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Sample records for human dystrophin exon

  1. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

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    Yanagawa, H.; Nishio, H.; Takeshima, Y. [Kobe Univ. School of Medicine (Japan)] [and others

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  2. Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

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    Fletcher Sue

    2007-07-01

    Full Text Available Abstract Background Antisense oligonucleotides (AOs can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. Results Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. Conclusion The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino

  3. Detection of an exon 53 polymorphism in the dystrophin gene.

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    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S

    1993-10-01

    We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.

  4. Exon exchange approach to repair Duchenne dystrophin transcripts.

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    Stéphanie Lorain

    Full Text Available BACKGROUND: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. METHODOLOGY/PRINCIPAL FINDINGS: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23. This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.

  5. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: Characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of Exons 3-7

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    Thanh, L. T.; Man, N. thi; Morris, G.E. (North East Wales Institute, Clwyd (United Kingdom)); Love, D.R.; Davies, K.E. (Institute of Molecular Medicine, John Radcliffe Hospital, Oxford (United Kingdom)); Helliwell, T.R. (Liverpool Univ. (United Kingdom))

    1993-07-01

    The first three exons of the human muscle dystrophin gene were expressed as a [beta]-galactosidase fusion protein. 1-his protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the distrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient. 27 refs., 6 figs.

  6. Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening.

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    Debra A O'Leary

    Full Text Available One therapeutic approach to Duchenne Muscular Dystrophy (DMD recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD, by employing antisense oligonucleotides (AONs targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2 were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

  7. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies.

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    Zhi Yon Charles Toh

    Full Text Available Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

  8. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

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    De Angelis, Fernanda Gabriella; Sthandier, Olga; Berarducci, Barbara; Toso, Silvia; Galluzzi, Giuliana; Ricci, Enzo; Cossu, Giulio; Bozzoni, Irene

    2002-01-01

    Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48–50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5′ and 3′ splice sites are coexpressed in the same cell. PMID:12077324

  9. 2′-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping

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    Tomoko Lee

    2017-02-01

    Full Text Available Duchenne muscular dystrophy (DMD is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51, which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA, which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2′-O-methyl RNA(2′-OMeRNA/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2′-OMeRNA/ENA chimera (AO85 had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2′-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD.

  10. Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

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    van Ommen Gert Jan B

    2011-04-01

    Full Text Available Abstract Background Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients. Methods We targeted myostatin exon 2 using antisense oligonucleotides (AON in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections. Results Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes. Conclusions We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

  11. Is the human dystrophin gene's intron structure related to its intron instability?

    Institute of Scientific and Technical Information of China (English)

    盛文利; 陈江瑛; 朱良付; 刘焯霖

    2003-01-01

    Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

  12. Targeted exon skipping to correct exon duplications in the dystrophin gene

    National Research Council Canada - National Science Library

    Greer, Kane L; Lochmüller, Hanns; Flanigan, Kevin; Fletcher, Susan; Wilton, Steve D

    2014-01-01

    .... Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide...

  13. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

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    HaiFang Yin

    2013-01-01

    Full Text Available We have recently reported that cell-penetrating peptides (CPPs and novel chimeric peptides containing CPP (referred as B peptide and muscle-targeting peptide (referred as MSP motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO and control peptide 3 (B-3-PMO and 3-B-PMO were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO, further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG, indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

  14. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.

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    Yin, Haifang; Boisguerin, Prisca; Moulton, Hong M; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew Ja

    2013-09-24

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013

  15. Becker Muscular Dystrophy (BMD) caused by duplication of exons 3-6 of the dystrophin gene presenting as dilated cardiomyopathy

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    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L. [Univ. of Toronto, Ontario (Canada)] [and others

    1994-09-01

    X-linked dilated cardiomyopathy (XLCM) is a progressive myocardial disease presenting with congestive heart failure in teenage males without clinical signs of skeletal myopathy. Tight linkage of XLCM to the DMD locus has been demonstrated; it has been suggested that, at least in some families, XLCM is a {open_quotes}dystrophinopathy.{close_quotes} We report a 14-year-old boy who presented with acute heart failure due to dilated cardiomyopathy. He had no history of muscle weakness, but physical examination revealed pseudohypertrophy of the calf muscles. He subsequently received a heart transplantation. Family history was negative. Serum CK level at the time of diagnosis was 10,416. Myocardial biopsy showed no evidence of carditis. Dystrophin staining of cardiac and skeletal muscle with anti-sera to COOH and NH{sub 2}termini showed a patchy distribution of positivity suggestive of Becker muscular dystrophy. Analysis of 18 of the 79 dystrophin exons detected a duplication that included exons 3-6. The proband`s mother has an elevated serum CK and was confirmed to be a carrier of the same duplication. A mutation in the muscle promotor region of the dystrophin gene has been implicated in the etiology of SLCM. However, Towbin et al. (1991) argued that other 5{prime} mutations in the dystrophin gene could cause selective cardiomyopathy. The findings in our patient support the latter hypothesis. This suggests that there are multiple regions in the dystrophin gene which, when disrupted, can cause isolated dilated cardiomyopathy.

  16. A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation.

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    Patria, S Y; Alimsardjono, H; Nishio, H; Takeshima, Y; Nakamura, H; Matsuo, M

    1996-07-01

    The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that

  17. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

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    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  18. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

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    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  19. Effective exon skipping and dystrophin restoration by 2'-o-methoxyethyl antisense oligonucleotide in dystrophin-deficient mice.

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    Lu Yang

    Full Text Available Antisense oligonucleotide (AO-mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2'-O-methoxyethyl oligonucleotides (MOE on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS AOs provided the greatest exon-skipping efficacy. When compared with 2'O methyl phosphorothioate (2'OmePS AOs, 25-mer MOE (PS AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

  20. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice

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    Sirsi Shashank R

    2008-04-01

    Full Text Available Abstract Background Exon skipping oligonucleotides (ESOs of 2'O-Methyl (2'OMe and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD. However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine (PEI and poly(ethylene glycol (PEG are regarded as effective agents for enhanced delivery of nucleic acids in various applications. Results We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 μg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG or adsorbtion of colloidal gold (CG, respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3–60 μg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal

  1. Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry

    OpenAIRE

    Brown, Kristy J; Marathi, Ramya; Fiorillo, Alyson A; Ciccimaro, Eugene F.; Sharma, Seema; Rowlands, David S.; Rayavarapu, Sree; Nagaraju, Kanneboyina; Eric P. Hoffman; Hathout, Yetrib

    2012-01-01

    Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based ...

  2. Early cardiac failure in a child with Becker muscular dystrophy is due to an abnormally low amount of dystrophin transcript lacking exon 13.

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    Ishigaki, C; Patria, S Y; Nishio, H; Yoshioka, A; Matsuo, M

    1997-12-01

    Two Japanese brothers with Becker muscular dystrophy were shown by polymerase chain reaction (PCR) and cDNA sequence analysis to produce a dystrophin gene transcript lacking a single exon: that is, number 13. Despite having the same deletion mutation, the brothers showed clearly different clinical phenotypes: the younger brother developed cardiac failure at the age of nine, while the elder brother was asymptomatic. As alternative splicing was not responsible for this clinical difference, the amount of dystrophin transcript was examined by using reverse transcription semi-nested and parallel PCR. The results showed that the amount of the dystrophin transcript in the younger brother was 20% of that of the elder brother. This finding suggested that lesser amount of dystrophin transcript in the younger brother was responsible for the early onset of cardiac failure. This would represent a novel molecular mechanism for dystrophinopathy.

  3. A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient cavalier king charles spaniels is amenable to exon 51 skipping.

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    Gemma L Walmsley

    Full Text Available BACKGROUND: Duchenne muscular dystrophy (DMD, which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD. The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE: Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

  4. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...... showed dystrophic changes. He had comorbidity with dystonia, slight mental retardation, low stature and neuropathy. The brother of the proband's mother came to medical attention when he was 43 years old. He complained about muscle pain. On examination, a MRC grade 4+ hip extention palsy and a discrete...

  5. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...... showed dystrophic changes. He had comorbidity with dystonia, slight mental retardation, low stature and neuropathy. The brother of the proband's mother came to medical attention when he was 43 years old. He complained about muscle pain. On examination, a MRC grade 4+ hip extention palsy and a discrete...

  6. Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy

    Science.gov (United States)

    Hendriksen, Ruben G. F.; Schipper, Sandra; Hoogland, Govert; Schijns, Olaf E. M. G.; Dings, Jim T. A.; Aalbers, Marlien W.; Vles, Johan S. H.

    2016-01-01

    Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

  7. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi (Kobe Univ. School of Medicine (Japan))

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  8. Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR

    Energy Technology Data Exchange (ETDEWEB)

    Risch, N. (Yale Univ., New Haven, CT (United States))

    1992-09-01

    Deletion mutations of the dystrophin gene may cause either the severe Duchenne muscular dystrophy (DMD) or the milder, allelic Becker muscular dystrophy (BMD) and are clustered in two high-frequency-deletion regions (HFDRs) located, respectively, 500 kb and 1,200 kb downstream from the 5[prime] end of the gene. Three PCR reactions described allowed the analysis of a total of 30 exons and led, to the identification of three additional deletions involving the following exons: (a) 42 only, (b) 28-42, and (c) 16 only, none of which were detected with the two original multiplex reactions. Therefore, the three modified multiplexes detected 95 of the 96 deletions identified among the 152 patients studied so far by using Southern analysis and cDNA probes. The only deletion that remained undetected with this system involves exons 22-25 and generates the junction fragment described elsewhere. The percentage of deletion mutations among DMS/BMD patients amounts to 63%, which is in agreement with similar estimates from other laboratories. When field-inversion gel electrophoresis is coupled to Southern analysis, the detection rate of deletion and duplication mutations reaches 65%.

  9. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

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    Alberto Malerba

    2012-01-01

    Full Text Available The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD. In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO. Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.

  10. Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

    Science.gov (United States)

    Serena, Elena; Zatti, Susi; Zoso, Alice; Lo Verso, Francesca; Tedesco, F Saverio; Cossu, Giulio; Elvassore, Nicola

    2016-12-01

    : Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from

  11. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    Science.gov (United States)

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

  12. Early-progressive dilated cardiomyopathy in a family with Becker muscular dystrophy related to a novel frameshift mutation in the dystrophin gene exon 27.

    Science.gov (United States)

    Tsuda, Takeshi; Fitzgerald, Kristi; Scavena, Mena; Gidding, Samuel; Cox, Mary O; Marks, Harold; Flanigan, Kevin M; Moore, Steven A

    2015-03-01

    We report a family in which two male siblings with Becker muscular dystrophy (BMD) developed severe dilated cardiomyopathy (DCM) and progressive heart failure (HF) at age 11 years; one died at age 14 years while awaiting heart transplant and the other underwent left ventricular assist device implantation at the same age. Genetic analysis of one sibling showed a novel frameshift mutation in exon 27 of Duchenne muscular dystrophy (DMD) gene (c.3779_3785delCTTTGGAinsGG), in which seven base pairs are deleted and two are inserted. Although this predicts an amino-acid substitution and premature termination (p.Thr1260Argfs*8), muscle biopsy dystrophin immunostaining instead indicates that the mutation is more likely to alter splicing. Despite relatively preserved skeletal muscular performance, both the siblings developed progressive HF secondary to early-onset DCM. In addition, their 7-year-old nephew with delayed gross motor development, mild proximal muscle weakness and markedly elevated serum creatine kinase level (>13 000 IU l(-1)) at 16 months was recently demonstrated to have the familial DMD mutation. Here, we report a novel genotype of BMD with early-onset DCM and progressive lethal HF during early adolescence.

  13. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

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    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  14. Exon skipping and Duchenne muscular dystrophy: Hope, hype and how feasible?

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    Wilton Steve

    2008-01-01

    Full Text Available Duchenne muscular dystrophy (DMD, the most common and serious form of childhood muscle wasting is generally caused by protein-truncating mutations in the large DMD gene. Specific removal of an exon from a defective DMD gene transcript has the potential to allow synthesis of a semi-functional dystrophin, thereby reducing the severity and presumably progression of muscle wasting. The efficacy of this treatment will vary greatly between the different mutations that preclude the synthesis of a functional dystrophin. Restoration of the reading frame from a large multi-exon genomic deletion, typically greater than 36 exons, may lead to synthesis of a protein with only partial function and limited clinical benefit, whereas excising a nonsense mutation in a redundant exon should generate a near normal dystrophin. A clinical trial has recently confirmed proof-of-principle that exclusion of Exon 51 from human dystrophin mRNAs, carrying frame-shifting deletions adjacent to this exon, results in dystrophin expression. No major side-effects after local administration of the antisense oligomer were reported. Additional trials are underway, targeting the same exon but using an oligomer of different backbone chemistry. If functional dystrophin synthesis is demonstrated, and safety issues are addressed, subsequent trials will involve systemic delivery. Great challenges are ahead, some technical; establishing an effective delivery regimen, some ethical; choosing subsequent targets for therapy, and others of an administrative and regulatory nature.

  15. Species-Specific Exon Loss in Human Transcriptomes

    OpenAIRE

    Wang, Jinkai; Lu, Zhi-xiang; Tokheim, Collin J.; Miller, Sara E.; Xing, Yi

    2014-01-01

    Changes in exon–intron structures and splicing patterns represent an important mechanism for the evolution of gene functions and species-specific regulatory networks. Although exon creation is widespread during primate and human evolution and has been studied extensively, much less is known about the scope and potential impact of human-specific exon loss events. Historically, transcriptome data and exon annotations are significantly biased toward humans over nonhuman primates. This ascertainm...

  16. Characteristics of transposable element exonization within human and mouse.

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    Noa Sela

    Full Text Available Insertion of transposed elements within mammalian genes is thought to be an important contributor to mammalian evolution and speciation. Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Elucidation of the evolutionary constraints that have shaped fixation of transposed elements within human and mouse protein coding genes and subsequent exonization is important for understanding of how the exonization process has affected transcriptome and proteome complexities. Here we show that exonization of transposed elements is biased towards the beginning of the coding sequence in both human and mouse genes. Analysis of single nucleotide polymorphisms (SNPs revealed that exonization of transposed elements can be population-specific, implying that exonizations may enhance divergence and lead to speciation. SNP density analysis revealed differences between Alu and other transposed elements. Finally, we identified cases of primate-specific Alu elements that depend on RNA editing for their exonization. These results shed light on TE fixation and the exonization process within human and mouse genes.

