Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA
2007; Elsevier BV; Volume: 81; Issue: 6 Linguagem: Inglês
10.1086/522376
ISSN1537-6605
AutoresAna María Rincón, C. Aguado, Lourdes R. Desviat, Rocío Sánchez-Alcudia, Magdalena Ugarte, Belén Pérez,
Tópico(s)RNA modifications and cancer
ResumoWe describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5′ or 3′ cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients’ fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of 14C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques. We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5′ or 3′ cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients’ fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of 14C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques. In the past few years, special attention has been given in the field of genetic diseases to research on mutations affecting splicing, which generally account for 10%–30% of the total mutant alleles1Krawczak M Ball EV Fenton I Stenson PD Abeysinghe S Thomas N Cooper DN Human gene mutation database—a biomedical information and research resource.Hum Mutat. 2000; 15: 45-51Crossref PubMed Scopus (203) Google Scholar and for which novel pharmacological and molecular therapies have begun to be tested.2Tazi J Durand S Jeanteur P The spliceosome: a novel multi-faceted target for therapy.Trends Biochem Sci. 2005; 30: 469-478Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Most of the mutations affecting splicing disrupt conserved sequences at the exon-intron junctions—namely, the 5′ donor site, the 3′ acceptor site, the polypyrimidine tract and the branch-point sequence—with different consequences (exon skipping, activation of cryptic splice sites, etc.) depending on the local sequence context.3Krawczak M Reiss J Cooper DN The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.Hum Genet. 1992; 90: 41-54Crossref PubMed Scopus (1121) Google Scholar, 4Buratti E Baralle M Baralle FE Defective splicing, disease and therapy: searching for master checkpoints in exon definition.Nucleic Acids Res. 2006; 34: 3494-3510Crossref PubMed Scopus (172) Google Scholar, 5Pagani F Baralle FE Genomic variants in exons and introns: identifying the splicing spoilers.Nat Rev Genet. 2004; 5: 389-396Crossref PubMed Scopus (463) Google Scholar Some mutations affect less well-conserved auxiliary splicing sequences—that is, exonic and intronic splicing enhancers or silencers—which are recognized by specific SR proteins.6Cartegni L Chew SL Krainer AR Listening to silence and understanding nonsense: exonic mutations that affect splicing.Nat Rev Genet. 2002; 3: 285-298Crossref PubMed Scopus (1742) Google Scholar Other types of mutations, rather than disrupting conserved splice sites, create novel ones that are erroneously used by the splicing machinery, resulting in the generation of aberrant transcripts.4Buratti E Baralle M Baralle FE Defective splicing, disease and therapy: searching for master checkpoints in exon definition.Nucleic Acids Res. 2006; 34: 3494-3510Crossref PubMed Scopus (172) Google Scholar, 5Pagani F Baralle FE Genomic variants in exons and introns: identifying the splicing spoilers.Nat Rev Genet. 2004; 5: 389-396Crossref PubMed Scopus (463) Google Scholar These mutant-activated splice sequences generally occur deep in introns, causing the abnormal inclusion of intron sequences (pseudoexons) in the mRNA.7Lacerra G Sierakowska H Carestia C Fucharoen S Summerton J Weller D Kole R Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.Proc Natl Acad Sci USA. 2000; 97: 9591-9596Crossref PubMed Scopus (127) Google Scholar, 8Vetrini F Tammaro R Bondanza S Surace EM Auricchio A De Luca M Ballabio A Marigo V Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.Hum Mutat. 