In-frame Transcripts Research Articles

Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms.

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10-8 Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.

Investigating Synthetic Oligonucleotide Targeting of Mir31 in Duchenne Muscular Dystrophy.

Exon-skipping via synthetic antisense oligonucleotides represents one of the most promising potential therapies for Duchenne muscular dystrophy (DMD), yet this approach is highly sequence-specific and thus each oligonucleotide is of benefit to only a subset of patients. The discovery that dystrophin mRNA is subject to translational suppression by the microRNA miR31, and that miR31 is elevated in the muscle of DMD patients, raises the possibility that the same oligonucleotide chemistries employed for exon skipping could be directed toward relieving this translational block. This approach would act synergistically with exon skipping where possible, but by targeting the 3’UTR it would further be of benefit to the many DMD patients who express low levels of in-frame transcript. We here present investigations into the feasibility of combining exon skipping with several different strategies for miR31-modulation, using both in vitro models and the mdx mouse (the classical animal model of DMD), and monitoring effects on dystrophin at the transcriptional and translational level. We show that despite promising results from our cell culture model, our in vivo data failed to demonstrate similarly reproducible enhancement of dystrophin translation, suggesting that miR31-modulation may not be practical under current oligonucleotide approaches. Possible explanations for this disappointing outcome are discussed, along with suggestions for future investigations.

Novel mutations in EVC cause aberrant splicing in Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome (EvC) is a rare autosomal recessive disorder characterized by disproportionate chondrodysplasia, postaxial polydactyly, nail dystrophy, dental abnormalities and in a proportion of patients, congenital cardiac malformations. Weyers acrofacial dysostosis (Weyers) is another dominantly inherited disorder allelic to EvC syndrome but with milder phenotypes. Both disorders can result from loss-of-function mutations in either EVC or EVC2 gene, and phenotypes associated with the two gene mutations are clinically indistinguishable. We present here a clinical and molecular analysis of a Chinese family manifested specific features of EvC syndrome. Sequencing of both EVC and EVC2 identified two novel heterozygous splice site mutations c.384+5G>C in intron 3 and c.1465-1G>A in intron 10 in EVC, which were inherited from mother and father, respectively. In vitro minigene expression assay, RT-PCR and sequencing analysis demonstrated that c.384+5G>C mutation abolished normal splice site and created a new cryptic acceptor site within exon 4, whereas c.1465-1G>A mutation affected consensus splice junction site and resulted in full exon 11 skipping. These two aberrant pre-mRNA splicing processes both produced in-frame abnormal transcripts that possibly led to abolishment of important functional domains. To our knowledge, this is the first report of EVC mutations that cause EvC syndrome in Chinese population. Our data revealed that EVC splice site mutations altered splicing pattern and helped elucidate the pathogenesis of EvC syndrome.

A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma.

NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1–585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3′ acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1–585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity.

Abstract 479: Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma

Abstract Ewing sarcoma (ES) is a highly aggressive cancer of the bone and soft tissue. In ∼85% of ES tumors the primary oncogenic event is a t(11:22)(q24:q12) translocation that generates a fusion of the 5′ end of EWSR1 and the 3′ end of FLI1 referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. The most common EWS-FLI1 transcripts fuse either exon 7 of EWSR1 to exon 6 of FLI1 (a type I or a 7/6 fusion), or fuse exon 7 of EWSR1 to exon 5 of FLI1 (a type II or 7/5 fusion). In an estimated 40% of EWS-FLI1 driven tumors the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. Using an assay of EWS-FLI1 activity and genome-wide siRNA screening we have identified RNA processing as a therapeutic vulnerability in ES. Parallel genome-wide siRNA-mediated RNAi screens were conducted in ES TC32 cell lines expressing a luciferase (luc) reporter protein driven by either the promoter of the EWS-FLI1 target gene NR0B1 (TC32-NR0B1-luc) or the CMV promoter (TC32-CMV-luc). The top gene ontology terms associated with the 28 priority candidate genes that when silenced induced a differential decrease in the TC32-NR0B1-luc signal versus the TC32-CMV-luc signal were mRNA splicing (p-value = 1.42E-08) and mRNA processing (p-value = 2.32E-07). To investigate the mechanistic basis for the identification of specific RNA processing proteins as required for the activity of EWS-FLI1 we focused on two lead candidate genes, the heterogeneous nuclear ribonucleoprotein H1, HNRNPH1, and the core splicing factor, SF3B1. Using PCR analysis we determined that HNRNPH1 is required for the splicing of EWS-FLI1 fusion transcripts expressed in ES cells in which the chromosome 22 breakpoint retains EWSR1 exon 8, specifically in TC32 and SKNMC ES cells. We also show ES cell lines harboring 7/ 6 (TC32, SKNMC, and TC71) or 7/ 5 (RD-ES) EWS-FLI1 fusions are all sensitive to the loss-of-function of SF3B1. Quantitative RT-PCR, immunoblot, and whole transcriptome analysis show that disrupted splicing of the EWS-FLI1 transcript alters its expression and reverses the expression of a significant proportion of genes that are targets of EWS-FLI1. These observations were confirmed in four ES cell lines using the splicing inhibitor Pladienolide B. Our results provide the basis for a novel strategy to target fusion oncogenes by interfering with RNA processing. This study has implications for the treatment of ES through inhibition of proteins required for expression of the EWS-FLI1 transcript and identifies a candidate lead compound for further clinical development. Our findings may also open up strategies for treatment of other cancers driven by fusion oncogenes. Citation Format: Patrick J. Grohar, Suntae Kim, Sara Haddock, Guillermo Rangel Rivera, Matt Harlow, Nichole K. Maloney, Konrad Huppi, Kristen Gehlhaus, Magdalena Grandin, Carleen Klumpp-Thomas, Eugen Buehler, Lee J. Helman, Scott E. Martin, Natasha J. Caplen. Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2015-479

