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.

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

CAVALLARI, Nicola
Primo
;
BALESTRA, Dario
Secondo
;
BRANCHINI, Alessio;MAESTRI, Iva;BERNARDI, Francesco
Penultimo
;
PINOTTI, Mirko
Ultimo
2012

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.
2012
Cavallari, Nicola; Balestra, Dario; Branchini, Alessio; Maestri, Iva; Chuamsunrit, A; Sasanakul, W; Mariani, G; Pagani, F; Bernardi, Francesco; Pinotti, Mirko
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1682571
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 17
  • ???jsp.display-item.citation.isi??? 17
social impact