2006; 27: 420-426Crossref PubMed Scopus (37) Google Scholar The true prevalence of this type of mutations is probably underestimated because few laboratories analyze intron sequences far from coding regions, and, in cDNA, the corresponding transcripts (usually with a frameshift and a premature termination codon [PTC]) are degraded by the nonsense-mediated mRNA decay (NMD) mechanism. NMD is a well-conserved mechanism that occurs naturally in cells and that actively degrades PTC-bearing transcripts, thus preventing the generation of truncated proteins that are potentially toxic to cells.9Maquat LE Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics.Nat Rev Mol Cell Biol. 2004; 5: 89-99Crossref PubMed Scopus (949) Google Scholar Alleles with intronic mutations activating cryptic splice sites are particularly amenable to therapeutic correction if use of the aberrant splice sites can be blocked, because the wild-type splice sites remain intact, thus retaining the potential for normal splicing. In this respect, antisense oligonucleotides have been used successfully to restore normal splicing in several disease models, such as β-thalassemia/HbE disorder,10Suwanmanee T Sierakowska H Fucharoen S Kole R Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder.Mol Ther. 2002; 6: 718-726Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar cystic fibrosis,11Friedman KJ Kole J Cohn JA Knowles MR Silverman LM Kole R Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides.J Biol Chem. 1999; 274: 36193-36199Crossref PubMed Scopus (122) Google Scholar ocular albinism type I,8Vetrini F Tammaro R Bondanza S Surace EM Auricchio A De Luca M Ballabio A Marigo V Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.Hum Mutat. 2006; 27: 420-426Crossref PubMed Scopus (37) Google Scholar and ataxia telangiectasia.12Du L Pollard JM Gatti RA Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides.Proc Natl Acad Sci USA. 2007; 104: 6007-6012Crossref PubMed Scopus (82) Google Scholar Antisense oligonucleotides modulate the splicing pattern by steric hindrance of the recognition and binding of the splicing apparatus to the selected cryptic sequences, thus forcing the machinery to use the natural sites. This strategy has also been used in Duchenne muscular dystrophy to force the skipping of a deleterious exon containing a premature stop codon.13Aartsma-Rus A Janson AA Kaman WE Bremmer-Bout M den Dunnen JT Baas F van Ommen GJ van Deutekom JC Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients.Hum Mol Genet. 2003; 12: 907-914Crossref PubMed Scopus (221) Google Scholar In this work, we report the identification of three novel deep intronic mutations that lead to the insertion of a pseudoexon or cryptic exon in the mRNA of patients with methylmalonic acidemia (MMA [MIM 251000]) or propionic acidemia (PA [MIM 606054]). These are the two most frequent organic acidemias affecting the propionate oxidation pathway in the catabolism of several amino acids, odd-chain fatty acids, and cholesterol.14Fenton WA Gravel RA Rosenberg LE Disorders of propionate and methylmalonate metabolism.in: Scriver CR Beaudet AL Sly W Valle D The metabolic and molecular bases of inherited disease. McGraw-Hill, New York2001: 2165-2190Google Scholar Both are life-threatening diseases that appear in the neonatal or infantile period and are caused by different gene defects inherited in autosomal recessive fashion and affecting the synthesis or function of two of the major enzymes of the pathway, propionyl coenzyme A (CoA) carboxylase (PCC [EC 6.4.1.3]) and methylmalonylCoA mutase (MCM [EC 5.4.99.2]), or of their coenzymes (biotin and adenosylcobalamin, respectively). Three consecutive enzymatic reactions are responsible for the conversion of proponylCoA to the succinylCoA that enters the Krebs cycle. The first reaction involves PCC that catalyzes the carboxylation of propionylCoA to d-methylmalonylCoA, which is then converted to the l form by a racemase, and, finally, the MCM enzyme catalyzes the isomerization of L-methylmalonylCoA to succinylCoA.14Fenton WA Gravel RA Rosenberg LE Disorders of propionate and methylmalonate metabolism.in: Scriver CR Beaudet AL Sly W Valle D The metabolic and molecular bases of inherited disease. McGraw-Hill, New York2001: 2165-2190Google Scholar Mutations in any of the two genes, PCCA or PCCB, which encode both subunits of the PCC enzyme, cause PA, whereas mutations in the MUT gene, which encodes the MCM enzyme, or in the genes MMAA and MMAB (responsible for the intramitochondrial synthesis of adenosylcobalamin) cause isolated MMA. The molecular bases of these disorders are well known, with >50 different mutations described for each of the PCCA, PCCB, and MUT genes. Missense mutations are the most frequent defects, followed by splicing mutations, which account for 15%–20% of the total alleles.15Desviat LR Perez B Perez-Cerda C Rodriguez-Pombo P Clavero S Ugarte M Propionic acidemia: mutation update and functional and structural effects of the variant alleles.Mol Genet Metab. 