Publicação
Splicing mutations: making sense with antisense therapy
| Resumo: | Galactose-1-phosphate uridylyltransferase (GALT) transfers a UMP group from UDP-glucose to Gal1P in the second step of the Leloir pathway for galactose metabolism. Pathogenic mutations in the GALT gene cause deficient or absent activity of the enzyme and result in Classical Galactosemia. Mutational analysis of 27 Portuguese patients confirmed Q188R as the prevalent molecular defect (≈60%), and revealed the intronic variation c.820+13a>g (IVS8+13a>g) as the second most frequent mutation, accounting for 12.5% of the mutant alleles. In silico analysis revealed that the presence of the c.820+13a>g variation activates a cryptic splicing donor site and seems to create a strong ESE motif for the binding of the SRSF1 protein. A minigene-based approach was used to analyze the effects of this presumptive pre-mRNA splicing mutation. The pSPL3 vector containing either the genomic wild-type or mutant fragments was transfected into COS-7 and Hek293 cell lines. 24h after transfection, RNA was purified and amplified by RT-PCR. Direct sequencing analysis of the reaction products clearly showed that c.820+13a>g favors the next GT dinucleotide (c.820+14_15) to be used as a new 5’ splice donor site, leading to the inclusion of the first 13 nucleotides of intron 8 in the coding sequence, thus inducing a frameshift which generates a premature stop codon 17 amino acids downstream (p.D274fsX291). Additionally, in vivo studies demonstrated that, contrarily to the control individuals, a homozygous patient for c.820+13a>g mutation only presented alternative transcripts with the inclusion of the first 13 nucleotides of intron 8. Accordingly, c.820+13a>g may be considered indeed a disease-causing mutation, being the first intronic variation in the GALT gene whose molecular mechanism was elucidated. In order to revert this alternative splicing, an antisense oligonucleotide approach was attempted, using a LNA (locked nucleic acid) that was cotransfected with the minigenes in COS-7 and Hek293 cells; however, this preliminary experience was not effective, so new alternatives will be developed. |
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| Autores principais: | Lourenço, Sílvia Pires, 1990- |
| Assunto: | Biologia molecular Mutação Splicing alternativo Teses de mestrado - 2012 |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Galactose-1-phosphate uridylyltransferase (GALT) transfers a UMP group from UDP-glucose to Gal1P in the second step of the Leloir pathway for galactose metabolism. Pathogenic mutations in the GALT gene cause deficient or absent activity of the enzyme and result in Classical Galactosemia. Mutational analysis of 27 Portuguese patients confirmed Q188R as the prevalent molecular defect (≈60%), and revealed the intronic variation c.820+13a>g (IVS8+13a>g) as the second most frequent mutation, accounting for 12.5% of the mutant alleles. In silico analysis revealed that the presence of the c.820+13a>g variation activates a cryptic splicing donor site and seems to create a strong ESE motif for the binding of the SRSF1 protein. A minigene-based approach was used to analyze the effects of this presumptive pre-mRNA splicing mutation. The pSPL3 vector containing either the genomic wild-type or mutant fragments was transfected into COS-7 and Hek293 cell lines. 24h after transfection, RNA was purified and amplified by RT-PCR. Direct sequencing analysis of the reaction products clearly showed that c.820+13a>g favors the next GT dinucleotide (c.820+14_15) to be used as a new 5’ splice donor site, leading to the inclusion of the first 13 nucleotides of intron 8 in the coding sequence, thus inducing a frameshift which generates a premature stop codon 17 amino acids downstream (p.D274fsX291). Additionally, in vivo studies demonstrated that, contrarily to the control individuals, a homozygous patient for c.820+13a>g mutation only presented alternative transcripts with the inclusion of the first 13 nucleotides of intron 8. Accordingly, c.820+13a>g may be considered indeed a disease-causing mutation, being the first intronic variation in the GALT gene whose molecular mechanism was elucidated. In order to revert this alternative splicing, an antisense oligonucleotide approach was attempted, using a LNA (locked nucleic acid) that was cotransfected with the minigenes in COS-7 and Hek293 cells; however, this preliminary experience was not effective, so new alternatives will be developed. |
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