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SMAD4 is a common transcription factor of the BMP\/TGF\u03b2 signalling pathway. In this study, we developed a cell-based functional assay to address the pathogenicity of <jats:italic>SMAD4<\/jats:italic> variants identified in the French HHT cohort.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                     <jats:italic>SMAD4<\/jats:italic> variants were generated by site-directed mutagenesis. A functional assay was developed in a cell line that does not express SMAD4, and the different <jats:italic>SMAD4<\/jats:italic> variants were tested for their capacity to activate the BMP and TGF\u03b2 response using luciferase reporter assays.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Twelve <jats:italic>SMAD4<\/jats:italic> variants were identified and studied. We were able to develop a robust functional assay for these variants. All the expressed variants resulted in loss of function (LOF) in response to BMP9 or TGF\u03b21 stimulation. <jats:italic>SMAD4<\/jats:italic> variants within the MH2 domain expressed SMAD4 mutated proteins that were unable to hetero-oligomerise with other SMADs, which could explain their LOF. Finally, we tested primary human endothelial cells isolated from patients with HHT carrying <jats:italic>SMAD4<\/jats:italic> heterozygous pathogenic variants and observed that they behaved like the control cells at rest or when stimulated with BMP9.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>We developed a SMAD4 functional assay that allows discrimination between benign and pathogenic <jats:italic>SMAD4<\/jats:italic> variants. We demonstrated that the underlying molecular mechanism of this pathogenicity is due mostly to a loss of hetero-oligomerisation. This assay will be transferable to clinical genetic laboratories and will improve the diagnosis of patients with HHT\u2013JPS.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jmg-2025-110797","type":"journal-article","created":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T07:10:15Z","timestamp":1755673815000},"page":"jmg-2025-110797","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":2,"title":["Development of a functional assay for the characterisation of <i>SMAD4<\/i> variants from the French haemorrhagic hereditary telangiectasia cohort"],"prefix":"10.1136","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-7963-3673","authenticated-orcid":false,"given":"Louane","family":"Despas","sequence":"first","affiliation":[{"name":"Biosant\u00e9 unit U1292, Grenoble Alpes University, INSERM, CEA, INSERM, Grenoble, 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