2004; 83: 28-37Crossref PubMed Scopus (70) Google Scholar, 16Worgan LC Niles K Tirone JC Hofmann A Verner A Sammak A Kucic T Lepage P Rosenblatt DS Spectrum of mutations in mut methylmalonic acidemia and identification of a common Hispanic mutation and haplotype.Hum Mutat. 2006; 27: 31-43Crossref PubMed Scopus (92) Google Scholar In this work, we describe three genomic alterations—one in the MUT gene, one in the PCCA gene, and one in the PCCB gene—that are responsible for the aberrant insertion of intronic sequences in patients’ mRNA. The intronic pseudoexons aberrantly inserted in the mRNA were targeted with antisense morpholino oligonucleotides (AMOs) that prevent aberrant splicing, thus generating normal mRNA, which is translated into functional protein, achieving therapeutic correction of the defect. The study included fibroblast cell lines from one Spanish patient with MMA described elsewhere17Martinez MA Rincon A Desviat LR Merinero B Ugarte M Perez B Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants.Mol Genet Metab. 2005; 84: 317-325Crossref PubMed Scopus (62) Google Scholar and from two patients from Turkey with PA, one PCCA deficient and the other PCCB deficient. Genetic analysis was performed using fibroblast cell lines as the source of mRNA and genomic DNA (gDNA). Total mRNA was isolated by Tripure Isolation reagent (Roche), and subsequent RT-PCR was done using primers and conditions described elsewhere.17Martinez MA Rincon A Desviat LR Merinero B Ugarte M Perez B Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants.Mol Genet Metab. 2005; 84: 317-325Crossref PubMed Scopus (62) Google Scholar, 18Perez B Desviat LR Rodriguez-Pombo P Clavero S Navarrete R Perez-Cerda C Ugarte M Propionic acidemia: identification of twenty-four novel mutations in Europe and North America.Mol Genet Metab. 2003; 78: 59-67Crossref PubMed Scopus (52) Google Scholar The PCR products were sequenced with the same primers used for amplification, with BigDye Terminator v.3.1 mix and subsequent analysis by capillary electrophoresis on an ABI Prism 3700 Genetic Analyzer (Applied Biosystems). BLAST analysis was used to localize the inserted sequence. Intronic gDNA was amplified using primers located in intron 14 (5′-GTAACCCGTTTACTAGTTGCC-3′ and 5′-CACTATAACATACCTGAAGGG-3′) for the PCCA gene insertion, primers located in intron 5 (5′-TATCTTTCCACAGATAATGCCTC-3′) and intron 6 (5′-AAGCAAGGTTTGAGATGAATGG-3′) for the PCCB gene insertion, and primers located in intron 11 (5′-GGCTTCCAGCTTCATCCATG-3′ and 5′-TGGCACGTGCCTGTAGTACC-3′) for the MUT gene insertion. The insertions and gDNA mutations were described as recommended by the Human Genome Variation Society (HGVS). The DNA mutations are numbered on the basis of cDNA sequence and intronic positions described in Ensembl. The genomic changes were studied in 300 control alleles by restriction analysis with use of NlaIII, BsaAI, and BbsI to detect the PCCA, PCCB, and MUT intronic mutations, respectively. Splice scores of the natural and cryptic donor and acceptor sites were determined using the analysis tools from the Berkeley Drosophila Genome Project (BDGP), and prediction of the presence of exonic splice enhancer or silencer sequences was performed using ESEfinder,6Cartegni L Chew SL Krainer AR Listening to silence and understanding nonsense: exonic mutations that affect splicing.Nat Rev Genet. 2002; 3: 285-298Crossref PubMed Scopus (1742) Google Scholar Rescue-ESE19Fairbrother WG Yeh RF Sharp PA Burge CB Predictive identification of exonic splicing enhancers in human genes.Science. 2002; 297: 1007-1013Crossref PubMed Scopus (847) Google Scholar (RESCUE-ESE Web Server), and PESX20Zhang XH Chasin LA Computational definition of sequence motifs governing constitutive exon splicing.Genes Dev. 2004; 18: 1241-1250Crossref PubMed Scopus (348) Google Scholar (PESXs Server). The 25-mer AMOs were designed, synthesized, and purified by Gene Tools and were targeted to donor or acceptor cryptic splice sites in the pre-mRNA for each of the intronic inserted sequences in accordance with the manufacturer’s criteria.21Morcos PA Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos.Biochem Biophys Res Commun. 