  17. Molecular Diagnosis of Duchenne/Becker Muscular Dystrophy: Analysis of Exons Deletion and Carrier Detection

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    Mohammad Taghi Akbari

    2010-01-01

    Full Text Available Objective: Duchenne and Becker Muscular Dystrophy (DMD and BMD are X-linked conditionsresulting from a defect in the dystrophin gene located at Xp21.2. DMD is the mostfrequent neuromuscular disease in humans (1/3500 male newborns. In approximately65% of DMD and BMD patients, deletions in the dystrophin gene have been identified asthe molecular determinant. The frequency and distribution of dystrophin gene deletions inDMD/BMD patients from different populations are different.The aim of this study was to delineate various types of deleted exons and their frequencyin affected male patients and identification of carrier females by linkage analysis.Materials and Methods: In this study 100 unrelated patients with DMD/BMD were studiedfor intragenic deletions in 28 exons and the promoter region of the dystrophin geneusing multiplex PCR. We also performed linkage analysis within the dystrophin gene utilizing8 short tandem repeat markers.Results: Fifty-two (52% patients showed intragenic deletions. A total of 81% of the deletionswere located at the distal hot spot region (44-55 exons and 19% of the deletionswere located at the proximal region (exon 2-19. The most frequent deleted exons were47(16%, 48 and 46 (11%.Most of the STR markers showed heterozygosity in the families studied. The linkageanalysis was useful for detecting carrier status.Conclusion: The present study suggests that intragenic dystrophin gene deletions occurwith the same frequency in Iranian patients compared with other ethnic groups.

  18. DMD transcript imbalance determines dystrophin levels.

    Science.gov (United States)

    Spitali, Pietro; van den Bergen, Janneke C; Verhaart, Ingrid E C; Wokke, Beatrijs; Janson, Anneke A M; van den Eijnde, Rani; den Dunnen, Johan T; Laros, Jeroen F J; Verschuuren, Jan J G M; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2013-12-01

    Duchenne and Becker muscular dystrophies are caused by out-of-frame and in-frame mutations, respectively, in the dystrophin encoding DMD gene. Molecular therapies targeting the precursor-mRNA are in clinical trials and show promising results. These approaches will depend on the stability and expression levels of dystrophin mRNA in skeletal muscles and heart. We report that the DMD gene is more highly expressed in heart than in skeletal muscles, in mice and humans. The transcript mutated in the mdx mouse model shows a 5' to 3' imbalance compared with that of its wild-type counterpart and reading frame restoration via antisense-mediated exon skipping does not correct this event. We also report significant transcript instability in 22 patients with Becker dystrophy, clarifying the fact that transcript imbalance is not caused by premature nonsense mutations. Finally, we demonstrate that transcript stability, rather than transcriptional rate, is an important determinant of dystrophin protein levels in patients with Becker dystrophy. We suggest that the availability of the complete transcript is a key factor to determine protein abundance and thus will influence the outcome of mRNA-targeting therapies.

  19. Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

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    Aziza Sbiti

    2002-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD and BMD are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn. Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.

  20. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

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    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  1. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Science.gov (United States)

    Saito, Takashi; Nakamura, Akinori; Aoki, Yoshitsugu; Yokota, Toshifumi; Okada, Takashi; Osawa, Makiko; Takeda, Shin'ichi

    2010-08-18

    Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  2. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

    Science.gov (United States)

    Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E; McIntosh, Mark A; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

  3. Computational study of the human dystrophin repeats: interaction properties and molecular dynamics.

    Science.gov (United States)

    Legrand, Baptiste; Giudice, Emmanuel; Nicolas, Aurélie; Delalande, Olivier; Le Rumeur, Elisabeth

    2011-01-01

    Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

  4. Computational study of the human dystrophin repeats: interaction properties and molecular dynamics.

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    Baptiste Legrand

    Full Text Available Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD. It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

  5. Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

    Science.gov (United States)

    Karpati, G; Carpenter, S; Morris, G E; Davies, K E; Guerin, C; Holland, P

    1993-03-01

    A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15-17-fold increase of DRP over normal in DMD/BMD and 4-10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.

  6. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    Science.gov (United States)

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-05-24

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

  7. Multiplex amplification of large sets of human exons.

    Science.gov (United States)

    Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

    2007-11-01

    A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

  8. Screening of Dystrophin Gene Deletions in Egyptian Patients with DMD/BMD Muscular Dystrophies

    Directory of Open Access Journals (Sweden)

    Laila K. Effat

    2000-01-01

    Full Text Available Duchenne muscular dystrophy (DMD and Becker muscular dystrophy (BMD are allelic disorders caused by mutations within the dystrophin gene. Our study has identified 100 Egyptian families collected from the Human Genetics Clinic, National Research Center, Cairo. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase enzyme (CPK levels and DNA analysis. Multiplex PCR using 18 pairs of specific primers were used for screening of deletion mutations within the dystrophin gene. A frequency of 55% among the families. Sixty per cent of detected deletions involved multiple exons spanning the major or the minor hot spot of the dystrophin gene. The remainder 40% which mainly involved exon 45. Comparing these findings with frequencies of other countries it was found that our figures fall within the reported range of 40%– for deletions. The distribution of deletions in our study and other different studies was variable and specific ethnic differences do not apparently account for specific deletions. In addition this study concluded that employment of the 18 exon analysis is a cost effective and a highly accurate (97% to launch a nationwide program.

  9. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  10. Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

    Directory of Open Access Journals (Sweden)

    Xuan Guan

    2014-03-01

    Full Text Available The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD. Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs. USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

  11. Comparison of multiple vertebrate genomes reveals the birth and evolution of human exons.

    Science.gov (United States)

    Zhang, Xiang H-F; Chasin, Lawrence A

    2006-09-05

    Orthologous gene structures in eight vertebrate species were compared on a genomic scale to detect the birth and maturation of new internal exons during the course of evolution. We found that 40% of new human exons are alternatively spliced, and most of these are cassette exons (exons that are either included or skipped in their entirety) with low inclusion rates. This proportion decreases steadily as older and older exons are examined, even as splicing efficiency increases. Remarkably, the great majority of new cassette exons are composed of highly repeated sequences, especially Alu. Many new cassette exons are 5' untranslated exons; the proportion that code for protein increases steadily with age. New protein-coding exons evolve at a high rate, as evidenced by the initially high substitution rates (K(s) and K(a)), as well as the SNP density compared with older exons. This dynamic picture suggests that de novo recruitment rather than shuffling is the major route by which exons are added to genes, and that species-specific repeats could play a significant role in recent evolution.

  12. Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

    Science.gov (United States)

    Prior, T W; Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Kissel, J T; Luquette, M H; Tsao, C Y; Mendell, J R

    1997-02-01

    The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

  13. Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase

    Science.gov (United States)

    Carnicer, Ricardo; Recalde, Alice; Muszkiewicz, Anna; Jayaram, Raja; Carena, Maria Cristina; Wijesurendra, Rohan; Stefanini, Matilde; Surdo, Nicoletta C.; Lomas, Oliver; Ratnatunga, Chandana; Sayeed, Rana; Krasopoulos, George; Rajakumar, Timothy; Bueno-Orovio, Alfonso; Verheule, Sander; Fulga, Tudor A.; Rodriguez, Blanca; Schotten, Ulrich

    2016-01-01

    Atrial fibrillation (AF) is a growing public health burden, and its treatment remains a challenge. AF leads to electrical remodeling of the atria, which in turn promotes AF maintenance and resistance to treatment. Although remodeling has long been a therapeutic target in AF, its causes remain poorly understood. We show that atrial-specific up-regulation of microRNA-31 (miR-31) in goat and human AF depletes neuronal nitric oxide synthase (nNOS) by accelerating mRNA decay and alters nNOS subcellular localization by repressing dystrophin translation. By shortening action potential duration and abolishing rate-dependent adaptation of the action potential duration, miR-31 overexpression and/or disruption of nNOS signaling recapitulates features of AF-induced remodeling and significantly increases AF inducibility in mice in vivo. By contrast, silencing miR-31 in atrial myocytes from patients with AF restores dystrophin and nNOS and normalizes action potential duration and its rate dependency. These findings identify atrial-specific up-regulation of miR-31 in human AF as a key mechanism causing atrial dystrophin and nNOS depletion, which in turn contributes to the atrial phenotype begetting this arrhythmia. miR-31 may therefore represent a potential therapeutic target in AF. PMID:27225184

  14. The first exon duplication mouse model of Duchenne muscular dystrophy: A tool for therapeutic development.

    Science.gov (United States)

    Vulin, Adeline; Wein, Nicolas; Simmons, Tabatha R; Rutherford, Andrea M; Findlay, Andrew R; Yurkoski, Jacqueline A; Kaminoh, Yuuki; Flanigan, Kevin M

    2015-11-01

    Exon duplication mutations account for up to 11% of all cases of Duchenne muscular dystrophy (DMD), and a duplication of exon 2 is the most common duplication in patients. For use as a platform for testing of duplication-specific therapies, we developed a mouse model that carries a Dmd exon 2 duplication. By using homologous recombination we duplicated exon 2 within intron 2 at a location consistent with a human duplication hotspot. mRNA analysis confirms the inclusion of a duplicated exon 2 in mouse muscle. Dystrophin expression is essentially absent by immunofluorescent and immunoblot analysis, although some muscle specimens show very low-level trace dystrophin expression. Phenotypically, the mouse shows similarities to mdx, the standard laboratory model of DMD. In skeletal muscle, areas of necrosis and phagocytosis are seen at 3 weeks, with central nucleation prominent by four weeks, recapitulating the "crisis" period in mdx. Marked diaphragm fibrosis is noted by 6 months, and remains unchanged at 12 months. Our results show that the Dup2 mouse is both pathologically (in degree and distribution) and physiologically similar to mdx. As it recapitulates the most common single exon duplication found in DMD patients, this new model will be a useful tool to assess the potential of duplicated exon skipping.

  15. Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

    Directory of Open Access Journals (Sweden)

    Kole Ryszard

    2011-10-01

    Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

  16. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    Energy Technology Data Exchange (ETDEWEB)

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E. [Wales Institute, Clwyd (United Kingdom)] [and others

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  17. Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Vassilia Partali

    2012-01-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs. The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC.

  18. Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate

    Directory of Open Access Journals (Sweden)

    de Torres Inés

    2009-06-01

    Full Text Available Abstract Background The human Sex Hormone Binding Globulin (SHBG gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the second begins with an alternative exon 1 sequence, which replaces the exon 1 present in liver transcripts. Alternative exon 1 transcription and translation has only been demonstrated in the testis of transgenic mice containing an 11-kb human SHBG transgene and in the human testis. Our goal has been to further characterize the 5' end of the SHBG gene and analyze the presence of the SHBG alternative transcripts in human prostate tissue and derived cell lines. Results Using a combination of in silico and in vitro studies, we have demonstrated that the SHBG gene, along with exon 1 and alternative exon 1 (renamed here exon 1A, contains four additional alternative first exons: the novel exons 1B, 1C, and 1E, and a previously identified exon 1N, which has been further characterized and renamed as exon 1D. We have shown that these four alternative first exons are all spliced to the same 3' splice site of SHBG exon 2, and that exon 1A and the novel exon 1B can be spliced to exon 1. We have also demonstrated the presence of SHBG transcripts beginning with exons 1B, 1C and 1D in prostate tissues and cell lines, as well as in several non-prostatic cell lines. Finally, the alignment of the SHBG mammalian sequences revealed that, while exons 1C, 1D and 1E are very well conserved phylogenetically through non-primate mammal species, exon 1B probably aroused in apes due to a single nucleotide change that generated a new 5' splice site in exon 1B. Conclusion The identification of multiple transcription start sites (TSS upstream of the annotated first exon of human SHBG, and the detection of the alternative transcripts in human prostate

  19. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    DEFF Research Database (Denmark)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most pr...... oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA)....

  20. The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

    Directory of Open Access Journals (Sweden)

    Seim Inge

    2009-05-01

    Full Text Available Abstract Background The murine ghrelin gene (Ghrl, originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0. We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. Findings 5' RACE revealed two transcription start sites (TSSs in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR, we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. Conclusion We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1 raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

  1. STUDY OF ECK GENE EXON-3 FROM HUMAN NORMAL TISSUE AND BREAST CANCER CELL LINE

    Institute of Scientific and Technical Information of China (English)

    李瑶琛; 孔令洪; 王一理; 司履生

    2003-01-01

    Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR-75-1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR-75-1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm-T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR-75-1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR-75-1 cell line respectively. ④ ZR-75-1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.

  2. Role of the intracellular receptor domain of gp130 (exon 17) in human inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Christoph J. Auernhammer; Thomas Ochsenkühn; Kathrin Zitzmann; Fabian Schnitzler; Julia Seiderer; Peter Lohse; George Vlotides; Dieter Engelhardt; Michael Sackmann; Burkhard G(o)ke

    2005-01-01

    AIM: To study the role of the intracellular receptor domain of gp130 in human inflammatory bowel disease (IBD).METHODS: We amplified and sequenced the complete exon 17 of the human gp130 gene in 146 patients with IBD. According to clinical and histopathological signs,the 146 patients with IBD were classified as having Crohn's disease (n = 73) or ulcerative colitis (n = 63),or as indeterminate status (n = 10).RESULTS: No mutations in exon 17 of the gp130 gene could be detected in any of the 146 patients with IBD examined.CONCLUSION: There is no evidence that mutations in exon 17 of the gp130 gene are involved in the pathogenesis of human IBD.

  3. Naturally occuring nucleosome positioning signals in human exons and introns

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves;

    1996-01-01

    alignments of internal exon and intron sequences corresponds to a periodic "in phase" bending potential towards the major groove of the DNA. The nucleosome positioning data show that the consensus triplets (and their complements) have a preference for locations on a bent double helix where the major groove...... of roughly ten nucleotides. The periodic pattern is also present in intron sequences, although the strength per nucleotide is weaker. Using two independent profile methods based on triplet bendability parameters from DNase I experiments and nucleosome positioning data, we show that the pattern in multiple...... faces inward and is compressed. The in-phase triplets are located adjacent to GCC/GGC triplets known to have the strongest bias in their positioning on the nucleosome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence...

  4. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    Science.gov (United States)

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

  5. Distribution of dystrophin- and utrophin-associated protein complexes (DAPC/UAPC) in human hematopoietic stem/progenitor cells.

    Science.gov (United States)

    Teniente-De Alba, Carmen; Martínez-Vieyra, Ivette; Vivanco-Calixto, Raúl; Galván, Iván J; Cisneros, Bulmaro; Cerecedo, Doris

    2011-10-01

    Hematopoietic stem cells (HSC) are defined by their cardinal properties, such as sustained proliferation, multilineage differentiation, and self-renewal, which give rise to a hierarchy of progenitor populations with more restricted potential lineage, ultimately leading to the production of all types of mature blood cells. HSC are anchored by cell adhesion molecules to their specific microenvironment, thus regulating their cell cycle, while cell migration is essentially required for seeding the HSC of the fetal bone marrow (BM) during development as well as in adult BM homeostasis. The dystrophin-associated protein complex (DAPC) is a large group of membrane-associated proteins linking the cytoskeleton to the extracellular matrix and exhibiting scaffolding, adhesion, and signaling roles in muscle and non-muscle cells including mature blood cells. Because adhesion and migration are mechanisms that influence the fate of the HSC, we explored the presence and the feasible role of DAPC. In this study, we characterized the pattern expression by immunoblot technique and, by confocal microscopy analysis, the cellular distribution of dystrophin and utrophin gene products, and the dystrophin-associated proteins (α-, β-dystroglycan, α-syntrophin, α-dystrobrevin) in relation to actin filaments in freshly isolated CD34+ cells from umbilical cord blood. Immunoprecipitation assays demonstrated the presence of Dp71d/Dp71Δ110m ∼DAPC and Up400/Up140∼DAPC. The subcellular distribution of the two DAPC in actin-based structures suggests their dynamic participation in adhesion and cell migration. In addition, the particular protein pattern expression found in hematopoietic stem/progenitor cells might be indicative of their feasible participation during differentiation.

