Assessing the clinical significance of a novel rare variant in Loeys-Dietz syndrome by combining AI-driven modelling and cell biology

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Abstract Loeys–Dietz syndrome (LDS) is an autosomal dominant connective-tissue disorder caused by genetic variants in TGF-β pathway genes, most often TGFBR1/2 . While pathogenic TGFBR2 mutations usually cluster in the kinase domain and disrupt SMAD signaling, the real challenge in accurate genetic testing is separating the variants that truly affect TGFBR2 function from the rare benign alterations that only look suspicious at first glance. Therefore, there is a pressing need to develop methods that can improve functional variant interpretation. Here, we describe and characterize the functional impact of a novel genetic variant in the TGFBR2 kinase domain (E431K), in a patient with the clinical diagnosis of syndromic genetic aortopathy. We assessed the structural and functional consequences of this variant using AI-driven molecular modelling and in vitro cell-based assays. A high-quality homology-based model of TGFBR2 was generated and computational mutagenesis based on the structural context and evolutionary conservation was used to forecast variant pathogenicity. Relative to wild type, the variant affects protein stability by disrupting intramolecular interactions and likely induces conformational changes that may affect kinase activity and thus TGF-β signalling. This was experimentally confirmed by showing abnormal protein level and alteration of canonical TGF-β pathway activation. Overall, our results establish that the E431K variant leads to aberrant TGF-β signalling and confirm the diagnosis of Loeys–Dietz syndrome type 2 in this patient.