Detection of a rare CDKN2A intronic mutation in a Hungarian melanoma-prone family and its role in splicing regulation

The genetic predisposition to melanoma is quite heterogeneous. The major locus for melanoma predisposition is the cell cycle regulatory CDKN2A gene on chromosome 9p21 though alterations in it have been detected in only 20 40% of melanoma prone families. However, with regard to the frequency of melanoma-prone families linked to 9p21, the frequency of germline coding mutations of the CDKN2A gene is lower than expected. We set out to investigate whether the rare IVS1+37 G/C intronic mutation of the CDKN2A gene recently identified in a Hungarian melanoma prone family, influences mRNA splicing regulation. To this end, CDKN2A minigenes containing the wild type and the mutant intronic sequence were created and transfected into HeLa cells with the aim of study of the mRNA transcripts. The results revealed the emergence of a differential splicing pattern from the wild type and the mutant minigene, suggesting that this mutation may alter the splicing of CDKN2A primary mRNA and therefore might have a pathogenetic role in familial melanoma. We believe that these results confirm the importance of the identification and characterization of CDKN2A intronic mutations with a view to improvement of our understanding of the pathogenesis and the explanation of why the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21

Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2

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Therapeutic Approach in the Treatment of Medication-Related Osteonecrosis of the Jaw: Case Series of 3 Patients and State of the Art on Surgical Strategies

Bisphosphonates and receptor activator of nuclear factor kappa-B ligand inhibitors are currently the most widely used antiresorptive therapies in bone metabolism diseases treatment. Unfortunately they can evoke medication-related osteonecrosis of the jaws. The present case series study proposes to evaluate clinical features, evolution and the surgical therapeutic approaches in three patients affected by medication-related osteonecrosis of the jaw and to review the state of art regarding the management of this complication in light of the most recent literature. Methods: Three cases of medication-related osteonecrosis of the jaws are discussed, two related to bisphosphonates therapy (ibandronic acid) and one due to denosumab. Results: All three patients were aged female and had probably a dental trigger agent. The lesions located in posterior mandible were treated in one case with the surgical approach alone and, in the other case, with surgical approach associated with Er:YAG laser. The lesion related to denosumab was treated with surgical approach and platelet rich fibrin application. A complete healing was always achieved. Conclusions: Dentists should be aware of the potential risk of developing medication-related osteonecrosis of the jaws for patients who take or had taken antiresorptive drugs. The side effects of denosumab and bisphosphonates are partly overlapping and currently there is still no consensus about the therapeutic surgical options. Prevention and early detection of the lesions should be the primary strategy

Rescue of spinal muscular atrophy mouse models with AAV9-Exon-specific U1 snRNA

Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA

Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

<div><p>Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects.</p></div

Identification of SRSF2 as the main splicing factor responsible for FIX exon 5 recognition.

<p>(A) Effect of overexpression of a panel of splicing factors on the splicing pattern in the FIX exon 5 wild-type and mutant minigenes (V107V and R116R). Minigenes were transfected in HeLa cells alone (NT) or with the indicated splicing factors and the splicing pattern evaluated by RT-PCR. The identity of the amplified bands with inclusion or exclusion of exon 5 is indicated. (B) Silencing of SRSF2 reduces the percentage of exon 5 inclusion in wild-type minigene. siRNA treated (+) and control HeLa cells (-) were transfected with wt minigenes and the resulting splicing pattern evaluated by RT-PCR. (C) Relative expression level of SRSF2 RNA, analyzed by qRT-PCR in siSRSF2 and control (si Luc) Hela cells. All data in the three panels represent the average of 3 replicates with error bars indicating SD.</p

Identification of splicing regulatory elements and exon skipping mutations in FIX exon 5.

<p>(A) Schematic representation of FIX exon 5 sequence showing the location of the deletions, the exonic mutations and <i>in silico</i> predicted binding sites for splicing factors. Nucleotide changes for exonic mutants are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006082#pgen.1006082.t001" target="_blank">Table 1</a>. (B) Splicing assays of minigenes carrying the 10bp deletions (Δ1 to Δ11). Minigenes were transfected in HeLa cells and the splicing pattern evaluated by RT-PCR. The levels of exon inclusion were quantified by ImageJ and compared to the wild-type. The identity of the amplified bands with inclusion or exclusion of exon 5 is indicated (C) Splicing assays of minigenes with 3 bp deletions (Δ9.1 to Δ10.4) (D) Minigene splicing assay of disease-causing exonic mutations. Data in all panels are the average of 3 replicates with error bars indicating SD.</p

V197V and R116R mutant RNAs affect binding to inhibitory splicing factors.

<p>(A) Western blot analysis of hnRNP A1, hnRNP H and DAZAP1 proteins identified by pull-down mass-spectroscopy analysis. (B) FIX exon 5 inclusion levels upon silencing of <i>HNRNPA1/2</i> and <i>DAZAP1</i>, in V107V and R116R mutated minigenes. Data represent the average of 3 replicates with error bars indicating SD. Student <i>t</i>-test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, <i>n</i>.<i>s</i>. not significant. (C) Efficiency of <i>HNRNPA1/2</i> and <i>DAZAP1</i> silencing analyzed by Western blot.</p

The effect of FIX exon 5 mutations on splicing and FIX secretion/activity explains the residual FIX levels in Hemophilia B patients and the therapeutic potential of a splicing-switching molecule.

<p>The upper box shows exon 5 splicing, secretion efficiency and specific activity levels of Factor IX in normal conditions. The lower box shows the three classes of Exonic Splicing Mutations (ESM) categorized according to their pathologic effect on exon 5 splicing, FIX secretion efficiency and FIX specific activity, and according to the therapeutic splicing rescue promoted by ExSpeU1. The graphs on the left of each class summarize the effect of each mutation on splicing, FIX secretion efficiency and FIX specific activity relative to the wild type as identified in this paper. Detail of the model are provided in discussion.</p