Splicing Alterations Associated with Multiple Homozygous TNXB Variants in a Patient with Suspected Classical-Like...

Ehlers-Danlos syndrome (EDS) comprises a heterogeneous group of heritable connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Classical-like EDS (clEDS) is typically associated with pathogenic biallelic variants in TNXB. The patient showed generalized joint hypermobility involving the knees, fingers, elbows, and feet, along with soft, pliable skin. She had a history of three dislocations of the right elbow and one dislocation of the left elbow. She also reported orthostatic dizziness and chronic constipation, suggesting possible autonomic nervous system involvement. Cardiovascular assessment detected mild mitral valve regurgitation. Based on these findings, hypermobility syndrome or EDS was suspected, and gene panel testing was conducted for classical and pseudo-classical EDS. The patient was found to be homozygous for multiple TNXB (NM_001365276.2) variants, including c.11,925 + 6 C > G, c.12,156 C > G (p. Arg4052=), c.12,210 + 5G > A, c.12,210 + 39dup, and c.12,210 + 43T > G, and also carried a mosaic variant c.8012 C > T (p. Ala2671Val). To investigate the potential effects of these variants on splicing, RT-PCR using peripheral blood RNA and in vitro minigene splicing assays were performed. Analysis of the patient-derived cDNA and cDNA derived from mutant minigenes revealed alternative splicing leading to intron 40 retention alongside the normal transcript. Intron 40 retention was more pronounced with multiple variants and was most prominently associated with c.12,210 + 5G > A in the minigene assays. These results suggest that c.12,210 + 5G > A can influence TNXB splicing, generating in-frame transcripts. The aberrant transcripts are predicted to affect the fibrinogen C-terminal domain. Although the clinical significance of these variants remains uncertain, these findings highlight the importance of integrating population frequency data, clinical assessment, and functional analyses when interpreting uncertain TNXB variants in patients with suspected clEDS.