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Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung’s disease

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Abstract

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract. The major susceptibility gene for the disease is the RET proto-oncogene, which encodes a receptor tyrosine kinase activated by the glial cell-derived neurotrophic factor (GDNF) family ligands. We analyzed the coding sequence of GDNF, NTRN, and, for the first time, ARTN and PSPN in HSCR patients and detected several novel variants potentially involved in the pathogenesis of HSCR. In vitro functional analysis revealed that the variant R91C in PSPN would avoid the correct expression and secretion of the mature protein. Moreover, this study also highlighted the role of both this variant and F127L in NRTN in altering RET activation by a significant reduction in phosphorylation. To support the role of PSPN R91C in HSCR phenotype, enteric nervous system (ENS) progenitors were isolated from human postnatal gut tissues and expression of GFRα4, the main co-receptor for PSPN, was demonstrated. This suggests that not only GDNF and NRTN but also PSPN might promote survival of precursor cells during ENS development. In summary, we report for the first time the association of PSPN gene with HSCR and confirm the involvement of NRTN in the disease, with the identification of novel variants in those genes. Our results suggest that the biological consequence of the mutations NTRN F127L and PSPN R91C would be a reduction in the activation of RET-dependent signaling pathways, leading to a defect in the proliferation, migration, and/or differentiation process of neural crest cells within the developing gut and thus to the typical aganglionosis of the HSCR phenotype.

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Acknowledgments

We would like to thank the patients and families that participated in this study. We also thank Dr. Borghini (from Dr. Ceccherini’s lab, Instituto G Gaslini) for providing the SK-N-MC cell line stably transfected with RET cDNA, Dr. Saarma (University of Helsinki) for the GFRα4 expression vector, and Dr. Bocciardi (Instituto G Gaslini) for technical assistance. This work was supported by Fondo de Investigación Sanitaria, Spain (PI070080 and PI071315 for the E-Rare project) and Consejería de Innovación Ciencia y Empresa (CTS 2590). The CIBER de Enfermedades Raras is an initiative of the ISCIII.

Disclosure of potential conflict of interests

The authors declare no conflict of interests related to this study.

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Authors and Affiliations

  1. Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Avda. Manuel Siurot s/n, 41013, Seville, Spain

    Macarena Ruiz-Ferrer, Ana Torroglosa, Berta Luzón-Toro, Raquel M. Fernández, Guillermo Antiñolo & Salud Borrego

  2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Seville, Spain

    Macarena Ruiz-Ferrer, Ana Torroglosa, Berta Luzón-Toro, Raquel M. Fernández, Guillermo Antiñolo & Salud Borrego

  3. Department of Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, Canada

    Lois M. Mulligan

Authors
  1. Macarena Ruiz-Ferrer
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  2. Ana Torroglosa
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  7. Salud Borrego
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Correspondence to Salud Borrego.

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Ruiz-Ferrer, M., Torroglosa, A., Luzón-Toro, B. et al. Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung’s disease. J Mol Med 89, 471–480 (2011). https://doi.org/10.1007/s00109-010-0714-2

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  • DOI: https://doi.org/10.1007/s00109-010-0714-2

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