  6. Analysis of dystrophin gene deletions by multiplex PCR in eastern India

    Directory of Open Access Journals (Sweden)

    Basak Jayasri

    2006-01-01

    Full Text Available The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63% showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e., between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.

  7. Mutations of p53 gene exons 4-8 in human esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Ya Li; Jin-Tian Tang; Li-Qun Jia; Pei-Wen Li

    2005-01-01

    AIM: To characterize the tumor suppressor gene p53 mutations in exon 4, esophageal cancer and adjacent noncancerous tissues.METHODS: We performed p53 (exons 4-8) gene mutation analysis on 24 surgically resected human esophageal cancer specimens by PCR, single-strand conformation polymorphism, and DNA sequencing. RESULTS: p53 gene mutations were detected in 9 of 22 (40.9%) esophageal cancer specimens and 10 of 17 (58.8%) adjacent non-cancerous tissues. Eight of sixteen (50.0%) point mutations detected were G-A transitions and 9 of 18 (50.0%) p53 gene mutations occurred in exon 4 in esophageal cancer specimens. Only 1 of 11 mutations detected was G-A transition and 4 of 11 (36.4%) p53 gene mutations occurred in exon 4 in adjacent non-cancerous tissues.CONCLUSION: Mutation of p53 gene in exon 4 may play an important role in development of esophageal cancer. The observation of p53 gene mutation in adjacent noncancerous tissues suggests that p53 gene mutation may be an early event in esophageal carcinogenesis. Some clinical factors, including age, sex, pre-operation therapy and location of tumors, do not influence p53 gene mutation rates.

  8. Short Tandem Repeats in Human Exons: A Target for Disease Mutations

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    Villesen Palle

    2008-09-01

    Full Text Available Abstract Background In recent years it has been demonstrated that structural variations, such as indels (insertions and deletions, are common throughout the genome, but the implications of structural variations are still not clearly understood. Long tandem repeats (e.g. microsatellites or simple repeats are known to be hypermutable (indel-rich, but are rare in exons and only occasionally associated with diseases. Here we focus on short (imperfect tandem repeats (STRs which fall below the radar of conventional tandem repeat detection, and investigate whether STRs are targets for disease-related mutations in human exons. In particular, we test whether they share the hypermutability of the longer tandem repeats and whether disease-related genes have a higher STR content than non-disease-related genes. Results We show that validated human indels are extremely common in STR regions compared to non-STR regions. In contrast to longer tandem repeats, our definition of STRs found them to be present in exons of most known human genes (92%, 99% of all STR sequences in exons are shorter than 33 base pairs and 62% of all STR sequences are imperfect repeats. We also demonstrate that STRs are significantly overrepresented in disease-related genes in both human and mouse. These results are preserved when we limit the analysis to STRs outside known longer tandem repeats. Conclusion Based on our findings we conclude that STRs represent hypermutable regions in the human genome that are linked to human disease. In addition, STRs constitute an obvious target when screening for rare mutations, because of the relatively low amount of STRs in exons (1,973,844 bp and the limited length of STR regions.

  9. A defect in dystrophin causes a novel porcine stress syndrome

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    Nonneman Dan J

    2012-06-01

    Full Text Available Abstract Background Losses of slaughter-weight pigs due to transport stress are both welfare and economic concerns to pork producers. Historically, the HAL-1843 mutation in ryanodine receptor 1 was considered responsible for most of the losses; however, DNA testing has effectively eliminated this mutation from commercial herds. We identified two sibling barrows in the USMARC swine herd that died from apparent symptoms of a stress syndrome after transport at 12 weeks of age. The symptoms included open-mouth breathing, skin discoloration, vocalization and loss of mobility. Results We repeated the original mating along with sire-daughter matings to produce additional offspring. At 8 weeks of age, heart rate and electrocardiographs (ECG were monitored during isoflurane anesthesia challenge (3% for 3 min. Four males from the original sire-dam mating and two males from a sire-daughter mating died after one minute of anesthesia. Animals from additional litters were identified as having a stress response, sometimes resulting in death, during regular processing and weighing. Affected animals had elevated plasma creatine phosphokinase (CPK levels before and immediately after isoflurane challenge and cardiac arrhythmias. A pedigree containing 250 pigs, including 49 affected animals, was genotyped with the Illumina PorcineSNP60 Beadchip and only one chromosomal region, SSCX at 25.1-27.7 Mb over the dystrophin gene (DMD, was significantly associated with the syndrome. An arginine to tryptophan (R1958W polymorphism in exon 41 of DMD was the most significant marker associated with stress susceptibility. Immunoblots of affected heart and skeletal muscle showed a dramatic reduction of dystrophin protein and histopathology of affected hearts indicated muscle fiber degeneration. Conclusions A novel stress syndrome was characterized in pigs and the causative genetic factor most likely resides within DMD that results in less dystrophin protein and cardiac

  10. TNF-α-Induced microRNAs Control Dystrophin Expression in Becker Muscular Dystrophy

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    Alyson A. Fiorillo

    2015-09-01

    Full Text Available The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45–47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31. microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.

  11. Dystrophin in frameshift deletion patients with Becker Muscular Dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Gangopadhyay, S.B.; Ray, P.N.; Worton, R.G.; Sherratt, T.G.; Heckmatt, J.Z.; Dubowitz, V.; Strong, P.N.; Miller, G. (Penn State College of Medicine, Hershey, PA (United States)); Shokeir, M. (Univ. Hospital, Saskatchewan (Canada))

    1992-09-01

    In a previous study the authors identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. They have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases they detected slightly lower-molecular-weight dystrophin in 12%-15% abundance relative to the normal. By sequencing amplified mRNA they have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternate splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame. 34 refs., 5 figs. 1 tab.

  12. Evolutionary study of vertebrate and invertebrate members of the dystrophin and utrophin gene family

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    Roberts, R.G.; Nicholson, L.; Bobrow, M. [Paediatric Research Unit, London (United Kingdom)] [and others

    1994-09-01

    Vertebrates express two members of the dystrophin gene family. The prototype, dystrophin, is expressed in muscle and neural tissue, and is defective in the human disorders Duchenne and Becker muscular dystrophy (DMD, BMD). The dystrophin homologue utrophin is more generally expressed but has not yet been associated with a genetic disorder. The function of neither protein is clear. A comparison of human utrophin with the known dystrophins (human, mouse, chicken, Torpedo) suggests that dystrophin and utrophin diverged before the vertebrate radiation. We have used reverse-transcript PCR (RT-PCR) directed by degenerate primers to characterize dystrophin and utrophin transcripts from a range of vertebrate and invertebrate animals. Our results suggest that the duplication leading to distinct dystrophin and utrophin genes occurred close to the point of divergence of urochordates from the cephalochordate-vertebrate lineage. This divergence may have occurred to fulfill a novel role which arose at this point, or may reflect a need for separate regulation of the neuromuscular and other functions of the ancient dystrophin. Our data include sequences of the first non-human utrophins to be characterized, and show these to be substantially more divergent than their cognate dystrophins. In addition, our results provide a large body of information regarding the tolerance of amino acid positions in the cysteine-rich and C-terminal domains to substitution. This will aid the interpretations of DMD and BMD missense mutations in these regions.

  13. Gene identification using exon amplification on human chromosome 18q21: implications for bipolar disorder.

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    Chen, H; Huo, Y; Patel, S; Zhu, X; Swift-Scanlan, T; Reeves, R H; DePaulo, R; Ross, C A; McInnis, M G

    2000-09-01

    We previously reported linkage between bipolar disorder and a region on human chromosome (HC) 18q21. To identify genes in this region, exon trapping was performed on cosmids isolated from an HC18-specific cosmid library (LL18NC02) using 47 sequence tagged site (STS) markers from 18q21 as hybridization probes. A total of 285 unique sequences (exons) were obtained from 850 sequenced clones. Homology searching of the databases using NCBI's BLAST algorithms revealed that 31 exons have identity to known genes and/or ESTs, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region.

  14. LACKING EXON5 OF VARIANT ESTROGEN RECEPTOR IN HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    Zhang Lei; Gong Ping; Sun Sulian; Dong Zhiwei

    1998-01-01

    Methods: The target sequence of ER RNA covering exon4~6(1082~1520bp) was amplified in 7 clinical human breast cancer tissues by reverse transcription and polymerase chain reaction (RT-PCR) techniques.Results: PCR products were transferred to nitrocellulose membranes and hybridized using a [r-32P]-ATP labeled ER 29 oligonulceotide probe representing the antisense strand of the ER Cdna sequence 1271~1299. Specific bands were found at 438 and 300 base pairs in two tumors. The 300 base pair of PCR product was recovered from ER+/PR+ and ER+/PR- tumor, respectively.Conclusion: Dideoxy sequence analysis revealed that they contained the variant ER completely missing exon 5.

  15. A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene.

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    Ishigaki, C; Patria, S Y; Nishio, H; Yabe, M; Matsuo, M

    1996-05-01

    We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases.

  16. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy.

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    Thibaut Larcher

    Full Text Available A few animal models of Duchenne muscular dystrophy (DMD are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

  17. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Larcher, Thibaut; Lafoux, Aude; Tesson, Laurent; Remy, Séverine; Thepenier, Virginie; François, Virginie; Le Guiner, Caroline; Goubin, Helicia; Dutilleul, Maéva; Guigand, Lydie; Toumaniantz, Gilles; De Cian, Anne; Boix, Charlotte; Renaud, Jean-Baptiste; Cherel, Yan; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio; Huchet, Corinne

    2014-01-01

    A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

  18. Spectrum of small mutations in the dystrophin coding region.

    Science.gov (United States)

    Prior, T W; Bartolo, C; Pearl, D K; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Mendell, J R

    1995-07-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.

  19. Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

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    van Ommen Gert-Jan B

    2007-07-01

    Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.

  20. Deletion and Mutation of WWOX Exons 6-8 in Human Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6-8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6-8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6 %) lung cancer samples showed loss of WWOX exons 6-8 transcript and the deletion was detected in only 3 of 44 (6.8 %) corresponding adjacent normal tissues (P<0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6-8 revealed no difference from the sequence of GenBank. Moreover, the deletion of WWOX exons 6-8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P<0.05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6-8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6-8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6-8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6-8.

  1. Exon Deletion Pattern in Duchene Muscular Dystrophy in North West of Iran

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    Mohammad BARZEGAR

    2015-01-01

    Full Text Available How to Cite This Article: Barzegar M, Habibi P, Bonyady M, Topchizadeh V, Shiva Sh. Exon Deletion Pattern in Duchene Muscular Dystrophy in North West of Iran. Iran J Child Neurol. 2015 Winter; 9(1: 42-48.AbstractObjectiveDuchene and Becker Muscular Dystrophy (DMD/ BMD are x-linked disorders that both are the result of heterogeneous mutations in the dystrophin gene. The frequency and distribution of dystrophin gene deletions in DMD/ BMD patients show different patterns among different populations. This study investigates the deletion rate, type, and distribution of this gene in the Azeri Turk population of North West Iran.Materials &MethodsIn this study, 110 patients with DMD/ BMD were studied for intragenic deletions in 24 exons and promoter regions of dystrophin genes by using multiplex PCR.ResultsDeletions were detected in 63 (57.3% patients, and around 83% localized in the mid-distal hotspot of the gene (on exons 44–52, 21 cases (33.3 % with singleexon deletions, and 42 cases (66.6% with multi-exonic deletions. The most frequent deleted exons were exon 50 (15 % and exon 49 (14%. No deletion was detected in exon 3.ConclusionThis study suggests that the frequency and pattern of dystrophin gene deletions in DMD/ BMD in the Azeri Turk population of North West Iran occur in the same pattern when compared with other ethnic groups.ReferencesEmery AE. Clinical and molecular studies in Duchenne muscular dystrophy. Prog Clin Biol Res 1989; 306:15-28.Moser H. Duchenne muscular dystrophy: pathogenic aspects and genetic prevention. Hum Genet 1984; 66(1:17-40.Emery AE. Population Frequencies of inherited neuromuscular diseases: a world survey Neuromuscul Disord 1991; I (I:19-29.Bushby KM, Thmabyayah M, Gardner M D. Prevalence and incidence of Becker muscular dystrophy. Lancet 1991; 337(8748:1022-1024.Koenig M, Hoffman EP, Bertelosn CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD DNA and

  2. A comparative study of N-glycolylneuraminic acid (Neu5Gc and cytotoxic T cell (CT carbohydrate expression in normal and dystrophin-deficient dog and human skeletal muscle.

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    Paul T Martin

    Full Text Available The expression of N-glycolylneuraminic acid (Neu5Gc and the cytotoxic T cell (CT carbohydrate can impact the severity of muscular dystrophy arising from the loss of dystrophin in mdx mice. Here, we describe the expression of these two glycans in skeletal muscles of dogs and humans with or without dystrophin-deficiency. Neu5Gc expression was highly reduced (>95% in muscle from normal golden retriever crosses (GR, n = 3 and from golden retriever with muscular dystrophy (GRMD, n = 5 dogs at multiple ages (3, 6 and 13 months when compared to mouse muscle, however, overall sialic acid expression in GR and GRMD muscles remained high at all ages. Neu5Gc was expressed on only a minority of GRMD satellite cells, CD8⁺ T lymphocytes and macrophages. Human muscle from normal (no evident disease, n = 3, Becker (BMD, n = 3 and Duchenne (DMD, n = 3 muscular dystrophy individuals had absent to very low Neu5Gc staining, but some punctate intracellular muscle staining was present in BMD and DMD muscles. The CT carbohydrate was localized to the neuromuscular junction in GR muscle, while GRMD muscles had increased expression on a subset of myofibers and macrophages. In humans, the CT carbohydrate was ectopically expressed on the sarcolemmal membrane of some BMD muscles, but not normal human or DMD muscles. These data are consistent with the notion that altered Neu5Gc and CT carbohydrate expression may modify disease severity resulting from dystrophin deficiency in dogs and humans.

  3. A comparative study of N-glycolylneuraminic acid (Neu5Gc) and cytotoxic T cell (CT) carbohydrate expression in normal and dystrophin-deficient dog and human skeletal muscle.