Abstract 4964: Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia

Abstract Mediator complex subunit 12 (MED12) is part of a Mediator, which is a large, highly conserved protein complex that participates in the regulation of general as well as gene-specific transcription. First somatic MED12 mutations were identified when exome sequencing revealed highly specific mutations in exon 2 in 70% of uterine leiomyomas studied. Additional mutations have subsequently been observed in exon 1, which further highlight the role of MED12 in these tumors. Functional analyses have indicated that exon 1 and exon 2 mutations lead to a diminished interaction between MED12 and other components of the Mediator kinase module, reduced Mediator-associated CDK kinase activity, and a unique global gene expression pattern. Recently, we observed similar mutations in chronic lymphocytic leukemia (CLL). Mutations were observed in 5% of the patients and the positive mutation status was associated with well-known poor prognosis markers in CLL. All the observed mutations both in uterine leiomyomas and CLL have been missense mutations or small insertions or deletions leading to an in-frame transcript. Interestingly, the first MED12 mutation in a patient with T-cell acute lymphoblastic leukemia (T-ALL) was recently identified through exome sequencing. This is a nonsense mutation affecting the mutation hotspot observed in CLL. It is also the first nonsense mutation identified in the MED12 N-terminal mutation hotspot area in any tumor type. The patient has received bone marrow transplantation and is now in complete remission, but small amounts of cancer cells were collected before the treatment. RNA was extracted from the cells, and cDNA sequencing revealed that only the mutant allele is expressed. We have now generated a construct with the MED12 specific mutation and transfected it into HEK293 Flp-In cells. Wild-type MED12 is being used as a control. Western blotting with N- and C-terminal MED12 antibodies indicates that the mutant protein is being produced but that the N-terminal part is lacking. We now aim to further characterize the function of this highly unusual mutation. Mutant and wild type protein will be produced for proteome-wide mass spectrometry analyses to detect possible effects on protein-protein interactions and also to gain more information on the translation start site. Also transcriptome-wide expression data will be produced to study the effect of the mutation on global gene expression profile. Overall, a mutation affecting the 5′ terminus of MED12 has now been identified in yet another cancer type, T-ALL. Our preliminary analyses indicate that the observed nonsense mutation escapes nonsense-mediated RNA decay and leads to a truncated protein product. Besides providing additional knowledge on the role of MED12 mutations in cancer, this exceptional mutation has potential to produce novel information on the normal MED12 function. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Salla Keskitalo, Mikko Turunen, Heikki Kuusanmäki, Netta Mäkinen, Satu Mustjoki, Caroline Heckman, Markku Varjosalo, Pia Vahteristo. Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4964. doi:10.1158/1538-7445.AM2015-4964

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis.

Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology (n = 26), short inserted sequences (n = 8), or paralogous repeats (n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX, SMOC2-PROX1, and PIEZO2-MTA1, which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0–4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation.

