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Blood transcriptomic markers of necrotizing enterocolitis in preterm pigs

Abstract

Background

Necrotizing enterocolitis (NEC), a severe gut disorder in preterm infants, is difficult to predict due to poor specificity and sensitivity of clinical signs and biomarkers. Using preterm piglets as a model, we hypothesized that early development of NEC affects blood gene expression, potentially related to early systemic immune responses.

Methods

A retrospective analysis of clinical, tissue, and blood data was performed on 129 formula-fed piglets with NEC diagnosis at necropsy on day 5. Subgroups of NEC (n = 20) and control piglets (CON, n = 19) were analyzed for whole-blood transcriptome.

Results

Preterm piglets had variable NEC lesions, especially in the colon region, without severe clinical signs (e.g. normal growth, activity, hematology, digestion, few piglets with bloody stools). Transcriptome analysis showed 344 differentially expressed genes (DEGs) between NEC and CON piglets. Validation experiment showed that AOAH, ARG2, FKBP5, PAK2, and STAT3 were among the genes affected by severe lesions on day 5, when analyzed in whole blood and in dried blood spots (DBS).

Conclusion

Whole-blood gene expressions may be affected in preterm pigs before clinical signs of NEC get severe. Blood gene expression analysis, potentially using DBS samples, is a novel tool to help identify new early biomarkers of NEC.

Impact

  • Preterm pig model was used to investigate if blood transcriptomics could be used to identify new early blood biomarkers of NEC progression.

  • Whole-blood transcriptome revealed upregulation of target genes in NEC cases when clinical symptoms are subtle, and mainly colon regions were affected.

  • Differential NEC-associated gene expressions could be detected also in dried blood spots, potentially allowing easy collection of small blood volumes in infants.

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Fig. 1: Representative photographs of small intestine and colon with NEC score.
Fig. 2: Whole-blood transcriptome in response to NEC.
Fig. 3: Expression of target DEGs in validation cohort.

Data availability

All sequencing and processed data are deposited in the Gene Expression Omnibus (GEO) with accession number GSE166152.

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Acknowledgements

We thank Thomas Thymann, Anders Brunse, Duc Ninh Nguyen, Jing Sun, Kristine Holgersen, Jane Povlsen, Elin Skytte, Kristina Møller (University of Copenhagen), and Jonas Bybjerg-Grauholma (Statens Serum Institut, Denmark) for their support to animal procedures and laboratory analyses. This work was supported by the Innovation Foundation Denmark NEOCOL project (to P.T.S.) and the Agricultural Science and Technology Innovation Program (ASTIP) of China (to F.G.).

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X.P. analyzed and interpreted the transcriptome data, and was the major contributor in writing the manuscript. T.M., S.R., and D.N.N. performed animal experiment and analysis. D.N.N., R.L.S., F.G., and P.T.S. took part in the main study design and critically reviewed the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Per Torp Sangild.

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Pan, X., Muk, T., Ren, S. et al. Blood transcriptomic markers of necrotizing enterocolitis in preterm pigs. Pediatr Res (2021). https://doi.org/10.1038/s41390-021-01605-4

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