    Science.gov (United States)

    Martin, Paul T; Golden, Bethannie; Okerblom, Jonathan; Camboni, Marybeth; Chandrasekharan, Kumaran; Xu, Rui; Varki, Ajit; Flanigan, Kevin M; Kornegay, Joe N

    2014-01-01

    The expression of N-glycolylneuraminic acid (Neu5Gc) and the cytotoxic T cell (CT) carbohydrate can impact the severity of muscular dystrophy arising from the loss of dystrophin in mdx mice. Here, we describe the expression of these two glycans in skeletal muscles of dogs and humans with or without dystrophin-deficiency. Neu5Gc expression was highly reduced (>95%) in muscle from normal golden retriever crosses (GR, n = 3) and from golden retriever with muscular dystrophy (GRMD, n = 5) dogs at multiple ages (3, 6 and 13 months) when compared to mouse muscle, however, overall sialic acid expression in GR and GRMD muscles remained high at all ages. Neu5Gc was expressed on only a minority of GRMD satellite cells, CD8⁺ T lymphocytes and macrophages. Human muscle from normal (no evident disease, n = 3), Becker (BMD, n = 3) and Duchenne (DMD, n = 3) muscular dystrophy individuals had absent to very low Neu5Gc staining, but some punctate intracellular muscle staining was present in BMD and DMD muscles. The CT carbohydrate was localized to the neuromuscular junction in GR muscle, while GRMD muscles had increased expression on a subset of myofibers and macrophages. In humans, the CT carbohydrate was ectopically expressed on the sarcolemmal membrane of some BMD muscles, but not normal human or DMD muscles. These data are consistent with the notion that altered Neu5Gc and CT carbohydrate expression may modify disease severity resulting from dystrophin deficiency in dogs and humans.

  4. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

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    Joshua D. Tompkins

    2016-02-01

    Full Text Available The directed differentiation of human cardiomyocytes (CMs from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.

  5. Dystrophin-Deficient Cardiomyopathy.

    Science.gov (United States)

    Kamdar, Forum; Garry, Daniel J

    2016-05-31

    Dystrophinopathies are a group of distinct neuromuscular diseases that result from mutations in the structural cytoskeletal Dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD and BMD female carriers. The primary presenting symptom in most dystrophinopathies is skeletal muscle weakness. However, cardiac muscle is also a subtype of striated muscle and is similarly affected in many of the muscular dystrophies. Cardiomyopathies associated with dystrophinopathies are an increasingly recognized manifestation of these neuromuscular disorders and contribute significantly to their morbidity and mortality. Recent studies suggest that these patient populations would benefit from cardiovascular therapies, annual cardiovascular imaging studies, and close follow-up with cardiovascular specialists. Moreover, patients with DMD and BMD who develop end-stage heart failure may benefit from the use of advanced therapies. This review focuses on the pathophysiology, cardiac involvement, and treatment of cardiomyopathy in the dystrophic patient. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  6. Characterization of a silencer element in the first exon of the human osteocalcin gene.

    Science.gov (United States)

    Li, Y P; Chen, W; Stashenko, P

    1995-01-01

    Osteocalcin, the major non-collagenous protein in bone, is transcribed in osteoblasts at the onset of extracellular matrix mineralization. In this study it was demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation-related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable in UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critical for silencing function. Mobility shift assays demonstrated that a nuclear factor bound to the OSE. The OSE binding protein was present in proliferating normal pre-osteoblasts and in UMR-106 and ROS 17/2.8 osteosarcoma cells, but was absent from post-proliferative normal osteoblasts. The binding protein was inhibited by fragments containing the +29/+35 sequence, but not by other promoter fragments or by the consensus oligomers of unrelated nuclear factors AP-1 and Sp1. DNase 1 footprinting demonstrated that the OSE binding-protein protected the +17 to +36 portion of the first exon, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that this silencer is activated by complexing of the OSE binding protein to the OSE during the osteoblast proliferation stage and that the OSE binding protein is down-regulated at the onset of extracellular matrix mineralization. Images PMID:8559666

  7. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

    Energy Technology Data Exchange (ETDEWEB)

    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y. [Tel Aviv Univ. (Israel)

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  8. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.

    Science.gov (United States)

    Cox, G A; Cole, N M; Matsumura, K; Phelps, S F; Hauschka, S D; Campbell, K P; Faulkner, J A; Chamberlain, J S

    1993-08-19

    Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked recessive diseases caused by defective expression of dystrophin. The mdx mouse, an animal model for DMD, has a mutation that eliminates expression of the 427K muscle and brain isoforms of dystrophin. Although these animals do not display overt muscle weakness or impaired movement, the diaphragm muscle of the mdx mouse is severely affected and shows progressive myofibre degeneration and fibrosis which closely resembles the human disease. Here we explore the feasibility of gene therapy for DMD by examining the potential of a full-length dystrophin transgene to correct dystrophic symptoms in mdx mice. We find that expression of dystrophin in muscles of transgenic mdx mice eliminates the morphological and immunohistological symptoms of muscular dystrophy. In addition, overexpression of dystrophin prevents the development of the abnormal mechanical properties associated with dystrophic muscle without causing deleterious side effects. Our results provide functional evidence for the feasibility of gene therapy for DMD.

  9. Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database

    Directory of Open Access Journals (Sweden)

    Nicolas Aurélie

    2012-07-01

    Full Text Available Abstract Background Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD and Becker (BMD muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. Methods and results We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. Conclusion This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

  10. NUCLEOTIDE-SEQUENCE OF THE LAST EXON OF THE GENE FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB AND ITS FLANKING REGIONS

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; BOKMA, E; REUVEKAMP, P; AGSTERIBBE, E; DEVRIES, H

    1991-01-01

    A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding re

  11. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  12. Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S; Western, L M; Bartolo, C; Moxley, R T; Mendell, J R

    1994-03-01

    Approximately one-third of the Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, we identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. We conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing.

  13. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R. [Ohio State Univ., Columbus, OH (United States); Moxley, R.T. [Univ. of Rochester Medical Center, NY (United States)

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  14. Identification and evolutionary analysis of novel exons and alternative splicing events using cross-species EST-to-genome comparisons in human, mouse and rat

    Directory of Open Access Journals (Sweden)

    Ho Jar-Yi

    2006-03-01

    Full Text Available Abstract Background Alternative splicing (AS is important for evolution and major biological functions in complex organisms. However, the extent of AS in mammals other than human and mouse is largely unknown, making it difficult to study AS evolution in mammals and its biomedical implications. Results Here we describe a cross-species EST-to-genome comparison algorithm (ENACE that can identify novel exons for EST-scanty species and distinguish conserved and lineage-specific exons. The identified exons represent not only novel exons but also evolutionarily meaningful AS events that are not previously annotated. A genome-wide AS analysis in human, mouse and rat using ENACE reveals a total of 758 novel cassette-on exons and 167 novel retained introns that have no EST evidence from the same species. RT-PCR-sequencing experiments validated ~50 ~80% of the tested exons, indicating high presence of exons predicted by ENACE. ENACE is particularly powerful when applied to closely related species. In addition, our analysis shows that the ENACE-identified AS exons tend not to pass the nonsynonymous-to-synonymous substitution ratio test and not to contain protein domain, implying that such exons may be under positive selection or relaxed negative selection. These AS exons may contribute to considerable inter-species functional divergence. Our analysis further indicates that a large number of exons may have been gained or lost during mammalian evolution. Moreover, a functional analysis shows that inter-species divergence of AS events may be substantial in protein carriers and receptor proteins in mammals. These exons may be of interest to studies of AS evolution. The ENACE programs and sequences of the ENACE-identified AS events are available for download. Conclusion ENACE can identify potential novel cassette exons and retained introns between closely related species using a comparative approach. It can also provide information regarding lineage- or species

  15. Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients.

    NARCIS (Netherlands)

    Helderman-van den Enden, A.T.; Straathof, C.S.; Aartsma-Rus, A.; Dunnen, J.T. den; Verbist, B.M.; Bakker, E.; Verschuuren, J.J.; Ginjaar, H.B.

    2010-01-01

    Theoretically, 13% of patients with Duchenne muscular dystrophy may benefit from antisense-mediated skipping of exon 51 to restore the reading frame, which results in the production of a shortened dystrophin protein. We give a detailed description with longitudinal follow up of three patients with B

  16. Becker muscular dystrophy in Indian patients: Analysis of dystrophin gene deletion patterns

    Directory of Open Access Journals (Sweden)

    Dastur Rashna

    2008-01-01

    Full Text Available Background: Becker muscular dystrophy (BMD is caused by mutations in the dystrophin gene with variable phenotypes. Becker muscular dystrophy patients have low levels of nearly full-length dystrophin and carry in-frame mutations, which allow partial functioning of the protein. Aim: To study the deletion patterns of BMD and to correlate the same with reading frame rule and different phenotypes. Setting: A tertiary care teaching hospital. Design: This is a prospective hospital-based study. Materials and Methods: Thirty-two exons spanning different "hot spot" regions using Multiplex PCR techniques were studied in 347 patients. Two hundred and twenty-two showed deletions in one or more of the 32 exons. Out of these, 46 diagnosed as BMD patients were analyzed. Results: Forty-six BMD patients showed deletions in both regions of the dystrophin gene. Out of these 89.1% (41/46 were in-frame deletions. Deletions starting with Exon 45 were found in 76.1% (35/46 of the cases. Mutations in the majority of cases i.e. 39/46 (84.8% were seen in 3′ downstream region (Exon 45-55, distal rod domain. Few, i.e. 5/46 (10.8% showed deletions in 5′ upstream region (Exons 3-20, N-terminus and proximal rod domain of the gene, while in 2/46 (4.4% large mutations (>40 bp spanning both regions (Exons 3-55 were detected. Conclusion: This significant gene deletion analysis has been carried out for BMD patients particularly from Western India using 32 exons.

  17. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    Science.gov (United States)

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  18. Exons I and VII of the gene (Ker10) encoding human keratin 10 undergo structural rearrangements within repeats.

    Science.gov (United States)

    Tkachenko, A V; Buchman, V L; Bliskovsky, V V; Shvets YuP; Kisselev, L L

    1992-07-15

    A genomic fragment containing the K51 gene previously isolated from a rat genomic library by hybridization with the v-mos probe in nonstringent conditions [Chumakov et al., Dokl. Akad. Nauk SSSR 290 (1986) 1252-1254], resembles a human keratin type-I-encoding gene [Shvets et al., Mol. Biol. 24 (1990) 663-677]. This genomic clone, K51, has been used as a probe to search for related human genes. A recombinant clone, HK51, with a 1.5-kb insert, was isolated from a human embryonic skin cDNA library, and its nucleotide (nt) sequence was determined. Analysis has shown that the cloned cDNA encodes human keratin 10 (Ker10). All presently known nt sequences of the human Ker10-encoding gene (Ker10) are not identical. Differences are concentrated in the 5'-end of the first exon and in the middle of the seventh exon within repeats. In spite of structural rearrangements in two of eight exons, the reading frame and position of the stop codon are preserved. The genetic rearrangements cause changes in hydrophobicity profiles of the N and C termini of Ker10. It was also noticed that insertion of one nt leads to the formation of an unusual 3'-end of the transcript.

  19. A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Sailesh Gopalakrishna-Pillai

    Full Text Available A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs relative to spontaneously differentiated cells (SDCs. Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

  20. Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure.

    Science.gov (United States)

    Wang, Juejin; Li, Guang; Yu, Dejie; Wong, Yuk Peng; Yong, Tan Fong; Liang, Mui Cheng; Liao, Ping; Foo, Roger; Hoppe, Uta C; Soong, Tuck Wah

    2017-09-26

    Recently, we reported that homozygous deletion of alternative exon 33 of CaV1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased CaV1.2Δ33 ICaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33(+/-) cardiomyocytes showed similar CaV1.2 channel properties as wild-type cardiomyocyte, even though CaV1.2Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of CaV1.2 exon 33. (149 words).

  1. Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

    Directory of Open Access Journals (Sweden)

    Johann Böhm

    2013-06-01

    Full Text Available Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM, a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM. However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD. Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

  2. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

    Directory of Open Access Journals (Sweden)

    Jean-Paul Iyombe-Engembe

    2016-01-01

    Full Text Available The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD patients, expression of dystrophin (DYS protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel, a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

  3. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles.

    Science.gov (United States)

    Pichavant, Christophe; Chapdelaine, Pierre; Cerri, Daniel G; Bizario, Joao C S; Tremblay, Jacques P

    2010-11-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin (427 kDa). An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophin cDNA. Because the phenotype of the dystrophic dog is closer to the human phenotype than is the mdx mouse phenotype, we have studied the electrotransfer of a plasmid carrying the full-length dog dystrophin (FLDYS(dog)) in dystrophic dog muscle. To achieve this nonviral delivery, the FLDYS(dog) cDNA was cloned in two plasmids containing either a cytomegalovirus or a muscle creatine kinase promoter. In both cases, our results showed that the electrotransfer of these large plasmids (∼17 kb) into mouse muscle allowed FLDYS(dog) expression in the treated muscle. The electrotransfer of pCMV.FLDYS(dog) in a dystrophic dog muscle also led to the expression of dystrophin. In conclusion, introduction of the full-length dog dystrophin cDNA by electrotransfer into dystrophic dog muscle is a potential approach to restore dystrophin in patients with DMD. However, the electrotransfer procedure should be improved before applying it to humans.

  4. Polymorphisms of Exon 17 of Insulin-Receptor Gene in Pathogenesis of Human Disorders With Insulin Resistance

    Institute of Scientific and Technical Information of China (English)

    LU WANG; JIE MI; XIAO-YUAN ZHAO; JIAN-XIN WU; HONG CHENG; ZHI-KUN ZHANG; XIU-YUAN DING; DONG-QING HOU; HUILI

    2004-01-01

    To investigate the relationship between polymorphisms of insulin-receptor (INSR) gene and insulin resistance in a population-based study in China. Methods Polymerase Chain Reaction (PCR) was used to the amplify Exon 17 of INSR gene and all amplified products were analyzed by direct sequencing. Results Six single-nucleotide polymorphisms (SNPs) were found at the following loci: T to TC at the locus of 10699 (Tyr984), G to GC at the locus of 10731 (Glu994), Deletion G at the locus of 10798 (Asp1017), C to T/TC at the locus of 10923 (His1058), C to CA at the locus of 10954 (Leu1069), and T to TA at the locus of 10961 (Phe1071), which might not change the amino acid sequence. The data were in agreement with the test of Hardy-Weinberg balance (P>0.05). Among the 345 cases, all clinical indices were higher in males than in females except for HDL cholesterol (P0.05). After sex stratification in analysis,all allele frequencies on the six loci of SNPs of Exon 17 had different distributions between the insulin resistant group and the control group, but P>0.05. Conclusion SNPs of Exon 17 of INSR gene are unlikely to play a direct role in the pathogenesis of human disorders with insulin resistance.

  5. Structure of the human zinc finger protein HIVEP3: molecular cloning, expression, exon-intron structure, and comparison with paralogous genes HIVEP1 and HIVEP2.