143. Off-Target Analysis of a rAAV-U7snRNA Vector Used for the Treatment of Duchenne Patients By Exon Skipping

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1 male in 5000 and caused by mutations in the gene encoding for the Dystrophin protein. So far, there are no strongly effective treatments but gene-based therapies are currently being developed. Among them, the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in shorter in-frame transcripts that are translated into functionally active Dystrophin. Exon skipping can be achieved using modified U7-small nuclear RNA (snRNA) in which a therapeutic antisense oligonucleotide (AO) is cloned. Such optimized U7snRNA, called U7.OPT, can be packaged in recombinant vectors derived from Adeno-Associated Virus (rAAV), allowing a long-lasting repair of the dystrophin messenger after one single administration.We recently published the high ability of a rAAV8.U7.OPT vector to transduce the skeletal muscle but also the liver and restore the open reading frame of the dystrophin transcript in GRMD dogs muscles injected in one forelimb. We are now moving forward to a phase I/II clinical trial in DMD patients treatable by skipping of the exon 53 of the dystrophin messenger.Because an abundant presence of the therapeutic U7.OPT RNA can in theory lead to partial hybridization to non-targeted messenger(s) with unwanted side effects, we looked for possible off-target events. Primary human myotubes and hepatocytes, untransduced or transduced with our clinical rAAV.U7.OPT vector, served as our in vitro model system. The skipping of exon 53 of the human dystrophin messenger upon vector transduction was validated. In silico analysis using the miRanda algorithm identified some gene candidates expressed in liver and muscle, i.e. messengers which could be the target of our dystrophin-specific AO. Three of them were analyzed by RT-PCR and RTqPCR and showed not detectable exon skipping events or differential expression. Other candidates are currently analyzed. Simultaneously, further investigation included a full transcriptomic analysis by RNA sequencing comparing the samples obtained from untransduced vs transduced primary cells. Preliminary results revealed differential gene expression patterns between the two types of samples. We are now exploring the respective impact of the rAAV transduction itself vs the AO effect per se to finally identity potential off-target events.The complete data will be presented and discussed in the context of our future clinical trial in DMD patients.This project is supported by AFM (Association Francaise contre les Myopathies) and ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by research and innovation aid from the French public agency, BpiFrance.

Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45.

Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone.

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Annotation of pseudogenous gene segments by high-throughput sequencing of rearranged lymphocyte receptor loci (TECH1P.844)

Abstract The adaptive immune system generates a huge breadth of antigen-specific T (TCRs) and B cell receptors (BCRs) by combinatoric shuffling of gene segments. Lymphocytes undergo somatic recombination of variable (V), diversity (D), and joining (J) region gene segments, with deletions and non-templated insertions at the junctions, collectively encoding the CDR3 region. Many of the gene segments are annotated as non-functional; however, full annotation of genes as functional, pseudogene or ORF has proven elusive. We used high-throughput sequencing to identify 100K’s of rearranged TCRB and IGH receptors from a large cohort of healthy adults. We then annotated V and J gene segments as functional or pseudogenes based on selection. Random chance predicts that less than one-third of somatic rearrangements will lead to in-frame transcripts, but only lymphocytes with a productive receptor are selected. Non-functional rearrangements (out-of-frame or pseudogene) persist only as a second allele in mature T and B cells. Based on these data we annotated the functional status of each gene segment based on the realized ratio of productive to non-productive rearrangements. Some gene segments are currently mis-annotated: in TCRB 2 presumed-functional gene segments are pseudogenes, in IgH 3 presumed-functional genes are pseudogenes and 1 annotated pseudogene is functional. Additionally, in both loci we identified cases of gene segment functional/pseudogene status segregating among healthy subjects.

Proteolytic Processing of the Extracellular Scaffolding Protein LEV-9 Is Required for Clustering Acetylcholine Receptors

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.

In vitro analysis of splice site mutations in the CLCN1 gene using the minigene assay

Mutations in the chloride channel gene CLCN1 cause the allelic disorders Thomsen (dominant) and Becker (recessive) myotonia congenita (MC). The encoded protein, ClC-1, is the primary channel that mediates chloride (Cl-) conductance in skeletal muscle. Mutations in CLCN1 lower the channel's threshold voltage, leading to spontaneous action potentials that are not coupled to neuromuscular transmission and resulting in myotonia. Over 120 mutations in CLCN1 have been described, 10% of which are splicing defects. Biological specimens suitable for RNA extraction are not always available, but obtaining genomic DNA for analysis is easy and non-invasive. This is the first study to evaluate the pathogenic potential of novel splicing mutations using the minigene approach, which is based on genomic DNA analysis. Splicing mutations accounted for 23% of all pathogenic variants in our cohort of MC patients. Four were heterozygous mutations in four unrelated individuals, belonging to this cohort: c.563G>T in exon 5; c.1169-5T>G in intron 10; c.1251+1G>A in intron 11, and c.1931-2A>G in intron 16. These variants were expressed in HEK 293 cells, and aberrant splicing was verified by in vitro transcription and sequencing of the cDNA. Our findings confirm the need to further investigate the nature of rearrangements associated with this class of mutations and their effects on mature transcripts. In particular, splicing mutations predicted to generate in-frame transcripts may generate out-of-frame mRNA transcripts that do not produce functional ClC-1. Clinically, incomplete molecular evaluation could lead to delayed or faulty diagnosis.

