Functional analysis of three splicing mutations identified in the PMM2 gene: Toward a new therapy for congenital disorder of glycosylation type Ia

Abstract

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG-type Ia-is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256-1G>C and c.640-9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640-15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256-1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640-9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640-15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen.

The Molecular Landscape of Phosphomannose Mutase Deficiency in Iberian Peninsula: Identification of 15 Population-Specific Mutations

“…A large variety of mutations have been identified in our Iberian cohort of unrelated PMM2-CDG patients: 25 (83%) are missense mutations (Briones et al 2002;Quelhas et al 2007), four of them newly reported here (p.Y102C, p.T118S, p.P184T, and p.D209G); one nonsense (p.R123X) (Briones et al 2002), three affecting the splicing of mRNA (IVS3+ 2T>C; IVS3-1G>C and IVS7-9T>C) (Briones et al 2002;Vega et al 2009), and a deletion mediated by an Alu retrotransposition displaying the complete loss of exon 8 (Schollen et al 2007a). Of these 30 identified mutations, 13 have only been reported in our series of patients.…”

“…The primers used for cDNA and gDNA amplifications were designed using the ENSEMBL database (http://www. ensembl.org/index.html; ENSG00000140650) and GenBank accession number NM_000303.2 as described in Vega et al 2009. Amplifications of exons and flanking intronic sequences were performed using the FastStart kit (Roche Applied Sciences, Indianapolis).…”

“…The majority of mutations (81%) are missense changes affecting different amino acid residues; 10% are nonsense or single or multiple base pair deletions which lead to early stop codons or frameshifts and thus to truncated proteins. Ten splicing defects have also been described due either to disruption of conserved sequences at the exon-intron junctions or at the polypyrimidine tract or to an intronic change that activates a pseudoexon sequence or to a deletion mediated by an Alu retrotransposition (Schollen et al 2007a;Vega et al 2009). The most common mutations are p.R141H and p.F119L, representing nearly 88% of mutant alleles described to date, both are probably founder mutations in Northern European countries (Bjursell et al 1998).…”

Comprehensive Genetic and Mutation Analysis of Familial Dementia with Lewy Bodies Linked to 2q35-q36

“…Once regarded as junk DNA, intronic and intergenic sequences are now perceived to contain crucial splicing machinery elements and other gene expression regulating motifs. Recently, several studies have shown that mutations disturbing these important elements can lead to disease [34][35][36][37]. Therefore, we expect that high throughput sequencing of the entire 3.3 Mb candidate region might reveal a more complex mutation associated with DLB in family DR246.…”

Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

“…This enzyme converts mannose-6-phosphate to mannose 1-phosphate and is a key enzyme in the generation of N-linked glycans. An intronic mutation in the PMM2 gene was shown to provoke the exonization of 123 bp of intronic sequence, which was successfully reverted with SSO (Vega et al, 2009). Other pseudoexons activated by mutations have also been described in different glycosylation defects such as TMEM165 deficiency (MIM 614727), affecting a protein located mainly in the Golgi compartment and involved in calcium and pH homeostasis necessary for correct processing of glycan chains (Foulquier et al, 2012).…”