    Science.gov (United States)

    Hicar, M D; Liu, Y; Allen, C E; Wu, L C

    2001-01-01

    Here we report the cloning and characterization of HIVEP3, the newest member in the human immunodeficiency virus type 1 enhancer-binding protein family that encodes large zinc finger proteins and regulates transcription via the kappaB enhancer motif. The largest open reading frame of HIVEP3 contains 2406 aa. and is approximately 80% identical to the mouse counterpart. The HIVEP3 gene is located in the chromosomal region 1p34 and is at least 300 kb with 10 exons. RNA studies show that multiple HIVEP3 transcripts are differentially expressed and regulated. Additionally, transcription termination occurs in the ultimate exon, exon 10, or in exon 6. Therefore, HIVEP3 may produce protein isoforms that contain or exclude the carboxyl DNA binding domain and the leucine zipper by alternative RNA splicing and differential polyadenylation. Sequence homologous to HIVEP3 exon 6 is not found in mouse nor are the paralogous genes HIVEP1 and HIVEP2. Zoo-blot analysis suggests that sequences homologous to the human exon 6 are present only in primates and cow. Therefore, a foreign DNA harboring a termination exon likely was inserted into the HIVEP3 locus relatively recently in evolution, resulting in the acquisition of novel gene regulatory mechanisms as well as the generation of structural and functional diversity. Copyright 2001 Academic Press.

  6. Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins

    DEFF Research Database (Denmark)

    Moghadaszadeh, Behzad; Albrechtsen, Reidar; Guo, Ling T;

    2003-01-01

    , and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin....... The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans....

  7. Dystrophin and the two related genetic diseases, Duchenne and Becker muscular dystrophies

    Directory of Open Access Journals (Sweden)

    Elisabeth Le Rumeur

    2015-07-01

    Full Text Available Mutations of the dystrophin DMD gene, essentially deletions of one or several exons, are the cause of two devastating and to date incurable diseases, Duchenne (DMD and Becker (BMD muscular dystrophies. Depending upon the preservation or not of the reading frame, dystrophin is completely absent in DMD, or present in either a mutated or a truncated form in BMD. DMD is a severe disease which leads to a premature death of the patients. Therapy approaches are evolving with the aim to transform the severe DMD in the BMD form of the disease by restoring the expression of a mutated or truncated dystrophin. These therapies are based on the assumption that BMD is a mild disease. However, this is not completely true as BMD patients are more or less severely affected and no molecular basis of this heterogeneity of the BMD form of the disease is yet understood. The aim of this review is to report for the correlation between dystrophin structures in BMD deletions in view of this heterogeneity and to emphasize that examining BMD patients in details is highly relevant to anticipate for DMD therapy effects.

  8. Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice.

    Science.gov (United States)

    Lee, Eun-Joo; Kim, Ah-Young; Lee, Eun-Mi; Lee, Myeong-Mi; Min, Chang-Woo; Kang, Kyung-Ku; Park, Jin-Kyu; Hwang, Meeyul; Kwon, Soon-Hak; Tremblay, Jacques P; Jeong, Kyu-Shik

    2014-08-01

    Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

  9. Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

    Science.gov (United States)

    Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier; Duclert, Aymeric; Mamchaoui, Kamel; Gouble, Agnés; Mouly, Vincent; Voit, Thomas; Pâques, Frédéric; Cédrone, Frédéric; Isman, Olga; Yáñez-Muñoz, Rafael J; Dickson, George

    2013-07-01

    Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

  10. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

    Directory of Open Access Journals (Sweden)

    Nayereh Nouri

    2014-01-01

    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  11. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  12. Becker muscular dystrophy due to an intronic splicing mutation inducing a dual dystrophin transcript.

    Science.gov (United States)

    Todeschini, Alice; Gualandi, Francesca; Trabanelli, Cecilia; Armaroli, Annarita; Ravani, Anna; Fanin, Marina; Rota, Silvia; Bello, Luca; Ferlini, Alessandra; Pegoraro, Elena; Padovani, Alessandro; Filosto, Massimiliano

    2016-10-01

    We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy.

    Science.gov (United States)

    Robinson-Hamm, Jacqueline N; Gersbach, Charles A

    2016-09-01

    Duchenne muscular dystrophy is one of the most common inherited genetic diseases and is caused by mutations to the DMD gene that encodes the dystrophin protein. Recent advances in genome editing and gene therapy offer hope for the development of potential therapeutics. Truncated versions of the DMD gene can be delivered to the affected tissues with viral vectors and show promising results in a variety of animal models. Genome editing with the CRISPR/Cas9 system has recently been used to restore dystrophin expression by deleting one or more exons of the DMD gene in patient cells and in a mouse model that led to functional improvement of muscle strength. Exon skipping with oligonucleotides has been successful in several animal models and evaluated in multiple clinical trials. Next-generation oligonucleotide formulations offer significant promise to build on these results. All these approaches to restoring dystrophin expression are encouraging, but many hurdles remain. This review summarizes the current state of these technologies and summarizes considerations for their future development.

  14. Loss of dystrophin is associated with increased myocardial stiffness in a model of left ventricular hypertrophy.

    Science.gov (United States)

    Donato, Martín; Buchholz, Bruno; Morales, Celina; Valdez, Laura; Zaobornyj, Tamara; Baratta, Sergio; Paez, Diamela T; Matoso, Mirian; Vaccarino, Guillermo; Chejtman, Demian; Agüero, Oscar; Telayna, Juan; Navia, José; Hita, Alejandro; Boveris, Alberto; Gelpi, Ricardo J

    2017-08-01

    Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.

  15. Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance.

    Science.gov (United States)

    Duning, K; Wennmann, D O; Bokemeyer, A; Reissner, C; Wersching, H; Thomas, C; Buschert, J; Guske, K; Franzke, V; Flöel, A; Lohmann, H; Knecht, S; Brand, S-M; Pöter, M; Rescher, U; Missler, M; Seelheim, P; Pröpper, C; Boeckers, T M; Makuch, L; Huganir, R; Weide, T; Brand, E; Pavenstädt, H; Kremerskothen, J

    2013-06-18

    The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca(2+) dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.

  16. Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance

    Science.gov (United States)

    Duning, K; Wennmann, D O; Bokemeyer, A; Reissner, C; Wersching, H; Thomas, C; Buschert, J; Guske, K; Franzke, V; Flöel, A; Lohmann, H; Knecht, S; Brand, S-M; Pöter, M; Rescher, U; Missler, M; Seelheim, P; Pröpper, C; Boeckers, T M; Makuch, L; Huganir, R; Weide, T; Brand, E; Pavenstädt, H; Kremerskothen, J

    2013-01-01

    The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca2+ dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved. PMID:23778582

  17. A Multi-Agent System for Exon Prediction in Human Sequences.

    Science.gov (United States)

    Vignal; Lisacek

    1997-01-01

    Given the problem of identifying exons in new genomic DNA, the sketch of a resolution process was drawn using sequence data and models of site/signal recognition. A multi-agent architecture is used to validate these models and test hypotheses on the chronology of events involved in gene splicing. Information is channelled through a hierarchy of agents. Each type of agent is the result of a successful step in the resolution process. The system does not rely on the compositional bias of coding sequences which is a key feature of current computer methods.

  18. Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

    Directory of Open Access Journals (Sweden)

    Rasic Milic V.

    2014-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation- dependent probe amplification (MLPA, polymerase chain reaction (PCR] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale. In 37 patients with an estimated full scale intelligence quotient (FSIQ, six (16.22% had borderline intelligence (70exons 30 and 45. However, FSIQ was statistically significantly associated with mutation location when we assumed their functional consequence on dystrophin isoforms and when mutations in the 5’-untranslated region (5’UTR of Dp140 (exons 45-50 were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients.

  19. Parental source effect of inherited mutations in the dystrophin gene of mice and men

    Energy Technology Data Exchange (ETDEWEB)

    Kress, W.; Grimm, T.; Mueller, C.R. [Institute of Human Genetics, Wuerburg (Germany); Bittner, R. [Institute of Anatomy, Wein (Australia)

    1994-09-01

    Skewed X-inactivation has been suspected the genetic cause for some manifesting female carriers of BMD and DMD. To test whether a parental source effect on the protein expression of the dystrophin gene exists, we have set up backcrosses of mdx mice to wild type strains, enabling us to study the effect of the well-defined origin of the mutation on the dystrophin expression. In skeletal muscle sections the immunohistological staining patterns of dystrophin antibodies were showing a significant difference in the proportion of dystrophin positive versus negative fibers, suggesting a lower expression of paternally inherited mdx mutations. These data are in concordance with the pyruvate kinase (PK) levels in the serum: PK levels were much higher when the mutation was of maternal origin as compared to PK levels in paternally derived mutations. In order to test this {open_quotes}paternal source effect{close_quotes} in humans, we checked obligatory carriers of Becker muscular dystrophy (BMD) for the origin of their mutations. Creatin kinase (CK) levels in 21 carriers with maternally derived mutations were compared to CK values from 8 heterozygotes with mutations of paternal origin: CK (mat) = 140.3 IU/1 versus CK (pat) = 48.6 IU/I. The difference is statistically significant at the 5% level. These observations suggest either a differential X-inactivation or an imprinting of the dystrophin gene in mice and men.

  20. Expression and new exon mutations of the human Beta defensins and their association on colon cancer development.

    Directory of Open Access Journals (Sweden)

    Abdelhabib Semlali

    Full Text Available The development of cancer involves genetic predisposition and a variety of environmental exposures. Genome-wide linkage analyses provide evidence for the significant linkage of many diseases to susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Human β-defensins (hBDs are important molecules of innate immunity. This study was designed to analyze the expression and genetic variations in hBDs (hBD-1, hBD-2, hBD-3 and hBD-4 and their putative association with colon cancer. hBD gene expression and relative protein expression were evaluated by Real-Time polymerase chain reaction (qPCR and immunohistochemistry, respectively, from 40 normal patients and 40 age-matched patients with colon cancer in Saudi Arabia. In addition, hBD polymorphisms were genotyped by exon sequencing and by promoter methylation. hBD-1, hBD-2, hBD-3 and hBD-4 basal messenger RNA expression was significantly lower in tumor tissues compared with normal tissues. Several insertion mutations were detected in different exons of the analyzed hBDs. However, no methylation in any hBDs promoters was detected because of the limited number of CpG islands in these regions. We demonstrated for the first time a link between hBD expression and colon cancer. This suggests that there is a significant link between innate immunity deregulation through disruption of cationic peptides (hBDs and the potential development of colon cancer.

  1. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Schipani, E.; Bergwitz, C.; Iida-Klein, A. [Massachusetts General Hospital, Boston, MA (United States)] [and others

    1995-05-01

    Pseudohypoparathyroidism type Ib (PHP-Ib) is thought to be from caused by a PTH/PTH-related peptide (PTHrP) receptor defect. To search for receptor mutations in genomic DNA from 17 PHP-Ib patients, three recently isolated human genomic DNA clones were further characterized by restriction enzyme mapping and nucleotide sequencing across intron/exon borders. Regions including all 14 coding exons and their splice junctions were amplified by polymerase chain reaction, and the products were analyzed by either temperature gradient gel electrophoresis or direct nucleotide sequencing. Silent polymorphisms were identified in exons G (1 of 17), M4 (1 of 17), and M7 (15 of 17). Two base changes were found in introns, 1 at the splice-donor site of the intron between exons E2 and E3 (1 of 17) and the other between exons G and M1 (2 of 17). Total ribonucleic acid from COS-7 cells expressing minigenes with or without the base change between exons E2 and E3 showed no difference by either Northern blot analysis or reverse transcriptase-polymerase chain reaction. Radioligand binding was indistinguishable for both transiently expressed constructs. A missense mutation (E546 to K546) in the receptor`s cytoplamic tail (3 of 17) was also found in 1 of 60 healthy individuals, and PTH/PTHrP receptors with this mutation were functionally indistinguishable from wild-type receptors. PHP-Ib thus appears to be rarely, if ever, caused by mutations in the coding exons of the PTH/PTHrP receptor gene. 58 refs., 4 figs., 4 tabs.

  2. Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.

    Science.gov (United States)

    Kotnis, Smita; Bingham, Brendan; Vasilyev, Dmitry V; Miller, Scott W; Bai, Yuchen; Yeola, Sarita; Chanda, Pranab K; Bowlby, Mark R; Kaftan, Edward J; Samad, Tarek A; Whiteside, Garth T

    2010-06-01

    P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

  3. High resolution profiling of human exon methylation by liquid hybridization capture-based bisulfite sequencing

    Directory of Open Access Journals (Sweden)

    Wang Junwen

    2011-12-01

    Full Text Available Abstract Background DNA methylation plays important roles in gene regulation during both normal developmental and disease states. In the past decade, a number of methods have been developed and applied to characterize the genome-wide distribution of DNA methylation. Most of these methods endeavored to screen whole genome and turned to be enormously costly and time consuming for studies of the complex mammalian genome. Thus, they are not practical for researchers to study multiple clinical samples in biomarker research. Results Here, we display a novel strategy that relies on the selective capture of target regions by liquid hybridization followed by bisulfite conversion and deep sequencing, which is referred to as liquid hybridization capture-based bisulfite sequencing (LHC-BS. To estimate this method, we utilized about 2 μg of native genomic DNA from YanHuang (YH whole blood samples and a mature dendritic cell (mDC line, respectively, to evaluate their methylation statuses of target regions of exome. The results indicated that the LHC-BS system was able to cover more than 97% of the exome regions and detect their methylation statuses with acceptable allele dropouts. Most of the regions that couldn't provide accurate methylation information were distributed in chromosomes 6 and Y because of multiple mapping to those regions. The accuracy of this strategy was evaluated by pair-wise comparisons using the results from whole genome bisulfite sequencing and validated by bisulfite specific PCR sequencing. Conclusions In the present study, we employed a liquid hybridisation capture system to enrich for exon regions and then combined with bisulfite sequencing to examine the methylation statuses for the first time. This technique is highly sensitive and flexible and can be applied to identify differentially methylated regions (DMRs at specific genomic locations of interest, such as regulatory elements or promoters.

  4. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    Science.gov (United States)

    Newton, Derek C; Bevan, Sian C; Choi, Stephen; Robb, G Brett; Millar, Adam; Wang, Yang; Marsden, Philip A

    2003-01-03

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

  5. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, M; Vissing, J

    2013-01-01

    Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic......, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest...

  6. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    Science.gov (United States)

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  7. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  8. Designing exons for human olfactory receptor gene subfamilies using a mathematical paradigm

    Indian Academy of Sciences (India)

    Sk Sarif Hassan; Pabitra Pal Choudhury; Amita Pal; R L Brahmachary; Arunava Goswami

    2010-09-01

    Ligands for only two human olfactory receptors are known. One of them, OR1D2, binds to Bourgeonal, a volatile chemical constituent of the fragrance of the mythical flower, Lily of the valley or Our Lady’s tears, Convallaria majalis (also the national flower of Finland). OR1D2, OR1D4 and OR1D5 are three full-length olfactory receptors present in an olfactory locus in the human genome. These receptors are more than 80% identical in DNA sequences and have 108 base pair mismatches among them. Apparently, these mismatch positions show no striking pattern using computer pattern recognition tools. In an attempt to find a mathematical rule in those mismatches, we find that an L-system generated sequence can be inserted into the OR1D2 subfamily-specific star model and novel full-length olfactory receptors can be generated. This remarkable mathematical principle could be utilized for making new subfamily olfactory receptor members from any olfactory receptor subfamily. The aroma and electronic nose industry might utilize this rule in future.

