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Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

Matos L, Canals I, Dridi L, Choi Y, Prata MJ, Jordan P, Desviat LR, Pérez B, Pshezhetsky AV, Grinberg D, Alves S, Vilageliu L - Orphanet J Rare Dis (2014)

Bottom Line: Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Research and Development Unit, INSA, Porto, Portugal. liliana.matos@insa.min-saude.pt.

ABSTRACT

Background: Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.

Methods: In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.

Results: Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.

Conclusions: We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

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Related in: MedlinePlus

Analysis of alternativeHGSNATtranscripts from the mRNA of five MPS IIIC patients by RT-PCR. RT-PCR amplification of mutations c.234 + 1G > A (A), c.633 + 1G > A (B), c.1542 + 4dupA (C), and c.372-2A > G (D), and of the WT allele, using the primers indicated in the schemes by arrows. The different splicing patterns are depicted in the schemes (right), in which the mutations are indicated by black circles. M: marker, C-: negative control, SFCP, −3, − 6, −13, −7: patients. Boxes in the schemes correspond to exons.
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Fig1: Analysis of alternativeHGSNATtranscripts from the mRNA of five MPS IIIC patients by RT-PCR. RT-PCR amplification of mutations c.234 + 1G > A (A), c.633 + 1G > A (B), c.1542 + 4dupA (C), and c.372-2A > G (D), and of the WT allele, using the primers indicated in the schemes by arrows. The different splicing patterns are depicted in the schemes (right), in which the mutations are indicated by black circles. M: marker, C-: negative control, SFCP, −3, − 6, −13, −7: patients. Boxes in the schemes correspond to exons.

Mentions: This study involved four splicing mutations found in five different patients: SFCP and SFC3 (both homozygous for c.234 + 1G > A), SFC6 (compound heterozygous for c.633 + 1G > A and the missense mutation c.1334T > C; p.L445P), SFC13 (compound heterozygous for c.1542 + 4dupA and the nonsense mutation c.1150C > T; p.R384*) and SFC7 (homozygous for c.372-2A > G) (see Table 1). For patients SFC3 (previously reported [25]) and SFCP (novel patient), the RT-PCR analysis of cDNA with primers in exon 1 and 3 revealed a fragment skipping exon 2 (252 bp) due to the presence of the c.234 + 1G > A mutation in homozygosity (Figure 1A). For patient SFC6 (previously described [25]) the cDNA analysis with primers in exons 5 and 7 showed two transcripts: one of a normal length of 172 bp arising from the missense mutation allele (c.1334T > C; p.L445P); and a second one resulting from the skipping of exon 6 (102 bp), due to the splicing alteration c.633 + 1G > A in the other allele (Figure 1B). For a novel patient, SFC13, bearing the intron 15 mutation c.1542 + 4dupA in compound heterozygosity with the c.1150C > T nonsense change in exon 11, the RT-PCR analysis with primers in exons 11 and 16 revealed two aberrant transcripts. The first one showed the skipping of exon 15 (395 bp), and in the second, exon 15 was extended for the first 5 nucleotides of intron 15 (478 bp) due to the presence of a cryptic ss in the beginning of this intron (Figure 1C). For the fifth patient, SFC7 (previously reported [25]), who is homozygous for the c.372-2A > G mutation, the transcript analysis using primers in exons 3 and 5 revealed the presence of two transcripts: one of lower size (79 bp) in which exon 4 was skipped; and the other of higher size (189 bp) corresponding to a fragment with the deletion of the first 12 nucleotides of exon 4 due to the use of a cryptic 3’ ss localized downstream of the constitutive one (Figure 1D), which gives rise to the p.[L125_R128del] mutant protein.Figure 1


Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

Matos L, Canals I, Dridi L, Choi Y, Prata MJ, Jordan P, Desviat LR, Pérez B, Pshezhetsky AV, Grinberg D, Alves S, Vilageliu L - Orphanet J Rare Dis (2014)

Analysis of alternativeHGSNATtranscripts from the mRNA of five MPS IIIC patients by RT-PCR. RT-PCR amplification of mutations c.234 + 1G > A (A), c.633 + 1G > A (B), c.1542 + 4dupA (C), and c.372-2A > G (D), and of the WT allele, using the primers indicated in the schemes by arrows. The different splicing patterns are depicted in the schemes (right), in which the mutations are indicated by black circles. M: marker, C-: negative control, SFCP, −3, − 6, −13, −7: patients. Boxes in the schemes correspond to exons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279800&req=5

Fig1: Analysis of alternativeHGSNATtranscripts from the mRNA of five MPS IIIC patients by RT-PCR. RT-PCR amplification of mutations c.234 + 1G > A (A), c.633 + 1G > A (B), c.1542 + 4dupA (C), and c.372-2A > G (D), and of the WT allele, using the primers indicated in the schemes by arrows. The different splicing patterns are depicted in the schemes (right), in which the mutations are indicated by black circles. M: marker, C-: negative control, SFCP, −3, − 6, −13, −7: patients. Boxes in the schemes correspond to exons.
Mentions: This study involved four splicing mutations found in five different patients: SFCP and SFC3 (both homozygous for c.234 + 1G > A), SFC6 (compound heterozygous for c.633 + 1G > A and the missense mutation c.1334T > C; p.L445P), SFC13 (compound heterozygous for c.1542 + 4dupA and the nonsense mutation c.1150C > T; p.R384*) and SFC7 (homozygous for c.372-2A > G) (see Table 1). For patients SFC3 (previously reported [25]) and SFCP (novel patient), the RT-PCR analysis of cDNA with primers in exon 1 and 3 revealed a fragment skipping exon 2 (252 bp) due to the presence of the c.234 + 1G > A mutation in homozygosity (Figure 1A). For patient SFC6 (previously described [25]) the cDNA analysis with primers in exons 5 and 7 showed two transcripts: one of a normal length of 172 bp arising from the missense mutation allele (c.1334T > C; p.L445P); and a second one resulting from the skipping of exon 6 (102 bp), due to the splicing alteration c.633 + 1G > A in the other allele (Figure 1B). For a novel patient, SFC13, bearing the intron 15 mutation c.1542 + 4dupA in compound heterozygosity with the c.1150C > T nonsense change in exon 11, the RT-PCR analysis with primers in exons 11 and 16 revealed two aberrant transcripts. The first one showed the skipping of exon 15 (395 bp), and in the second, exon 15 was extended for the first 5 nucleotides of intron 15 (478 bp) due to the presence of a cryptic ss in the beginning of this intron (Figure 1C). For the fifth patient, SFC7 (previously reported [25]), who is homozygous for the c.372-2A > G mutation, the transcript analysis using primers in exons 3 and 5 revealed the presence of two transcripts: one of lower size (79 bp) in which exon 4 was skipped; and the other of higher size (189 bp) corresponding to a fragment with the deletion of the first 12 nucleotides of exon 4 due to the use of a cryptic 3’ ss localized downstream of the constitutive one (Figure 1D), which gives rise to the p.[L125_R128del] mutant protein.Figure 1

Bottom Line: Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Research and Development Unit, INSA, Porto, Portugal. liliana.matos@insa.min-saude.pt.

ABSTRACT

Background: Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.

Methods: In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.

Results: Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.

Conclusions: We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

Show MeSH
Related in: MedlinePlus