A novel deep intronic COL5A1 variant in an Ehlers-Danlos syndrome family: functional characterization by minigene assay

This study aimed to identify the genetic cause of Ehlers-Danlos syndrome (EDS) in a Chinese family and to evaluate the functional impact of a deep intronic COL5A1 variant using a minigene assay. The genetic analysis was performed using medical whole-exome sequencing, covering 5,398 genes associated with human genetic diseases. A candidate variant was prioritized based on its segregation and potential effect on splicing. To confirm functional effects, wild-type and mutant minigene constructs were created and transfected into HEK293T cells. RT-PCR and sequencing were used to analyze splicing products. A heterozygous intronic COL5A1 variant (NM_000093.5: c.1774–95 C > T) was found in both the proband and her father, but not in the mother. Clinically, affected individuals showed features typical of classic Ehlers-Danlos syndrome (cEDS), including hyperextensible skin, atrophic scars, easy bruising, and joint hypermobility. Functional minigene assays demonstrated that the variant causes abnormal splicing, leading to pseudoexon inclusion, a frameshift (p.Gly592CysfsTer2), and the production of a truncated protein. Our findings provide functional evidence that the deep intronic COL5A1 variant c.1774–95 C > T disrupts normal splicing and is likely pathogenic in cEDS. This discovery broadens the mutational range of COL5A1, highlights the diagnostic value of deep intronic variants, and points to a possible therapeutic target for splice-modulating strategies.