  9. Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6.

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    Chiharu Sogawa

    Full Text Available BACKGROUND: The transporters for dopamine (DAT and norepinephrine (NET are members of the Na+- and Cl--dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT and placenta (NET, both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms. CONCLUSIONS/SIGNIFICANCE: The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.

  10. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    Directory of Open Access Journals (Sweden)

    Dario Balestra

    2016-01-01

    Full Text Available In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1 can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G or 3′ (c.392-8T > G splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss splicing-competent human factor IX (hFIX cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml, leading to a striking shortening (from ≃100 seconds of untreated mice to ≃80 seconds of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

  11. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Richard S Finkel

    Full Text Available BACKGROUND: Approximately 13% of boys with Duchenne muscular dystrophy (DMD have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124 enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. METHODS: This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6 received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20 was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12 was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. FINDINGS: Twenty three of 38 (61% subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. INTERPRETATION: Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. TRIAL REGISTRATION: ClinicalTrials.gov NCT00264888.

  12. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy.

    Science.gov (United States)

    Finkel, Richard S; Flanigan, Kevin M; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H Lee; Reha, Allen; Northcutt, Valerie J; Elfring, Gary; Barth, Jay; Peltz, Stuart W

    2013-01-01

    Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. ClinicalTrials.gov NCT00264888.

  13. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartolo, C; Mendell, J R

    1993-01-01

    Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

  14. Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9

    Directory of Open Access Journals (Sweden)

    Hongmei Lisa Li

    2015-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

  15. Laryngeal Muscles Are Spared in the Dystrophin Deficient "mdx" Mouse

    Science.gov (United States)

    Thomas, Lisa B.; Joseph, Gayle L.; Adkins, Tracey D.; Andrade, Francisco H.; Stemple, Joseph C.

    2008-01-01

    Purpose: "Duchenne muscular dystrophy (DMD)" is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the…

  16. ExonMiner: Web service for analysis of GeneChip Exon array data

    Directory of Open Access Journals (Sweden)

    Imoto Seiya

    2008-11-01

    Full Text Available Abstract Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1 data normalization, (2 statistical analysis based on two-way ANOVA, (3 finding transcripts with significantly different splice patterns, (4 efficient visualization based on heatmaps and barplots, and (5 meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL http://ae.hgc.jp/exonminer. Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers.

  17. ExonMiner: Web service for analysis of GeneChip Exon array data

    Science.gov (United States)

    Numata, Kazuyuki; Yoshida, Ryo; Nagasaki, Masao; Saito, Ayumu; Imoto, Seiya; Miyano, Satoru

    2008-01-01

    Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1) data normalization, (2) statistical analysis based on two-way ANOVA, (3) finding transcripts with significantly different splice patterns, (4) efficient visualization based on heatmaps and barplots, and (5) meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL . Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers. PMID:19036125

  18. Sensitivity and Frequencies of Dystrophin Gene Mutations in Thai DMD/BMD Patients As Detected by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Thanyachai Sura

    2008-01-01

    Full Text Available Background: Duchenne muscular dystrophy (DMD, a lethal X-linked disease affecting 1 in 3500 male births, and its more benign variant, Becker muscular dystrophy (BMD, are caused by mutations in the dystrophin gene. Because of its large size, analysing the whole gene is impractical. Methods have been developed to detect the commonest mutations i.e. the deletions of the exons. Although these tests are highly specific, their sensitivity is inherently limited by the prevalence of deletions, which differs among different populations.

  19. Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues [v2; ref status: indexed, http://f1000r.es/2dl

    Directory of Open Access Journals (Sweden)

    Liliana Florea

    2013-11-01

    Full Text Available Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads.  Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60% were novel, involving new exons (~3000, new introns (~16000, or both. When tracing these events across the sixteen tissues, only a small number (4-7% appeared to be differentially expressed (‘switched’ between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared.  Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository (http://zenodo.org/record/7068; doi:10.5281/zenodo.7068 and from our web site http://ccb.jhu.edu/software/ASprofile.

  20. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

    Science.gov (United States)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker muscular dystrophy (BMD) is caused by a dystrophin insufficiency or expression of a partially functional protein product. Both of these dystrophinopathies are most commonly studied using the mdx mouse and a golden r...

  1. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9.

    Science.gov (United States)

    Chen, Yongchang; Zheng, Yinghui; Kang, Yu; Yang, Weili; Niu, Yuyu; Guo, Xiangyu; Tu, Zhuchi; Si, Chenyang; Wang, Hong; Xing, Ruxiao; Pu, Xiuqiong; Yang, Shang-Hsun; Li, Shihua; Ji, Weizhi; Li, Xiao-Jiang

    2015-07-01

    CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

  2. Leptospira Immunoglobulin-Like Protein B Interacts with the 20th Exon of Human Tropoelastin Contributing to Leptospiral Adhesion to Human Lung Cells

    Directory of Open Access Journals (Sweden)

    Ching-Lin Hsieh

    2017-05-01

    Full Text Available Leptospira immunoglobulin-like protein B (LigB, a surface adhesin, is capable of mediating the attachment of pathogenic leptospira to the host through interaction with various components of the extracellular matrix (ECM. Human tropoelastin (HTE, the building block of elastin, confers resilience and elasticity to lung, and other tissues. Previously identified Ig-like domains of LigB, including LigB4 and LigB12, bind to HTE, which is likely to promote Leptospira adhesion to lung tissue. However, the molecular mechanism that mediates the LigB-HTE interaction is unclear. In this study, the LigB-binding site on HTE was further pinpointed to a N-terminal region of the 20th exon of HTE (HTE20N. Alanine mutants of basic and aromatic residues on HTE20N significantly reduced binding to the LigB. Additionally, HTE-binding site was narrowed down to the first β-sheet of LigB12. On this binding surface, residues F1054, D1061, A1065, and D1066 were critical for the association with HTE. Most importantly, the recombinant HTE truncates could diminish the binding of LigB to human lung fibroblasts (WI-38 by 68%, and could block the association of LigA-expressing L. biflexa to lung cells by 61%. These findings should expand our understanding of leptospiral pathogenesis, particularly in pulmonary manifestations of leptospirosis.

  3. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Bengtsson, Niclas E; Hall, John K; Odom, Guy L; Phelps, Michael P; Andrus, Colin R; Hawkins, R David; Hauschka, Stephen D; Chamberlain, Joel R; Chamberlain, Jeffrey S

    2017-02-14

    Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx(4cv) mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

  4. Chemical and mechanistic toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice.

    Science.gov (United States)

    Sazani, Peter; Ness, Kirk P Van; Weller, Doreen L; Poage, Duane; Nelson, Keith; Shrewsbury, And Stephen B

    2011-05-01

    AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce skipping of dystrophin exon 51 and restore its expression in patients with Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in the dystrophic mdx mouse model requires skipping of exon 23, achieved with the mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological consequences of exon skipping and dystrophin restoration in mdx mice using AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals were generally limited to mild renal tubular basophilia/vacuolation, without any significant changes in renal function and with evidence of reversing. No toxicity associated with the mechanism of action of AVI-4225 in a dystrophic animal was observed.

  5. Quantitative analysis of the dystrophin gene by real-time PCR

    Directory of Open Access Journals (Sweden)

    Maksimovic Nela

    2012-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD/BMD are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt. The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective.

  6. Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle

    DEFF Research Database (Denmark)

    Hjortkjær, Camilla Brolin; Shiraishi, Takehiko; Hojman, Pernille;

    2015-01-01

    and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice...... switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find...... that electroporation can enhance PNA antisense effects in muscle tissue....

  7. Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

    Indian Academy of Sciences (India)

    MARYAM HAGHSHENAS; MOHAMMAD TAGHI AKBARI; SHOHREH ZARE KARIZI; FARAVAREH KHORDADPOOR DEILAMANI; SHAHRIAR NAFISSI; ZIVAR SALEHI

    2016-06-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progres-sive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletionsor duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to eval-uate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show anylarge deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependentprobe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 wassequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed fournonsense, one frameshift and two splice site mutations as well as two missense variants

  8. Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

    Science.gov (United States)

    Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

    2016-06-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

  9. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    Science.gov (United States)

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  10. Remarkable selective constraints on exonic dinucleotide repeats.

    Science.gov (United States)

    Haasl, Ryan J; Payseur, Bret A

    2014-09-01

    Long dinucleotide repeats found in exons present a substantial mutational hazard: mutations at these loci occur often and generate frameshifts. Here, we provide clear and compelling evidence that exonic dinucleotides experience strong selective constraint. In humans, only 18 exonic dinucleotides have repeat lengths greater than six, which contrasts sharply with the genome-wide distribution of dinucleotides. We genotyped each of these dinucleotides in 200 humans from eight 1000 Genomes Project populations and found a near-absence of polymorphism. More remarkably, divergence data demonstrate that repeat lengths have been conserved across the primate phylogeny in spite of what is likely considerable mutational pressure. Coalescent simulations show that even a very low mutation rate at these loci fails to explain the anomalous patterns of polymorphism and divergence. Our data support two related selective constraints on the evolution of exonic dinucleotides: a short-term intolerance for any change to repeat length and a long-term prevention of increases to repeat length. In general, our results implicate purifying selection as the force that eliminates new, deleterious mutants at exonic dinucleotides. We briefly discuss the evolution of the longest exonic dinucleotide in the human genome--a 10 x CA repeat in fibroblast growth factor receptor-like 1 (FGFRL1)--that should possess a considerably greater mutation rate than any other exonic dinucleotide and therefore generate a large number of deleterious variants. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

  11. The N-terminally truncated µ3 and µ3-like opioid receptors are transcribed from a novel promoter upstream of exon 2 in the human OPRM1 gene.

    Directory of Open Access Journals (Sweden)

    Sonja Andersen

    Full Text Available The human µ opioid receptor gene, OPRM1, produces a multitude of alternatively spliced transcripts encoding full-length or truncated receptor variants with distinct pharmacological properties. The majority of these transcripts are transcribed from the main promoter upstream of exon 1, or from alternate promoters associated with exons 11 and 13. Two distinct transcripts encoding six transmembrane domain (6TM hMOR receptors, µ3 and µ3-like, have been reported, both starting with the first nucleotide in exon 2. However, no mechanism explaining their initiation at exon 2 has been presented. Here we have used RT-PCR with RNA from human brain tissues to demonstrate that the µ3 and µ3-like transcripts contain nucleotide sequences from the intron 1-exon 2 boundary and are transcribed from a novel promoter located upstream of exon 2. Reporter gene assays confirmed the ability of the novel promoter to drive transcription in human cells, albeit at low levels. We also report the identification of a "full-length" seven transmembrane domain (7TM version of µ3, hMOR-1A2, which also contains exon 1, and a novel transcript, hMOR-1Y2, with the potential to encode the previously reported hMOR-1Y receptor, but with exon Y spliced to exon 4 instead of exon 5 as in hMOR-1Y. Heterologous expression of GFP-tagged hMOR variants in HEK 293 cells showed that both 6TM receptors were retained in the intracellular compartment and were unresponsive to exogenous opioid exposure as assessed by their ability to redistribute or affect cellular cAMP production, or to promote intracellular Ca(2+ release. Co-staining with an antibody specific for endoplasmic reticulum (ER indicated that the µ3-like receptor was retained at the ER after synthesis. 7TM receptors hMOR-1A2 and hMOR-1Y2 resided in the plasma membrane, and were responsive to opioids. Notably, hMOR-1A2 exhibits novel functional properties in that it did not internalize in response to the opioid peptide [D-Ala2, N

  12. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu-Ge; Ying-Nian Yu; Yu-Li Qian; Xin Li

    2002-01-01

    AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

  13. A marginal level of dystrophin partially ameliorates hindlimb muscle passive mechanical properties in dystrophin-null mice.

    Science.gov (United States)

    Hakim, Chady H; Duan, Dongsheng

    2012-12-01

    The goal of this study was to determine whether a minimal level of dystrophin expression improves the passive mechanical properties of skeletal muscle in the murine Duchenne muscular dystrophy model. We compared the elastic and viscous properties of the extensor digitorum longus muscle (EDL) in mdx3cv and mdx4cv mice at 6, 14, and 20 months of age. Both strains are on the C57Bl/6 background, and both lose the full-length dystrophin protein. Interestingly, mdx3cv mice express a near full-length dystrophin at ≈ 5% of the normal level. We found that the stress-strain profile and the stress relaxation rate of the EDL in mdx3cv mice were partially preserved in all age groups compared with age-matched mdx4cv mice. Our results suggest that a low level of dystrophin expression may treat muscle stiffness in Duchenne muscular dystrophy. Copyright © 2012 Wiley Periodicals, Inc.

  14. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype

    Science.gov (United States)

    Pierson, Christopher R.; Dulin-Smith, Ashley N.; Durban, Ashley N.; Marshall, Morgan L.; Marshall, Jordan T.; Snyder, Andrew D.; Naiyer, Nada; Gladman, Jordan T.; Chandler, Dawn S.; Lawlor, Michael W.; Buj-Bello, Anna; Dowling, James J.; Beggs, Alan H.

    2012-01-01

    X-linked myotubular myopathy (MTM) is a severe neuromuscular disease of infancy caused by mutations of MTM1, which encodes the phosphoinositide lipid phosphatase, myotubularin. The Mtm1 knockout (KO) mouse has a severe phenotype and its short lifespan (8 weeks) makes it a challenge to use as a model in the testing of certain preclinical therapeutics. Many MTM patients succumb early in life, but some have a more favorable prognosis. We used human genotype–phenotype correlation data to develop a myotubularin-deficient mouse model with a less severe phenotype than is seen in Mtm1 KO mice. We modeled the human c.205C>T point mutation in Mtm1 exon 4, which is predicted to introduce the p.R69C missense change in myotubularin. Hemizygous male Mtm1 p.R69C mice develop early muscle atrophy prior to the onset of weakness at 2 months. The median survival period is 66 weeks. Histopathology shows small myofibers with centrally placed nuclei. Myotubularin protein is undetectably low because the introduced c.205C>T base change induced exon 4 skipping in most mRNAs, leading to premature termination of myotubularin translation. Some full-length Mtm1 mRNA bearing the mutation is present, which provides enough myotubularin activity to account for the relatively mild phenotype, as Mtm1 KO and Mtm1 p.R69C mice have similar muscle phosphatidylinositol 3-phosphate levels. These data explain the basis for phenotypic variability among human patients with MTM1 p.R69C mutations and establish the Mtm1 p.R69C mouse as a valuable model for the disease, as its less severe phenotype will expand the scope of testable preclinical therapies. PMID:22068590

  15. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

    Directory of Open Access Journals (Sweden)

    Thomas C. Roberts

    2016-03-01

    Full Text Available Duchenne muscular dystrophy (DMD is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1 the determination of gene expression changes associated with dystrophic pathology, (2 identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3 investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis, and (4 prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO with the accession number GSE64420.