2007; 358: 521-527Crossref PubMed Scopus (111) Google Scholar The sequence of the AMOs used is shown in figure 1. Endo-Porter (Gene Tools) was used as the delivery mechanism. For AMO treatment, 4–5×105 fibroblast cells were grown in 6-well plates, and, after overnight culture, different concentrations of AMO with 6-8 βl/ml of Endo Porter were added to the culture medium. Cells were harvested at different times, and mRNA was isolated as described above. As we have described elsewhere,17Martinez MA Rincon A Desviat LR Merinero B Ugarte M Perez B Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants.Mol Genet Metab. 2005; 84: 317-325Crossref PubMed Scopus (62) Google Scholar the affected patient with an MUT mutation is compound heterozygous for the intronic insertion and a splicing mutation in the last nucleotide of exon 10 (c.1808G→A), which produces two aberrant transcripts as a result of the use of cryptic splice sites. For RT-PCR in this patient, we used a forward primer placed at the junction of exons 10 and 11 (5′-GCTATCAAGAGGGTTCATAAATT-3′) and a reverse primer located in exon 13 (5′-CTTAGAAGAAGAGATTTT-3′) to amplify only the allele corresponding to the pseudoexon insertion between exons 11 and 12. In some cases, a forward primer placed at the junction of exon 11 and inserted intronic sequence (5′-TCTTTTCCAGAGTCTCGCTCTTT-3′) was used to selectively amplify the cDNA containing the intronic insertion. For RT-PCR analysis of PCCA- and PCCB-deficient cell lines, we used primers described elsewhere.18Perez B Desviat LR Rodriguez-Pombo P Clavero S Navarrete R Perez-Cerda C Ugarte M Propionic acidemia: identification of twenty-four novel mutations in Europe and North America.Mol Genet Metab. 2003; 78: 59-67Crossref PubMed Scopus (52) Google Scholar PCC activity was assayed as described elsewhere,22Suormala T Wick H Bonjour JP Baumgartner ER Rapid differential diagnosis of carboxylase deficiencies and evaluation for biotin-responsiveness in a single blood sample.Clin Chim Acta. 1985; 145: 151-162Crossref PubMed Scopus (75) Google Scholar and 14C propionate incorporation into acid-precipitable material was determined in intact cells grown in basal medium as propionate metabolism via MCM.23Perez-Cerda C Merinero B Sanz P Jimenez A Garcia MJ Urbon A Diaz Recasens J Ramos C Ayuso C Ugarte M Successful first trimester diagnosis in a pregnancy at risk for propionic acidaemia.J Inherit Metab Dis. 1989; 12: 274-276Crossref PubMed Scopus (15) Google Scholar For the detection of biotin-bound proteins, fibroblasts were harvested by trypsinization and were freeze thawed, and protein concentration in cell extracts was determined by the Bradford assay. Equal amounts of total protein (30–50 βg) from each sample were loaded onto a denaturing 6% polyacrylamide gel. After electrophoresis, proteins were transferred to PVDF membranes (Immobilon-P [Millipore]), and the biotin-containing proteins were detected with an avidin alkaline phosphatase conjugate as described elsewhere.24Clavero S Martinez MA Perez B Perez-Cerda C Ugarte M Desviat LR Functional characterization of PCCA mutations causing propionic acidemia.Biochim Biophys Acta. 2002; 1588: 119-125Crossref PubMed Scopus (33) Google Scholar For evaluation of in vitro splicing, the pSPL3 vector (Life Technologies [Gibco BRL], kindly provided by Dr. B. Andresen) was used. Gene fragments corresponding to each pseudoexon and flanking regions were amplified from patients and from control DNA and were cloned into the TOPO vector (Invitrogen). For the PCCB minigene, the amplified fragment included exon 6. The insert was excised with EcoRI and subsequently was cloned into pSPL3. Clones containing the desired normal and mutant inserts in the correct orientation were identified by restriction-enzyme analysis and automated DNA sequencing. Samples of 2 βg of the wild-type or mutant minigenes were transfected into Hep3B cells by use of Jetpei (Polyplus transfections), in accordance with the manufacturer’s recommendations. At 24–48 h after transfection, cells were harvested, RNA was purified, and RT-PCR analysis was performed using the pSPL3-specific primers SD6 and SA2 (Exon Trapping System [Gibco BRL]). Amplified products were separated by agarose gel electrophoresis and were further analyzed by excising the bands from the gel by means of the Qiaex Gel extraction kit (Qiagen) and subsequent