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Title: Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant
Authors: Nicola Cavallari
Dario Balestra
Alessio Branchini
Iva Maestri
Ampaiwan Chuamsunrit
Werasak Sasanakul
Guglielmo Mariani
Franco Pagani
Francesco Bernardi
Mirko Pinotti
University of Ferrara
Mahidol University
Universita degli Studi dell'Aquila
International Centre for Genetic Engineering and Biotechnology
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jul-2012
Citation: Biochimica et Biophysica Acta - Molecular Basis of Disease. Vol.1822, No.7 (2012), 1109-1113
Abstract: 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 30. bp 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, 1. pM), which could trigger coagulation. The low but detectable thrombin generation (352. ±. 55. nM) 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. © 2012 Elsevier B.V.
ISSN: 1879260X
Appears in Collections:Scopus 2011-2015

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