Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria)

Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957–898A>G and c.1957–920C>A identified in the methylmalonyl–coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957–898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957–920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation. Hum Mutat 30:1–7, 2009. © 2009 Wiley-Liss, Inc.     

Assessment of the potential pathogenicity of missense mutations identified in the GTPase‐activating protein (GAP)‐related domain of the neurofibromatosis type‐1 (NF1) gene

Neurofibromatosis type‐1 (NF1) is caused by constitutional mutations of the NF1 tumor‐suppressor gene. Although ～85% of inherited NF1 microlesions constitute truncating mutations, the remaining ～15% are missense mutations whose pathological relevance is often unclear. The GTPase‐activating protein‐related domain (GRD) of the NF1‐encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras‐MAPK (mitogen‐activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1‐GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1‐GRD expression vector and activated Ras was assayed by an enzyme‐linked immunosorbent assay (ELISA). Ten NF1‐GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP‐bound Ras in comparison to wild‐type NF1 protein. The remaining five NF1‐GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild‐type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1‐GRD structure and function. Hum Mutat 33:1687–1696, 2012. © 2012 Wiley Periodicals, Inc.     

AG‐exclusion zone revisited: Lessons to learn from 91 intronic NF1 3′ splice site mutations outside the canonical AG‐dinucleotides

Uncovering frequent motives of action by which variants impair 3′ splice site (3′ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini‐gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722‐11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG‐exclusion zone (AGEZ) between the authentic 3′ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3′ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single‐nucleotide variants upstream of positions ‐3 interrupt the AGEZ. Of these AGEZ‐interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3′ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3′ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position ‐3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice‐neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.     

Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene

Missense, iso‐semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5′ or 3′ splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5′ or 3′ splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.     

Neurofibromin (NF1) genetic variant structure–function analyses using a full‐length mouse cDNA

Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full‐length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full‐length murine cDNA produces a > 250 kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1^?/? cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant‐directed therapeutics.     

Assessment of the functional impact on the pre‐mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.     

Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption

We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.     

U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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

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. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch‐point mapping strategy was also used to investigate further a putative branch‐point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. Hum Mutat 31:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Neurofibromatosis type 1 in two siblings due to maternal germline mosaicism

Neurofibromatosis type 1 (NF1) is caused by loss of function mutations of the NF1 gene, which are de novo in 50% of cases. Although this gene shows one of the highest mutation rates in the human genome, germline mosaicism is very rare in this condition. We describe the molecular analysis of a family in which neurofibromatosis type 1 occurred in two out of four siblings born to unaffected parents. Molecular analysis of the NF1 gene identified in both patients the same splicing mutation c.1392+1G>A, which was absent in parental lymphocytes. Microsatellite analysis showed that the two affected siblings shared the same maternal allele, however a specific PCR‐RFLP assay excluded the presence of the NF1 splicing mutation in multiple maternal tissues. Our molecular and clinical findings are consistent with a germline mosaicism for the NF1 splicing mutation. This is the first case of maternal germline mosaicism for a NF1 mutation characterized so far at the molecular level. Our data confirm that germline mosaicism is rare in neurofibromatosis 1, but it has important implications for genetic counseling.     

Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles

Canals I, Elalaoui SC, Pineda M, Delgadillo V, Szlago M, Jaouad IC, Sefiani A, Chabás A, Coll MJ, Grinberg D, Vilageliu L. Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles. The Sanfilippo syndrome type C [mucopolysaccharidosis IIIC (MPS IIIC)] is caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. We report the first molecular study on several Spanish Sanfilippo syndrome type C patients. Seven Spanish patients, one Argentinean and three Moroccan patients were analysed. All mutant alleles were identified and comprised nine distinct mutant alleles, seven of which were novel, including four missense mutations (p.A54V, p.L113P, p.G424V and p.L445P) and three splicing mutations due to two point mutations (c.633+1G>A and c.1378‐1G>A) and an intronic deletion (c.821‐31_821‐13del). Furthermore, we found a new single nucleotide polymorphism (SNP) (c.564‐98T>C). The two most frequent changes were the previously described c.372‐2A>G and c.234+1G>A mutations. All five splicing mutations were experimentally confirmed by studies at the RNA level, and a minigene experiment was carried out in one case for which no fibroblasts were available. Expression assays allowed us to show the pathogenic effect of the four novel missense mutations and to confirm that the already known c.710C>A (p.P237Q) is a non‐pathogenic SNP. Haplotype analyses suggested that the two mutations (c.234+1G>A and c.372‐2A>G) that were present in more than one patient have a common origin, including one (c.234+1G>A) that was found in Spanish and Moroccan patients.