direct sequencing. The three patients with MMA and PA exhibited aberrantly spliced mRNA with an amplified band that was larger than normal after RT-PCR analysis. Direct sequencing of the products obtained by RT-PCR and subsequent BLAST search revealed that the insertions corresponded to intronic sequences flanked by cryptic 5′ and 3′ splice sites, thus resembling a pseudoexon (fig. 1). In all three patients the presence of intronic mutations presumably activates the pseudoexon (table 1), whereas the naturally used adjacent splice sites of the surrounding exons remain functional (fig. 1). These intronic variants were not present in the National Center for Biotechnology (NCBI) dbSNP, and none were found in 300 control alleles analyzed.Table 1Inserted Intronic Sequences, Genomic Intronic Variations, and in Silico Analysis of Genomic ChangeGene (IDaGene identification (ID) numbers correspond to the NCBI Entrez Gene database.)Origin of Inserted SequencemRNA ChangebMutation nomenclature is as recommended by HGVS. GenBank accession numbers NT_007592 (MUT), NM_000282.2 (PCCA), and NM_000532.3 (PCCB) were used. The genomic and transcript sequences were obtained using Ensembl.gDNA ChangebMutation nomenclature is as recommended by HGVS. GenBank accession numbers NT_007592 (MUT), NM_000282.2 (PCCA), and NM_000532.3 (PCCB) were used. The genomic and transcript sequences were obtained using Ensembl.In Silico Effect of gDNA ChangecIn silico effect analyzed by ESEfinder and BDGP.MUT (4594)Intron 11r.1957ins76C→A in position +7 of 5′ donor site of pseudoexon (IVS11-891C→A)Increase in splicing score (from .98 to .99)PCCA (5095)Intron 14r.1284ins84A→G in the middle of pseudoexon (IVS14-1416A→G)Creates SRp40 binding site and eliminates SRp55 binding sitePCCB (5096)Intron 6r.654ins72A→G in position +5 of 5′ donor site of pseudoexon (IVS6+462A→G)Increase in splicing score (from .93 to 1) in 5′ donor site of pseudoexona Gene identification (ID) numbers correspond to the NCBI Entrez Gene database.b Mutation nomenclature is as recommended by HGVS. GenBank accession numbers NT_007592 (MUT), NM_000282.2 (PCCA), and NM_000532.3 (PCCB) were used. The genomic and transcript sequences were obtained using Ensembl.c In silico effect analyzed by ESEfinder and BDGP. Open table in a new tab In the MUT-deficient affected patient, we had previously detected a 76-bp insertion between exons 11 and 12 (r.1957ins76) in the heterozygous state and corresponding to an exon-like region in intron 11.17Martinez MA Rincon A Desviat LR Merinero B Ugarte M Perez B Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants.Mol Genet Metab. 2005; 84: 317-325Crossref PubMed Scopus (62) Google Scholar In controls and patients with other mutations, the insertion transcript could be detected using a specific primer.17Martinez MA Rincon A Desviat LR Merinero B Ugarte M Perez B Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants.Mol Genet Metab. 2005; 84: 317-325Crossref PubMed Scopus (62) Google Scholar The patient’s DNA was found to have a new C→A change in intron 11 at position +7 relative to the inserted sequence (IVS11-891C→A) (fig. 1). The scores of the 5′ and 3′ cryptic splice sites were 0.72 and 0.98, respectively, and the C→A mutation increased the 5′ cryptic splice site to 0.99. Additional in silico analysis revealed no significant changes in splicing regulatory sequences caused by the mutation (no exonic splicing enhancer [ESE] or exonic splicing suppressor [ESS] predicted by Rescue-ESE and PESX programs and just a slight decrease in the scores for SC35, SRp40, and SRp55 by ESEfinder analysis). In the PCCA-deficient affected patient, we have identified an 84-bp insertion between exons 14 and 15 of the PCCA gene (r.1284ins84) in a homozygous state. This 84-bp insertion corresponds to a pseudoexon in intron 14 and is readily detected in patients with mRNA-destabilizing mutations and even at low levels in control cell lines25Campeau E Dupuis L Leclerc D Gravel RA Detection of a normally rare transcript in propionic acidemia patients with mRNA destabilizing mutations in the PCCA gene.Hum Mol Genet. 