The Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn’s Disease study data from 164 pediatric patients with Chrohn’s disease and 74 controls was accessed from GSE112611 for this analysis. Study subjects were from North America and Crohn’s disease patients had repeated blood draws with DNA methylation measures 11. Samples for the Crohn’s cases were taken at diagnosis and again 1-3 years later. Treatment approaches between baseline and follow up samples varied among patients. Study subject demographics and clinical characteristics are provided in Table 1.
Table 1. Study population demographic and disease characteristics.
Characteristics
|
Cases
|
Controls
|
Total Subjects
|
164
|
74
|
Total Samples
|
328
|
74
|
Age, mean (range)
|
13.8 (4.5-20.8)
|
12.0 (4.5-16.8)
|
Sex, n (%)
|
|
|
Male
|
96 (59)
|
40 (54)
|
Female
|
68 (41)
|
34 (46)
|
Stage at Baseline, n (%)
|
|
|
Stage B1
|
150 (91)
|
NA
|
Stage B2
|
14 (9)
|
NA
|
Stage at Follow Up, n (%)
|
|
|
Stage B1
|
95 (58)
|
NA
|
Stage B2
|
69 (42)
|
NA
|
Immune cell proportions were quantified using blood DNA methylation data for all samples and subjects with the Flow Sorted. Blood Extended. EPIC R package 14. A univariate comparison for each of the twelve cell types between treatment-naive Chrohn’s patients at diagnosis and controls revealed that basophil and neutrophil levels were significantly elevated in Crohn’s (PBas = 0.02 and PNeu = 8.05 x 10-9). In addition, memory B-cells, memory and naive CD4 cells, memory and naive CD8 cells, eosinophils, and natural killers cells were all significantly lower in Crohn’s patients at baseline compared with controls (PBmem = 5.6×10−7, PCD4mem = 3.1×10−7, PCD4nv = 8.3×10−7, PCD8mem = 2.5×10−6, PCD8nv = 3.6×10−10, PEos = 1.2×10−4, PNK = 2.9×10−13) (Figure 1). After adjusting for patient age and sex using a multivariable logistic regression model, the proportions of CD4 memory and naive, CD8 naive, natural killer cell, and neutrophil proportions in cases remained statistically significantly different from controls (PCD4mem = 2.11 × 10−3, PCD4nv = 1.83 × 10−2, PCD8nv = 2.41 × 10−5, PNK = 8.43 × 10−13, PNeu = 1.03 × 10−5).
Next, we compared immune cell proportions in Crohn’s cases by disease stage at diagnosis (Supplementary Figure 1) and at follow-up (Supplementary Figure 2). No statistically significant differences were observed for cell type proportions or NLR in cases by disease stage at baseline or follow up.
Paired sample t-tests were performed to investigate the change in cell type proportion between patients at diagnosis and at follow-up (Figure 2 - C). It was found that memory B-cell, naive B-cell, memory CD4 cell, naive CD4 cell, naive CD8 cell, and eosinophil proportions significantly decreased in patients at follow-up while neutrophil proportions significantly increased.
At diagnosis, the neutrophil-to-lymphocyte ratio (NLR) was significantly higher in Crohn’s disease patients than in the controls. However, after treatment in the follow-up samples we observed significant attenuation of the NLR in cases where levels were similar to those observed in controls. After adjusting for patient age and sex using a multivariable logistic regression model, the NLR of patients at diagnosis remained statistically significantly different from controls (P = 1.97 × 10−5). The difference between control and follow-up did not remain significantly different after adjusting for age and sex.
The epigenome-wide association analysis revealed 397 CpG sites that were associated with Crohn’s disease at diagnosis from the blood when using an FDR cutoff of 0.05. This analysis was performed using 74 non-IBD controls and 164 patients at diagnosis and was corrected for age, sex, neutrophils, monocytes, natural killer cells, naive B-cells, memory B-cells, naive CD4 T-cells, memory CD4 T-cells, naive CD8 T-cells, basophils, and eosinophils. When compared to a model that only uses the six cell type proportions, the extended cell type model results in fewer CpG sites associated with Crohn’s disease. Of the 397 CpG sites, 219 are hypermethylated and 178 are hypomethylated. Additionally, 296 intersect with the 1189 sites found by Somineni et al., 67 percent of which were hypermethylated, and 101 are novel, 37 percent of which are hypermethylated.
The genomic context enrichment analysis demonstrates that CpGs associated with Crohn’s disease are more likely to be in open sea regions (OR = 2.38, P = 7.96x10-16), enhancers (OR = 3.43, P = 5.15x10-11), DNase I hypersensitive sites (OR = 1.41, P = 1.33 x10-3), introns (OR = 1.60, P = 5.84x10-6), and gene bodies (OR = 1.35, P = 3.07x10-3), and less likely to be located in north shores (OR = 0.49, P = 6.41x10-4), promoters (OR = 0.48, P = 1.41x10-9), and 5’ untranslated regions (OR = 0.70, P = 4.11x10-2). To view the details of the genomic-context enrichment analysis of the Crohn's–associated CpGs, see Supplementary Table 1. The CpGs mapped to 226 distinct genes, 45 of which intersect with those mapped by Somineni et al. We replicated some of the findings and found that SBNO2 and RPS6KA2 were associated with Crohn’s 13. However, it was not found to be associated with others that were previously reported such as VMP1, ITGB2, TXK, TNF, JAK3, IL12B, IL23A, IL1R1 11,13. To view the details of the genetic context of the Crohn's–associated CpGs, see Supplementary Table 2. Looking at genetic pathways associated with Crohn’s the only pathway to approach significance after FDR correction is the regulation of arginine metabolic processes. To view the details of the genetic pathway GO terms associated with Crohn's, see Supplementary Table 3.