Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant

• Published in: Biochimica et biophysica acta 2012-07, Vol.1822 (7), p.1109-1113
• Main Authors: Cavallari, Nicola, Balestra, Dario, Branchini, Alessio, Maestri, Iva, Chuamsunrit, Ampaiwan, Sasanakul, Werasak, Mariani, Guglielmo, Pagani, Franco, Bernardi, Francesco, Pinotti, Mirko
• Format: Article
• Language: English
• Subjects: Aberrant splicing; Alternative Splicing - genetics; Blood Coagulation - genetics; Child; Coagulation factor deficiency; DNA, Complementary - genetics; DNA, Complementary - metabolism; Exons - genetics; Factor VII - analysis; Factor VII - genetics; Factor VII Deficiency - genetics; Female; Frameshift Mutation - genetics; Genes, Lethal; Homozygote; Human genetic disease; Humans; Male; Point Mutation - genetics; Residual gene expression; RNA Splice Sites - genetics; RNA Splicing - genetics; Thrombin - analysis; Thrombin - genetics; Young Adult
• ISSN: 0925-4439; 0006-3002; 1879-260X
• DOI: 10.1016/j.bbadis.2012.03.001

Changes at the invariable donor splice site +1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6+1G>T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6+1G>T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30bp deleted transcript (8±2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2±0.04%) of protein with appreciable specific activity (48±16% of wt-FVII). Altogether these data indicate that the IVS6+1G>T mutation is compatible with the synthesis of functional FVII molecules (~0.01% of normal, 1pM), which could trigger coagulation. The low but detectable thrombin generation (352±55nM) measured in plasma from an IVS6+1G>T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition. ► +1G donor splice site mutations virtually suppress correct gene expression. ► Complete deficiency of coagulation factor VII (FVII) is lethal. ► The F7 IVS6+1G/T induces exon skipping and use of an in-frame cryptic 5'ss. ► The latter transcript encoded a protein lacking 10 residues in the activation domain. ► The residual activity of this variant might have reverted a lethal condition.