Journal
HUMAN MUTATION
Volume 36, Issue 5, Pages 504-512Publisher
WILEY-BLACKWELL
DOI: 10.1002/humu.22762
Keywords
SPINK5; LEKTI; ESE; ESS; splicing defect; U1 snRNA
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Funding
- Telethon Foundation [GGP14190]
- Italian Ministry of Health (Ricerca Finalizzata grant ) [RF-2011-02347596]
- Association Athina Ichtyose Monaco
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The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2 splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes.
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