  16. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  17. The "alternative" choice of constitutive exons throughout evolution.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2007-11-01

    Full Text Available Alternative cassette exons are known to originate from two processes-exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5' splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.

  18. Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    Directory of Open Access Journals (Sweden)

    Caples Matt

    2004-07-01

    Full Text Available Abstract Background Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. Results We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. Conclusion These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.

  19. Bortezomib (PS-341 treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

    Directory of Open Access Journals (Sweden)

    Karla P C Araujo

    Full Text Available Golden retriever muscular dystrophy (GRMD is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs with the proteasome inhibitor bortezomib, and three were control dogs (CD. Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some

  20. Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

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    Christa L Tanganyika-de Winter

    2012-01-01

    Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

  1. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

    OpenAIRE

    Zhao, Lei; Chao-ping HU; Wang, Yi; Shui-zhen ZHOU; Shi, Yi-Yun; Xi-hua LI

    2015-01-01

    Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC) from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD) is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles o...

  2. The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

    Science.gov (United States)

    Manoni, M; Tribioli, C; Lazzari, B; DeBellis, G; Patrosso, C; Pergolizzi, R; Pellegrini, M; Maestrini, E; Rivella, S; Vezzoni, P

    1991-03-01

    An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.

  3. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2.

    Science.gov (United States)

    Witting, N; Duno, M; Vissing, J

    2013-01-01

    Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest that this unusual phenotype is caused by translation re-initiation downstream from the mutation site. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Lubahn, D.B.; Simental, J.A.; Higgs, H.N.; Wilson, E.M.; French, F.S. (Univ. of North Carolina, Chapel Hill (USA)); Brown, T.R.; Migeon, C.J. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1989-12-01

    Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. The authors have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46, XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

  5. Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure.

    Science.gov (United States)

    Rafael, J A; Townsend, E R; Squire, S E; Potter, A C; Chamberlain, J S; Davies, K E

    2000-05-22

    The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.

  6. The roles of dystrophin and dystrobrevin : in synaptic signaling in drosophila

    NARCIS (Netherlands)

    Potikanond, Saranyapin

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a disease, characterized by progressive muscle wasting, caused by the lack of Dystrophin. A subset of DMD patients also have cognitive deficits likely due to the absence of Dystrophin from brain synapses where it is usually localized. Dystrophin and a number of o

  7. Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization

    Directory of Open Access Journals (Sweden)

    Graham Ian R

    2010-06-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology. Results Using RNA interference (RNAi to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (Pvalb and Casq1, or cardiomyopathy (Obscurin, Tcap. In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels in vivo and in vitro. Conclusion Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage

  8. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    Full Text Available Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

  9. Disodium cromoglycate protects dystrophin-deficient muscle fibers from leakiness.

    Science.gov (United States)

    Marques, Maria Julia; Ventura Machado, Rafael; Minatel, Elaine; Santo Neto, Humberto

    2008-01-01

    In dystrophin-deficient fibers of mdx mice and in Duchenne dystrophy, the lack of dystrophin leads to sarcolemma breakdown and muscle degeneration. We verified that cromolyn, a mast-cell stabilizer agent, stabilized dystrophic muscle fibers using Evans blue dye as a marker of sarcolemma leakiness. Mdx mice (n=8; 14 days of age) received daily intraperitoneal injections of cromolyn (50 mg/kg body weight) for 15 days. Untreated mdx mice (n=8) were injected with saline. Cryostat cross-sections of the sternomastoid, tibialis anterior, and diaphragm muscles were stained with hematoxylin and eosin. Cromolyn dramatically reduced Evans blue dye-positive fibers in all muscles (P<0.05; Student's t-test) and led to a significant increase in the percentage of fibers with peripheral nuclei. This study supports the protective effects of cromolyn in dystrophic muscles and further indicates its action against muscle fiber leakiness in muscles that are differently affected by the lack of dystrophin.

  10. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Verheul, Ruurd C.; van Deutekom, Judith C. T.; Datson, Nicole A.

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  11. Differentiated evolutionary rates in alternative exons and the implications for splicing regulation

    Directory of Open Access Journals (Sweden)

    Eyras Eduardo

    2006-06-01

    Full Text Available Abstract Background Alternatively spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. Two contradicting properties have been associated to evolutionary conserved alternative exons: higher sequence conservation and higher rate of non-synonymous substitutions, relative to constitutive exons. In order to clarify this issue, we have performed an analysis of the evolution of alternative and constitutive exons, using a large set of protein coding exons conserved between human and mouse and taking into account the conservation of the transcript exonic structure. Further, we have also defined a measure of the variation of the arrangement of exonic splicing enhancers (ESE-conservation score to study the evolution of splicing regulatory sequences. We have used this measure to correlate the changes in the arrangement of ESEs with the divergence of exon and intron sequences. Results We find evidence for a relation between the lack of conservation of the exonic structure and the weakening of the sequence evolutionary constraints in alternative and constitutive exons. Exons in transcripts with non-conserved exonic structures have higher synonymous (dS and non-synonymous (dN substitution rates than exons in conserved structures. Moreover, alternative exons in transcripts with non-conserved exonic structure are the least constrained in sequence evolution, and at high EST-inclusion levels they are found to be very similar to constitutive exons, whereas alternative exons in transcripts with conserved exonic structure have a dS significantly lower than average at all EST-inclusion levels. We also find higher conservation in the arrangement of ESEs in constitutive exons compared to alternative ones. Additionally, the sequence conservation at flanking introns remains constant for constitutive exons at all ESE-conservation values, but increases for

  12. Transposable elements in disease-associated cryptic exons.

    Science.gov (United States)

    Vorechovsky, Igor

    2010-02-01

    Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon-intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.

  13. Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin.

    Science.gov (United States)

    Tachi, N; Ohya, K; Chiba, S; Matsuo, M; Patria, S Y; Matsumura, K

    1997-08-01

    Primary deficiency of merosin is the cause of the classic form of congenital muscular dystrophy (CMD) accompanied by brain white matter abnormalities. We report a female infant with dystrophinopathy who was deficient in merosin in skeletal muscle. The patient had a phenotype of typical CMD and white matter abnormalities on brain MRI. Merosin was greatly reduced in the biopsied skeletal muscle. However, the expression of dystroglycan and syntrophin was also greatly reduced, and the immunoreactivity for the antibodies against the cysteine-rich/C-terminal domains of dystrophin was absent in the sarcolemma. Reverse transcriptase polymerase chain reaction analysis of the dystrophin gene revealed a complete lack of exons 71 through 74. In skeletal muscle, only the mutant gene was expressed. These results suggest that the patient is a symptomatic Duchenne muscular dystrophy carrier with skewed X-inactivation. This patient illustrates for the first time that a dystrophin abnormality can cause a secondary deficiency of merosin in dystrophinopathy. The reduction of merosin may account for the clinical phenotype of CMD and correlate with the white matter abnormalities in our patient.

  14. Deletion analysis of the dystrophin gene in Duchenne and Becker muscular dystrophy patients: Use in carrier diagnosis

    Directory of Open Access Journals (Sweden)

    Kumari D

    2003-04-01

    Full Text Available The dystrophin gene was analyzed in 8 Duchenne muscular dystrophy (DMD and 10 Becker muscular dystrophy (BMD unrelated families (22 subjects: 18 index cases and 4 sibs for the presence of deletions by multiplex polymerase chain reaction (mPCR; 27 exons and Southern hybridization using 8 cDMD probes. Deletions were identified in 5 DMD and 7 BMD patients (6 index cases and 1 sib. The concordance between the clinical phenotype and 'reading frame hypothesis' was observed in 11/12 patients (92%. The female relatives of DMD/BMD patients with identifiable deletions were examined by quantitative mPCR. Carriers were identified in 7 families. We also describe a variation in the HindIII pattern with cDNA probe 8 and 11-14. Molecular characterization of the dystrophin gene in this study has been helpful in advising the patients concerning the inheritance of the condition, and carrier diagnosis of female relatives, and should also prove useful for prenatal diagnosis.

  15. Dystrophin, utrophin and {beta}-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Kawajiri, Masakazu; Mitsui, Takao; Kawai, Hisaomi [Univ. of Tokushima (Japan)] [and others

    1996-08-01

    The precise localization and semiquantitative correlation of dystrophin, utrophin and {beta}-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of {beta}-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of {beta}-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of {beta}-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to {beta}-dystroglycan. 35 refs., 3 figs., 1 tab.

  16. A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin.

    Science.gov (United States)

    Legardinier, Sébastien; Legrand, Baptiste; Raguénès-Nicol, Céline; Bondon, Arnaud; Hardy, Serge; Tascon, Christophe; Le Rumeur, Elisabeth; Hubert, Jean-François

    2009-03-27

    Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.

  17. Canine disorder mirrors human disease: exonic deletion in HES7 causes autosomal recessive spondylocostal dysostosis in miniature Schnauzer dogs.

    Directory of Open Access Journals (Sweden)

    Cali E Willet

    Full Text Available Spondylocostal dysostosis is a congenital disorder of the axial skeleton documented in human families from diverse racial backgrounds. The condition is characterised by truncal shortening, extensive hemivertebrae and rib anomalies including malalignment, fusion and reduction in number. Mutations in the Notch signalling pathway genes DLL3, MESP2, LFNG, HES7 and TBX6 have been associated with this defect. In this study, spondylocostal dysostosis in an outbred family of miniature schnauzer dogs is described. Computed tomography demonstrated that the condition mirrors the skeletal defects observed in human cases, but unlike most human cases, the affected dogs were stillborn or died shortly after birth. Through gene mapping and whole genome sequencing, we identified a single-base deletion in the coding region of HES7. The frameshift mutation causes loss of functional domains essential for the oscillatory transcriptional autorepression of HES7 during somitogenesis. A restriction fragment length polymorphism test was applied within the immediate family and supported a highly penetrant autosomal recessive mode of inheritance. The mutation was not observed in wider testing of 117 randomly sampled adult miniature schnauzer and six adult standard schnauzer dogs; providing a significance of association of Praw = 4.759e-36 (genome-wide significant. Despite this apparently low frequency in the Australian population, the allele may be globally distributed based on its presence in two unrelated sires from geographically distant locations. While isolated hemivertebrae have been observed in a small number of other dog breeds, this is the first clinical and genetic diagnosis of spontaneously occurring spondylocostal dysostosis in a non-human mammal and offers an excellent model in which to study this devastating human disorder. The genetic test can be utilized by dog breeders to select away from the disease and avoid unnecessary neonatal losses.

  18. The dystrophin gene and cognitive function in the general population

    NARCIS (Netherlands)

    D. Vojinovic (Dina); H.H.H. Adams (Hieab); S. van der Lee (Sven); C.A. Ibrahim-Verbaas (Carla); R.W.W. Brouwer (Rutger); M.C.G.N. van den hout (Mirjam); E. Oole (Edwin); J. van Rooij (Jeroen); A.G. Uitterlinden (André); A. Hofman (Albert); W.F.J. van IJcken (Wilfred); A. Aartsma-Rus (Annemieke); G.-J.B. Van Ommen (Gert-Jan B.); M.A. Ikram (Arfan); C.M. van Duijn (Cornelia M.); N. Amin (Najaf)

    2015-01-01

    textabstractThe aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterd

  19. Role of dystrophins and utrophins in platelet adhesion process.

    Science.gov (United States)

    Cerecedo, Doris; Mondragón, Ricardo; Cisneros, Bulmaro; Martínez-Pérez, Francisco; Martínez-Rojas, Dalila; Rendón, Alvaro

    2006-07-01

    Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

  20. Ocular and neurodevelopmental features of Duchenne muscular dystrophy: a signature of dystrophin function in the central nervous system.

    Science.gov (United States)

    Ricotti, Valeria; Jägle, Herbert; Theodorou, Maria; Moore, Anthony T; Muntoni, Francesco; Thompson, Dorothy A

    2016-04-01

    Multiple isoforms of dystrophin (Dp427, Dp260, Dp140, Dp71) are expressed differentially in the central nervous system (CNS) including the retinal layers. Disruption of these protein products is responsible for cognitive dysfunction, electroretinogram (ERG) abnormalities and behavioural disorders in Duchenne muscular dystrophy (DMD). We studied the ocular characteristics and neuropsychiatric profile of 16 DMD boys. The ISCEV standard, full-field flash ERGs were assessed. Intellectual ability and behavioural disturbances were measured. All genotypes were associated with mildly abnormal photopic ERG a:b-wave amplitude ratios. In addition, we identified the following genotype/phenotype correlations: boys with mutations upstream of exon 30 (ie, isolated Dp427 altered expression) showed normal scotopic a:b ratios, abnormal photopic oscillatory potential OP2 and normal scotopic OP2. Conversely, all boys with DMD mutations downstream of exon 30 showed profoundly 'negative' scotopic ERGs (a:b ratios >1). In these patients, the involvement of Dp260 isoform resulted in the absence of slow rod pathway signalling in15 Hz scotopic flicker ERGs. These boys had abnormal scotopic OP2 and normal photopic OP2. Finally, children with mutations also affecting Dp71 were associated with more pronounced electronegative ERGs. When correlating ERGs to neurodevelopmental outcome, we found a positive correlation between negative scotopic ERGs and neurodevelopmental disturbances, and the most severe findings were in boys with Dp71 disruption. These findings suggest a strong association between DMD mutations affecting different DMD isoforms with characteristically abnormal scotopic ERGs and severe neurodevelopmental problems. The role of the ERG as a potential biomarker for dystrophin function in the CNS and response to novel genetic therapies warrants further exploration.

  1. The role of D4 receptor gene exon III polymorphisms in shaping human altruism and prosocial behavior

    Directory of Open Access Journals (Sweden)

    Yushi eJiang

    2013-05-01

    Full Text Available Human beings are an extraordinarily altruistic species often willing to help strangers at a considerable cost (sometimes life itself to themselves. But as Darwin noted …he who was ready to sacrifice his life, as many a savage has been, rather than betray his comrades, would often leave no offspring to inherit his noble nature. Hence, this is the paradox of altruism. Twin studies have shown that altruism and other prosocial behavior show considerable heritability and more recently a number of candidate genes have been identified with this phenotype. Among these first provisional findings are genes encoding elements of dopaminergic transmission. In this article we will review the evidence for the involvement of one of these, the dopamine D4 receptor (DRD4 gene, in shaping human prosocial behavior and consider the methodologies employed in measuring this trait, specific molecular genetic findings and finally, evidence from several Gene x Environment (GxE studies that imply differential susceptibility of this gene to environmental influences.