What Can We Learn From Clinical Trials of Exon Skipping for DMD?

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK's Phase III clinical trial ({"type":"clinical-trial","attrs":{"text":"NCT01254019","term_id":"NCT01254019"}}NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to placebo.1 On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA) has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature.2 The news came as a great disappointment to the scientific community and more specifically to the families and foundations that had followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA's view of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching impact beyond exon skipping therapy in DMD.

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2 : Use of potential alternative start sites and implications for predicting variant pathogenicity

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site.

P.20.12 Gene therapy of Duchenne Muscular Dystrophy using rAAV vectors: Patterns of dystrophin expression and histological improvements

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. About 60% of the affected boys are treatable by exon skipping, i.e. the selective removal of exons flanking an out-of frame mutation in the dystrophin messenger, which results in shorter in-frame transcripts that are translated into functionally active dystrophin. Recombinant AAV-derived vectors carrying modified U7snRNA sequences are promising tools to allow an efficient exon skipping. In order to prepare a clinical trial in non-ambulant DMD patients using a rAAV8-U7 vector injected in the forearm, we injected in one forelimb a total of 21 Golden Retriever Muscular Dystrophy (GRMD) dogs. All muscles of the limbs were observed about 14weeks later for dystrophin expression and histological patterns Depending on the injected dose and the injection protocol, we showed low to high (up to 80%) dystrophin expression levels in muscles of the injected legs depending of the vector dose. Improvement of the histological pattern was assessed by histomorphological analysis of the immunological detection of dystrophin expression, sclerosis (immunopexoxydase against Collagen I), regeneration (developmental Myosin Heavy Chain), calcium accumulation, anisocytosis, macrophagic infiltration, and lymphocytic infiltration. The dystrophin expression was characterized by a diffuse pattern with single or small groups of positive fibers in most fascicles. A significant decrease of sclerosis in muscles exhibiting more than 60% of dystrophin positive fibers and an important decrease of regeneration (in muscles containing up to 30% of dystrophin), was observed. These results show the efficiency of the rAAV8-U7 strategy to restrict the natural degradation of the dystrophic muscle. Supported by the Association Française contre les Myopathies and Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by OSEO.

P.20.13 Gene therapy of Duchenne Muscular Dystrophy using rAAV vectors: Exon skipping and microdystrophin approaches in GRMD dogs

The Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. About 60% of the affected boys are treatable by exon skipping, i.e. the selective removal of exons flanking an out-of frame mutation in the dystrophin messenger, which results in shorter in-frame transcripts that are translated into functionally active dystrophin. Recombinant adeno-associated virus (rAAV) derived vectors carrying modified U7snRNA sequences are promising tools to allow an efficient exon skipping. In order to prepare a clinical trial in non-ambulant DMD patients using a rAAV8-U7 vector injected in the forearm, we injected in one forelimb a total of 21 Golden Retriever Muscular Dystrophy (GRMD) dogs to determine the therapeutic dose of the product and establish safety of the treatment. Results demonstrated high dystrophin expression levels (up to 80%) in muscles of dogs injected with 2.5E13 vg/kg. Strength improvement and correction of histological, NMR imaging and spectroscopy indices were demonstrated in muscles with more than 40% dystrophin expression. Finally, since a proportion of DMD patients are not amenable to treatment by exon skipping, we also evaluated a therapy based on the transfer of a rAAV8 vector encoding a sequence-optimized microdystrophin (MD1), which could eventually benefit all DMD patients. Three GRMD dogs were injected in one forelimb with this vector, exactly as in the U7-GRMD dogs. Treated limbs of these dogs exhibited consistent MD1 expression, which correlated with local correction of the pathology and strength improvement. The complete results obtained during these two pre-clinical trials will be presented and a comparison of these two therapeutic approaches will be discussed. Supported by the Association Francaise contre les Myopathies, the UK Muscular Dystrophy Campaign and by Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by OSEO.

Inversion-mediated gene fusions involving NAB2-STAT6 in an unusual malignant meningioma

Background:Meningiomas are the most common primary intracranial tumours, with ∼3% meeting current histopathologic criteria for malignancy.Methods:In this study, we explored the transcriptome of meningiomas using RNA-Seq.Results:Inversion-mediated fusions between two adjacent genes, NAB2 and STAT6, were detected in one malignant tumour, creating two novel in-frame transcripts that were validated by RT–PCR and Sanger sequencing.Conclusion:Gene fusions of NAB2-STAT6 were recently implicated in the pathogenesis of solitary fibrous tumours; our study suggested that similar fusions may also have a role in a malignant meningioma with unusual histopathologic features.