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A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo- exon in CF patients  Lucy Costantino, Laura Claut, Valentina Paracchini,

Published byLeonardo de Santarém Sintra Modified over 6 years ago

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Presentation on theme: "A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo- exon in CF patients  Lucy Costantino, Laura Claut, Valentina Paracchini,"— Presentation transcript:

1 A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo- exon in CF patients  Lucy Costantino, Laura Claut, Valentina Paracchini, Domenico A. Coviello, Carla Colombo, Luigi Porcaro, Patrizia Capasso, Maddalena Zanardelli, Giovanna Pizzamiglio, Dario Degiorgio, Manuela Seia  Journal of Cystic Fibrosis  Volume 9, Issue 6, Pages (December 2010) DOI: /j.jcf Copyright © 2010 European Cystic Fibrosis Society Terms and Conditions

2 Fig. 1 RT-PCR analysis of CFTR mRNA from nasal brushing of two patients and two control subjects. After amplification of cDNA, only one band of 604bp was obtained from the control subjects n. 1, 4 while an additional band of 708bp was obtained from CFTR patients n. 2, 3. (a) The 708bp RT-PCR product was excised from agarose gel and the sequence determined and cloned in the plasmid vector. Sequencing results reveal an aberrant mRNA with a 104bp insertion between exon 10 and 11 that resembles a cryptic exon. (b) The ladder used in this experiment was the 50bp DNA Ladder, Invitrogen. Journal of Cystic Fibrosis 2010 9, DOI: ( /j.jcf ) Copyright © 2010 European Cystic Fibrosis Society Terms and Conditions

3 Fig. 2 The c bp A>G substitution is indicated by the letter “g”. Boxes containing lined bars represent the 104 bp inserted sequences of intron 10. The intronic sequence is represented by 5′-a-b-c-d-3′. The mutation creates the putative donor splice site that is underlined. Journal of Cystic Fibrosis 2010 9, DOI: ( /j.jcf ) Copyright © 2010 European Cystic Fibrosis Society Terms and Conditions

4 Fig. 3 The figure shows the results obtained by ESEfinder 3 web interface (Cartegni at al, 2003). We analyzed the new mutation c bp A>G using four sequence motifs that predict functional ESEs recognized by the SR proteins (SF2/ASF, SC35, SRp40, SRp55) and the splicing factors (5SS_U2_human, 3SS_U2_human, BranchSite). The mutation occurring in intron 10 generates: a) a new SRp55 motif (from 4.55 to 5.16) and b) a new 5SS_U2_human (from to ), the other motif score remains unchanged. ⋆Indicates the sequence (atgaatagtacgtac) of putative donor splice site. Journal of Cystic Fibrosis 2010 9, DOI: ( /j.jcf ) Copyright © 2010 European Cystic Fibrosis Society Terms and Conditions

5 Fig. 4 Agarose gel electrophoresis of amplified cDNA fragment spanning exons 7–13 PCR, in patients (1–7) and normal controls (8–14). The 730bp fragment lacks 183 nucleotides of exon 9. Patients 4 and 5 carrying the new mutation c bp A>G presented the extra band and did not reveal the band without exon skipping. The ladder used in this experiment was the 50bp DNA Ladder, Invitrogen. Journal of Cystic Fibrosis 2010 9, DOI: ( /j.jcf ) Copyright © 2010 European Cystic Fibrosis Society Terms and Conditions

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