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DNER as a novel protein contributes to HSCR pathogenesis: multi-omics combined Mendelian randomization analysis

Pediatric Research (2026)Cite this article

Abstract

Background

Hirschsprung disease (HSCR) is a congenital disorder characterized by the aganglionosis, which remains disputable and limited biomarkers about pathogenesis.

Methods

This study employed a multidimensional approach to explore the potential new targets and molecular mechanism for HSCR, including proteome-wide Mendelian randomization (MR) analysis, single-cell RNA sequencing (scRNA-seq), bioinformatics, and experimental validation through cellular and tissue studies.

Results

Our results confirmed RET and AGRN as key proteins implicated in HSCR genesis and development, further underscoring their indispensable role. Notably, for the first time, we identified DNER as a novel protein significantly contributing to HSCR pathogenesis. MR and Bayesian colocalization analysis revealed a robust causal relationship between DNER and HSCR risk (OR = 0.12, 95% CI: 0.04–0.39, P = 4.54 × 10-4) ScRNA-seq data analysis showed that DNER expression increased during enteric neural crest cell (ENCC) development, consistent along with western blotting result in colon tissue at different developmental stages. Meanwhile we demonstrated DNER protein expression in enteric neurons and observed a marked decreased expression of DNER in the colon of HSCR models. Moreover, down-regulation of DNER not only impeded ENCC migration but also compromised their subsequent differentiation into neurons and synaptic maturation. All above indicated the role in ENS formation and that DNER contribute crucially to aganglionosis, the hallmark of HSCR pathology. Functional enrichment analysis revealed the Notch signaling pathway as a potential downstream mechanism.

Conclusion

Our study emphasized the significance of DNER/Notch signaling pathway and provided new insights into the HSCR pathogenesis. Potentially, DNER seemed to be a novel biomarker and therapeutic target for HSCR, and future research should focus on the detailed molecular mechanisms how DNER/Notch roles in HSCR.

Impact

  • This study identifies DNER as a novel causal protein in Hirschsprung disease (HSCR) pathogenesis through integrated proteome-wide Mendelian randomization and Bayesian colocalization.

  • We demonstrate that DNER regulates enteric neural crest cell proliferation, migration, and differentiation via the Notch signaling pathway, with dynamic expression during ENS development revealed by single-cell RNA sequencing.

  • Our findings establish a novel DNER-Notch signaling axis in gut neural development and reveal that DNER deficiency disrupts ENCC colonization, neuronal differentiation, and neural network assembly.

  • This work provides fundamental insights into HSCR pathogenesis, highlighting a previously underappreciated molecular pathway in enteric nervous system development.

Potential new targets for HSCR: from a whole proteome-wide Mendelian randomization analysis to experiment in vivo and in vitro.

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Fig. 1: Flowchart of the study design.
Fig. 2: Mendelian randomization and colocalization analysis in HSCR.
Fig. 3: Protein-protein interaction network and functional enrichment analysis of DNER.
Fig. 4: Expression and pseudotime analysis of protein-coding genes identified by proteome-wide Mendelian randomization in single-cell types from the intestines of Ednrb-/- mice at postnatal day 14 (P14).
Fig. 5: DNER protein expression in ENCCs, neurons, and glial cells, with reduced expression in the dilated segment of the colon in HSCR rat and mouse models.
Fig. 6: Limited proliferation and migration abilities of ENCCs with low DNER expression.

Data availability

The datasets analyzed in the current study are available in the Decode database (https://www.decode.com/summarydata/), the UK Biobank-PPP (https://www.synapse.org/Synapse:syn51365301), and the NHGRI-EBI GWAS Catalog (https://www.ebi.ac.uk/gwas/). Single-cell sequencing data are available in the GEO database (GSE199683). Databases used for drug availability assessment include DGIdb (https://www.dgidb.org/), ChEMBL (https://www.ebi.ac.uk/chembl/), and DrugBank (https://go.drugbank.com/).

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Acknowledgements

We sincerely thank the scientists who shared their data on the public database. We thank the creators of the packages TwoSampleMR, MR-PRESSO, qvalue, ggplot2, coloc, clusterProfiler, Seurat, scrublet, Scanpy, and Monocle 3.

Funding

The study was supported by grants from the National Natural Science Foundation of China (No: 82300575, 82071692, 82170531), Xi’an Jiaotong University (No: YXJLRH2022053, xzy012024111) and the General Project of Shaanxi Science and Technology Department (No: 2022SF-133/033).

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Author notes

  1. These authors contributed equally: Wei Liu, Wenyao Xu.

Authors and Affiliations

  1. Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China

    Wei Liu, Wenyao Xu, Jingjing Huang, Zihao Fu, Hanlei Yan, Wanying Jia, Hui Yu, Baijun Zheng, Weikang Pan, Ya Gao & Donghao Tian

  2. Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, Xi’an, China

    Wei Liu, Jingjing Huang, Zihao Fu, Hanlei Yan, Wanying Jia, Hui Yu, Xinlin Chen & Donghao Tian

  3. National & Local Joint Engineering Research Center of Biodiagnosis and Biotherapy, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China

    Jiayi Xu

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Contributions

Wei Liu: conceptualized this study design, acquired, analyzed and visualized data, interpreted the results and wrote the manuscript with all authors providing feedback for revision. Wenyao Xu and Jingjing Huang : Western Boltting, acquired, analyzed and visualized data and interpreted the results. Jiayi Xu, Zihao Fu,Wanying Jia and Hanlei Yan: acquired, analyzed and visualized data. Hui Yu and Weikang Pan contributed to data analysis, visualization and curation. Xinlin Chen and Baijun Zheng performed supervision. Ya Gao: wrote the manuscript with all authors providing feedback for revision. Donghao Tian: conceptualized this study design, interpreted the results and wrote the manuscript with all authors providing feedback for revision. All authors read and approved the final report.

Corresponding authors

Correspondence to Ya Gao or Donghao Tian.

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The authors declare no competing interests.

Ethics approval and consent to participate

All the animal procedures were handled in accordance with the guidelines outlined by the Animal Care and Ethics Committee of Xi’an Jiaotong University (No. 2018-2148). Clinical trial number with the Clinical Trial Registry: not applicable.

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Liu, W., Xu, W., Huang, J. et al. DNER as a novel protein contributes to HSCR pathogenesis: multi-omics combined Mendelian randomization analysis. Pediatr Res (2026). https://doi.org/10.1038/s41390-026-04789-9

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