Unusual splice site mutations disrupt FANCA exon 8 definition.
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.
Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology Trieste, Italy.
Chiara
C
Mattioli
Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology Trieste, Italy.
Giulia
G
Pianigiani
Institute for Maternal and Child Health - IRCCS Burlo Garofolo Trieste, Italy.
Daniela
D
De Rocco
Institute for Maternal and Child Health - IRCCS Burlo Garofolo Trieste, Italy.
Anna Monica Rosaria
AM
Bianco
Clinical and Experimental Hematology Unit, G. Gaslini Children's Hospital Genoa, Italy;
Enrico
E
Cappelli
Institute for Maternal and Child Health - IRCCS Burlo Garofolo Trieste, Italy; Department of Medical Sciences, University of Trieste, Trieste, Italy.
Anna
A
Savoia
Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology Trieste, Italy. Electronic address: pagani@icgeb.org.
Franco
F
Pagani
D001483:Base Sequence; D045744:Cell Line, Tumor; D018389:Codon, Nonsense; D005091:Exons; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing; D017411:Ribonucleoproteins, Small Nuclear
D018389:Codon, Nonsense; C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D022821:RNA Splice Sites; D017411:Ribonucleoproteins, Small Nuclear
Fanconi anemia; Nonsense codon; Nonsense mediated mRNA decay; RNA splice sites; RNA splicing; U1 small nuclear ribonucleoprotein