from non-White racial and ethnic groups. In eligible pediatric asthma patients, a larger proportion at 24.1% were from American Indian, Asian, Black, or Hispanic groups. In patients with allergic conditions, 10.3% of the adult patients were from non-White only groups, compared to 21.9% in pediatric patients. Among all eligible patients, White-alone patients were more likely to enroll in the study compared to their counterparts from non-White groups, further skewing the racial and ethnic distribution in the enrolled cohorts. As a result, less than 10% of enrolled adult patients were from non-White background in both allergy and asthma cohorts. Among the enrolled pediatric asthma and allergy patients, 16.9% and 10.3% respectively were from non-White groups. We also identified sex and age dependent enrollment behavior across different study cohorts. In adult groups, there were more female eligible patients (allergy: 66.7% female; asthma: 66.7% female) than male patients. Female adult patients were significantly more likely to participate, thus constituting 79.6% and 75.8% of the enrolled allergy and asthma cohorts. In the pediatric groups, male patients represented a larger share of eligible patients (allergy: 58.6%; asthma 64.7%). Female pediatric patients were still more likely to participate in the study. Hence, male and female enrolled patients were closer to equal distribution (allergy: 50.0% male; asthma 59.7% male). Conclusion The non-even distribution of demographics in eligible patients contributed to biased distribution in enrolled patients. Existing outreach approaches were not effective in reversing enrollment rates in underrepresented racial and ethnic groups.