TY - JOUR
T1 - Unusual splice site mutations disrupt FANCA exon 8 definition
AU - Mattioli, Chiara
AU - Pianigiani, Giulia
AU - De Rocco, Daniela
AU - Bianco, Anna Monica Rosaria
AU - Cappelli, Enrico
AU - Savoia, Anna
AU - Pagani, Franco
PY - 2014
Y1 - 2014
N2 - The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position - 3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition.
AB - The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position - 3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition.
KW - Fanconi anemia
KW - Nonsense codon
KW - Nonsense mediated mRNA decay
KW - RNA splice sites
KW - RNA splicing
KW - U1 small nuclear ribonucleoprotein
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U2 - 10.1016/j.bbadis.2014.03.014
DO - 10.1016/j.bbadis.2014.03.014
M3 - Article
C2 - 24704046
AN - SCOPUS:84904785868
VL - 1842
SP - 1052
EP - 1058
JO - Biochimica et Biophysica Acta - Molecular Basis of Disease
JF - Biochimica et Biophysica Acta - Molecular Basis of Disease
SN - 0925-4439
IS - 7
ER -