1999; 8: 107-113Crossref PubMed Scopus (20) Google Scholar but never in a homozygous state. No other sequence changes were detected in the amplified cDNA from the patient. The pseudoexon was amplified from gDNA of the patient and was sequenced, revealing an A→G substitution (IVS14-1416A→G) in the middle of the inserted sequence. In silico analysis with ESEfinder prediction software showed that the change created an SRp40 binding site and eliminated an SRp55 binding site. Rescue-ESE predicted loss of an ESE sequence and creation of two novel ones. No change was predicted by the PESX program. In the PCCB-deficient affected patient, we have identified a new 72-bp insertion between exons 6 and 7 in the PCCB gene (r.654ins72) in a homozygous state, corresponding to an intron 6 region resembling an exon with 3′ and 5′ splice sites with high scores (fig. 1). Direct sequencing of the genomic region identified an A→G substitution at position +5 relative to the inserted sequence (IVS6+462A→G), increasing the cryptic 5′ donor splicing score from 0.93 to 1. Additional in silico analysis predicted creation of an SRp55 site (ESEfinder) and elimination of a putative silencer (PESX). Rescue-ESE predicted no changes. To provide evidence that the observed intronic changes are the cause of the pseudoexon inclusion in the patients’ mRNA, the splicing pattern associated with these changes was further evaluated using minigenes. Minigene constructs with wild-type and mutant pseudoexons and flanking sequences were generated in the pSPL3 vector. The results of splicing analysis after transfection in Hep3B cells are shown in figure 2. The wild-type PCCA and PCCB minigene constructs showed practically total absence of pseudoexon inclusion. The MUT wild-type minigene produced two bands corresponding to the pseudoexon insertion and to a splicing event between the vector splice sites. The mutant constructs resulted in the pseudoexon inclusion in all cases, representing the major transcript for the MUT and PCCB minigenes and part of the transcripts for the PCCA minigene. Sequence analysis confirmed the identity of the transcripts in all cases. The major transcript obtained from the PCCB mutant minigene corresponds to the pseudoexon inclusion along with the insertion of a vector sequence caused by the cloning-derived creation of a 3′ splice acceptor site, which was chosen along with a cryptic 5′ splice site present in the vector (Exon Trapping System [Life Technologies]). To demonstrate that these changes are disease-causing mutations in the patients and to try to rescue PCCA, PCCB, and MUT expression in the patients’ fibroblasts, we have investigated the possibility of redirecting transcript processing by modified AMOs. The AMOs used were complementary to the 5′ or 3′ cryptic splice sites of the intronic sequences inserted (fig. 1), to block access of the splicing machinery to the pre-mRNA. In all three cases, RT-PCR analysis demonstrated that the AMO used abolished the alternatively spliced transcript induced by the mutations (fig. 3). The optimal conditions for AMO treatments were determined in each fibroblast cell line. The exclusion of intronic sequences in all cases was oligonucleotide sequence–specific, since AMO targeted to another gene had no effect on pseudoexon inclusion (data not shown). In untreated cell lines, practically no correctly spliced mRNA was detected, as shown by the lack of band comigrating with that of the control one (fig. 3). The correctly spliced fragment was generated 24 h after treatment of the cell lines with AMO complexed with peptide carrier. The products of aberrant splicing were either absent or present at much lower levels (fig. 3). The identity of the PCR products was always confirmed by sequencing. In the MUT-mutated fibroblast cell line, AMO targeted to the 5′ splice site prevented the inclusion of the intronic sequence 24 h after treatment. The experiments showed dose-dependent correction of splicing (fig. 3A). The larger band containing the intronic insertion was detected with 10 βM AMO but not with 15 and 20 βM AMO. Using a primer located in the junction of exon 11 and the inserted pseudoexon to rescue the larger band, we observed essentially the same results (data not shown). To test the stability of the restored, correctly spliced MUT mRNA, the fibroblast cell line was treated with AMO and was harvested at 1–25 d. In this experiment, the correctly spliced mRNA was still at high levels at 10 d, and trace levels of aberrantly spliced mRN
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