514 - Targeted RNA sequencing identified gene expression profiles linked to severe necrosis in preterm infants with surgical necrotizing enterocolitis.

Poster Number: 514
Publication Number: 514.2117

Background: Genetic factors are known to affect necrotizing enterocolitis (NEC) development in preterm infants, however the mechanisms underlying NEC are not fully understood.

Objective: To identify immune gene expression changes in surgical NEC infants with and without severe necrosis detected on the small intestine histopathology.

Design/Methods: RNA was isolated from 10um section of NEC formalin-fixed, paraffin-embedded (FFPE) human intestine specimens (N=36) obtained following laparotomy Targeted RNA sequencing using the AmpliSeq Illumina Immune Response Panel was utilized for targeted quantitative expression of genes linked to immune system interactions which included 400 genes. Raw reads were de-multiplexed and aligned to human reference genome assembly (hg19) using RNA Amplicon Application (along with custom panel manifest) available on Illumina BaseSpace Computing Platform (DRAGEN APP; https://basespace.illumina.com/). Aligned reads for each gene were normalized to count per million for downstream analysis. DeSeq2 was used to analyze differential expression between mild- severe necrosis while accounting for other covariates such as gender and death/discharge disposition.

Results: Differential expression analysis was used to investigate whether the stage of necrosis affects gene expression in preterm infants. The analysis showed 35 genes being significantly differentially expressed (p < 0.05) between Mild-Medium Necrosis (Score 0-2) and Severe Necrosis (Score of 3 and 4). The majority of the amplicons included in the panel were detected. Genes related to altered host defense, natural killer cell signaling and development, apoptosis etc. were overexpressed in severe necrosis (IGJ, GZMA, TNFSF10, KLRB1, CD160). Expression of leukocytes antigens (ITGAM, ITGAX); cytokines and chemokines receptors (such as IL1A, IL1B, CCL2, CCL3) were upregulated in patients with mild necrosis. See Figure 1.

Conclusion(s): NEC infants with severe necrosis on based on intestinal pathology were associated with overexpressed gene related to host defense, natural killer cell signaling and development, apoptosis. The understanding of these pathogenic pathways would guide us to develop the prognostic and treatment pathways.