Coexpression of Normal and Mutated CD40 Ligand with Deletion of a Putative RNA Lariat Branchpoint Sequence in X-Linked Hyper-IgM Syndrome

We describe a novel CD40 ligand (CD40L) splicing mutation in a patient with X-linked hyper-IgM syndrome (X-HIM) associated with alternate splicing of exon 3, resulting in the expression of both full-length and exon-3-skipped CD40L mRNA. The mutation is an 8-bp deletion 25 bp upstream of the intron 2/exon 3 junction which overlaps a putative RNA branchpoint, suggesting that it may impair RNA lariat formation. The exon-3-skipped CD40L transcript encodes a truncated protein (CD40LΔE3) encompassing the cytoplasmic, transmembrane, and extracellular stalk domains, but lacking the CD40L receptor binding domain. CD40LΔE3 protein expression was readily detectable in transfected Cos cells by immunofluorescence. In cells cotransfected with CD40LΔE3 and wild-type CD40L, expression of CD40LΔE3 did not inhibit the expression of wild-type CD40L monomers, but strongly inhibited staining by the conformationally sensitive anti-CD40L mAb 5c8, suggesting that CD40LΔE3 acts in a dominant negative manner to inhibit the assembly of functional CD40L trimers. This mechanism may contribute to the pathophysiology of CD40L deficiency in X-HIM patients with leaky splice site mutations.