  2. Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248-249

    Energy Technology Data Exchange (ETDEWEB)

    Zelinka, L. [Biomedical Sciences Program, Kent State University, Kent, OH (United States); McCann, S.; Budde, J.; Sethi, S.; Guidos, M.; Giles, R. [Center for Applied Chemical Biology, Department of Biological Sciences, Youngstown State University, One University Plaza, Youngstown, OH 44555 (United States); Walker, G.R., E-mail: grwalker@ysu.edu [Center for Applied Chemical Biology, Department of Biological Sciences, Youngstown State University, One University Plaza, Youngstown, OH 44555 (United States); Biomedical Sciences Program, Kent State University, Kent, OH (United States)

    2011-08-05

    Highlights: {yields} Affinity purification of the autoimmune rippling muscle disease immunogenic domain of titin. {yields} Partial sequence analysis confirms that the peptides is in the I band region of titin. {yields} This region of the human titin shows high degree of homology to mouse titin N2-A. -- Abstract: Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: (EU428784)) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.

  3. Deletion of the Chd6 exon 12 affects motor coordination.

    Science.gov (United States)

    Lathrop, Melissa J; Chakrabarti, Lisa; Eng, Jeremiah; Rhodes, C Harker; Lutz, Thomas; Nieto, Amelia; Liggitt, H Denny; Warner, Sandra; Fields, Jennifer; Stöger, Reinhard; Fiering, Steven

    2010-04-01

    Members of the CHD protein family play key roles in gene regulation through ATP-dependent chromatin remodeling. This is facilitated by chromodomains that bind histone tails, and by the SWI2/SNF2-like ATPase/helicase domain that remodels chromatin by moving histones. Chd6 is ubiquitously expressed in both mouse and human, with the highest levels of expression in the brain. The Chd6 gene contains 37 exons, of which exons 12-19 encode the highly conserved ATPase domain. To determine the biological role of Chd6, we generated mouse lines with a deletion of exon 12. Chd6 without exon 12 is expressed at normal levels in mice, and Chd6 Exon 12 -/- mice are viable, fertile, and exhibit no obvious morphological or pathological phenotype. Chd6 Exon 12 -/- mice lack coordination as revealed by sensorimotor analysis. Further behavioral testing revealed that the coordination impairment was not due to muscle weakness or bradykinesia. Histological analysis of brain morphology revealed no differences between Chd6 Exon 12 -/- mice and wild-type (WT) controls. The location of CHD6 on human chromosome 20q12 is overlapped by the linkage map regions of several human ataxias, including autosomal recessive infantile cerebellar ataxia (SCAR6), a nonprogressive cerebrospinal ataxia. The genomic location, expression pattern, and ataxic phenotype of Chd6 Exon 12 -/- mice indicate that mutations within CHD6 may be responsible for one of these ataxias.

  4. Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies

    Directory of Open Access Journals (Sweden)

    Caroline Perronnet

    2010-01-01

    Full Text Available Two decades of molecular, cellular, and functional studies considerably increased our understanding of dystrophins function and unveiled the complex etiology of the cognitive deficits in Duchenne muscular dystrophy (DMD, which involves altered expression of several dystrophin-gene products in brain. Dystrophins are normally part of critical cytoskeleton-associated membrane-bound molecular scaffolds involved in the clustering of receptors, ion channels, and signaling proteins that contribute to synapse physiology and blood-brain barrier function. The utrophin gene also drives brain expression of several paralogs proteins, which cellular expression and biological roles remain to be elucidated. Here we review the structural and functional properties of dystrophins and utrophins in brain, the consequences of dystrophins loss-of-function as revealed by numerous studies in mouse models of DMD, and we discuss future challenges and putative therapeutic strategies that may compensate for the cognitive impairment in DMD based on experimental manipulation of dystrophins and/or utrophins brain expression.

  5. Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

    Science.gov (United States)

    Datson, N A; van de Vosse, E; Dauwerse, H G; Bout, M; van Ommen, G J; den Dunnen, J T

    1996-03-15

    To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

  6. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  7. Evolutionary history of exon shuffling.

    Science.gov (United States)

    França, Gustavo S; Cancherini, Douglas V; de Souza, Sandro J

    2012-06-01

    Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.

  8. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Meng, Jinhong; Counsell, John R; Reza, Mojgan; Laval, Steven H; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E

    2016-01-27

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

  9. A nonsense mutation (Arg-196-Term) in exon 6 of the human TP53 gene identified in small cell lung carcinoma

    NARCIS (Netherlands)

    Hayes, VM; Oosthuizen, CJJ; Kotze, MJ; Marx, MP; Buys, CHCM

    1996-01-01

    In a search for mutations of the TP53 tumour suppressor gene in lung cancer samples from gold miners from the Witwatersrand, South Africa, using heteroduplex and single strand conformation polymorphism (SSCP) analysis, a nonsense mutation was found in exon 6, consisting of a C to T transition and re

  10. 14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

    Science.gov (United States)

    Noutsios, Georgios T; Ghattas, Paul; Bennett, Stephanie; Floros, Joanna

    2015-07-15

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

  11. Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

    Science.gov (United States)

    Jirka, Silvana M G; Heemskerk, Hans; Tanganyika-de Winter, Christa L; Muilwijk, Daan; Pang, Kar Him; de Visser, Peter C; Janson, Anneke; Karnaoukh, Tatyana G; Vermue, Rick; 't Hoen, Peter A C; van Deutekom, Judith C T; Aguilera, Begoña; Aartsma-Rus, Annemieke

    2014-02-01

    Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

  12. EasyExonPrimer: automated primer design for exon sequences.

    Science.gov (United States)

    Wu, Xiaolin; Munroe, David J

    2006-01-01

    EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. Xiaolin Wu (forestwu@mail.nih.gov).

  13. Wild-type mouse models to screen antisense oligonucleotides for exon-skipping efficacy in Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Limin Cao

    Full Text Available A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD, a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.

  14. The influence of low dystrophin levels on disease pathology in mouse models for Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    Putten, Maaike van

    2013-01-01

    Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature de

  15. The first constant-domain (CH1) exon of human IGHG2 is polymorphic and in strong linkage disequilibrium with the CH2 exon polymorphism encoding the G2m(n+) allotype in Caucasians

    DEFF Research Database (Denmark)

    Hougs, L; Svejgaard, A; Barington, T

    2001-01-01

    Here we describe a hitherto unknown proline/threonine polymorphism at residue 72 of the human IgG2 CH1 domain (EU numbering 189) and show that it is linked to the known valine/methionine polymorphism at residue 52 of CH2 (EU numbering 282) defining the G2m(n+)/G2m(n-) allotypes. We sequenced...

  16. Carrier detection of duchenne and becker muscular dystrophy using muscle dystrophin immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Acary S. Bulle Oliveira

    1992-12-01

    Full Text Available To ascertain whether dystrophin immunohistochemistry could improve DMD/ BMD carrier detection, we analyzed 14 muscle biopsies from 13 DMD and one BMD probable and possible carriers. All women were also evaluated using conventional methods, including genetic analysis, clinical and neurological evaluation, serum CK levels, KMG, and muscle biopsy. In 6 cases, there was a mosaic of dystrophin-positive and dystrophin-deficient fibers that allowed to make the diagnosis of a carrier state. Comparing dystrophin immunohistochemistry to the traditional methods, it was noted that this method is less sensitive than serum CK measuremens, but is more sensitive than EMG and muscle biopsy. The use of dystrophin immunohistochemistry in addition to CK, EMG and muscle biopsy improved the accuracy of carrier detection. This method is also helpful to distinguish manifesting DMD carriers from patients with other neuromuscular diseases like limb-girdle muscular dystrophy and spinal muscular atrophy.

  17. Role of dystrophin in airway smooth muscle phenotype, contraction and lung function.

    Directory of Open Access Journals (Sweden)

    Pawan Sharma

    Full Text Available Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh when compared to genetic control BL10ScSnJ mice (wild-type. In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.

  18. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  19. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  20. Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary.

    Science.gov (United States)

    Jenkins, C; Michael, D; Mahendroo, M; Simpson, E

    1993-11-01

    Estrogens are synthesized from C19 steroids by a unique form of cytochrome P450, aromatase cytochrome P-450 (P-450AROM; the product of the CYP19 gene). We have shown that tissue-specific expression of human P-450AROM is determined, in part, by the use of alternative promoters. Previous methods of analysis for determining the specific 5'-termini of the different transcripts included S1 nuclease protection, primer extension, and Northern analysis. In the present study we have used the RACE procedure (rapid amplification of cDNA ends) to amplify and clone the 5' termini of P-450AROM transcripts expressed in human corpus luteum (CL). Sequencing of the resulting clones supports the results of the previously performed studies. Specifically, the proximal promoter, PII, is the predominant promoter utilized in CL, such that the start of transcription occurs 26 bp downstream of the putative TATA sequence. A minority of the clones possess an alternative 5'-end, namely I.3. Exon-specific Northern analysis confirms that the majority of the P-450AROM transcripts in CL tissue contain sequence specific for promoter II. Similarly, exon-specific Northern analysis indicates that transcripts in human follicles, as well as granulosa cells in culture, contain primarily sequence specific for promoter II.

  1. Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

    OpenAIRE

    Datson, N.A.; Vosse, E van de; Dauwerse, H.G.; Bout, M; van Ommen, G J; J T den Dunnen

    1996-01-01

    To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is de...

  2. Dystrophin is required for the normal function of the cardio-protective K(ATP channel in cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Laura Graciotti

    Full Text Available Duchenne and Becker muscular dystrophy patients often develop a cardiomyopathy for which the pathogenesis is still unknown. We have employed the murine animal model of Duchenne muscular dystrophy (mdx, which develops a cardiomyopathy that includes some characteristics of the human disease, to study the molecular basis of this pathology. Here we show that the mdx mouse heart has defects consistent with alteration in compounds that regulate energy homeostasis including a marked decrease in creatine-phosphate (PC. In addition, the mdx heart is more susceptible to anoxia than controls. Since the cardio-protective ATP sensitive potassium channel (K(ATP complex and PC have been shown to interact we investigated whether deficits in PC levels correlate with other molecular events including K(ATP ion channel complex presence, its functionality and interaction with dystrophin. We found that this channel complex is present in the dystrophic cardiac cell membrane but its ability to sense a drop in the intracellular ATP concentration and consequently open is compromised by the absence of dystrophin. We further demonstrate that the creatine kinase muscle isoform (CKm is displaced from the plasma membrane of the mdx cardiac cells. Considering that CKm is a determinant of K(ATP channel complex function we hypothesize that dystrophin acts as a scaffolding protein organizing the K(ATP channel complex and the enzymes necessary for its correct functioning. Therefore, the lack of proper functioning of the cardio-protective K(ATP system in the mdx cardiomyocytes may be part of the mechanism contributing to development of cardiac disease in dystrophic patients.

  3. Proteins, exons and molecular evolution.

    Science.gov (United States)

    Holland, S K; Blake, C C

    1987-01-01

    The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.

  4. Tdp-43 cryptic exons are highly variable between cell types.

    Science.gov (United States)

    Jeong, Yun Ha; Ling, Jonathan P; Lin, Sophie Z; Donde, Aneesh N; Braunstein, Kerstin E; Majounie, Elisa; Traynor, Bryan J; LaClair, Katherine D; Lloyd, Thomas E; Wong, Philip C

    2017-02-02

    TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle. In the present work, we investigated TDP-43's function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function. Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific. Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways.

  5. Sequence Analysis of Hoxc8 Exon-1 and Exon-2 of Multi-Vertebrae Mongolia Sheep%多脊椎蒙古羊Hoxc8 exon-1和exon-2的序列分析

    Institute of Scientific and Technical Information of China (English)

    陈琦; 赵静; 张立岭; 马月辉

    2011-01-01

    参考牛的Hoxc8基因序列设计引物,扩增正常蒙古羊(胸椎数13)和多脊椎蒙古羊(胸椎数14)Hoxc8的exon-1和exon-2基因,对得到的序列进行生物信息学分析。结果表明,经序列比对二者的DNA序列,除两侧个别碱基有差异外,中间序列完全一致。蒙古羊Hoxc8的exon-1和exon-2序列分别与其他物种进行同源性比对,蒙古羊Hoxc8 exon-1与人、小鼠、大鼠、犬的同源性达到96%以上,与斑马鱼的同源性为75.8%;exon-2与大猩猩、犬、人、小鼠、大鼠的同源性达到91%以上,与斑马鱼的同源性%In our study,according to the Hoxc8 sequence of cow,the specific primers were designed,and the sequences of Hoxc8 exon-1(432 bp)and exon-2(273 bp)of normal and multi-thoracic vertebrae mongolia sheep were obtained(Genebank accession number: EU817489 and FJ905472).Alignment results of them indicated that the sequences were conformity except a little difference in two sides of sequences.Hoxc8 exon-1 and exon-2 were aligned with other species and the results showed that compared with other mammals(human,dog,mouse,rat and chimpanzee),the homology were above 96%(exon-1) and 91%(exon-2);compared with zebra fish,the homology were 75.8% and 74%.

  6. Rodent-specific alternative exons are more frequent in rapidly evolving genes and in paralogs

    Directory of Open Access Journals (Sweden)

    Mironov Andrey A

    2009-06-01

    Full Text Available Abstract Background Alternative splicing is an important mechanism for generating functional and evolutionary diversity of proteins in eukaryotes. Here, we studied the frequency and functionality of recently gained, rodent-specific alternative exons. Results We projected the data about alternative splicing of mouse genes to the rat, human, and dog genomes, and identified exons conserved in the rat genome, but missing in more distant genomes. We estimated the frequency of rodent-specific exons while controlling for possible residual conservation of spurious exons. The frequency of rodent-specific exons is higher among predominantly skipped exons and exons disrupting the reading frame. Separation of all genes by the rate of sequence evolution and by gene families has demonstrated that rodent-specific cassette exons are more frequent in rapidly evolving genes and in rodent-specific paralogs. Conclusion Thus we demonstrated that recently gained exons tend to occur in fast-evolving genes, and their inclusion rate tends to be lower than that of older exons. This agrees with the theory that gain of alternative exons is one of the major mechanisms of gene evolution.

  7. Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification (MLPA) effects of detection of gene mutations. Methods: Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspicion of DMD or BMD were tested by MLPA and multiplex PCR. Results: The mean DQs for all peak of 20 control male samples was 1.02 (range from 0.83 to 1.21) by MLPA. Deletions or duplications were identified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions: dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions: MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detection of DMD and BMD.

  8. Restoring Dystrophin Expression in Duchenne Muscular Dystrophy Muscle: Progress in Exon Skipping and Stop Codon Read Through

    OpenAIRE

    Hoffman, Eric P.; Bronson, Abby; Levin, Arthur A.; Takeda, Shin'ichi; Yokota, Toshifumi; Baudy, Andreas R.; Connor, Edward M.

    2011-01-01

    The identification of the Duchenne muscular dystrophy gene and protein in the late 1980s led to high hopes of rapid translation to molecular therapeutics. These hopes were fueled by early reports of delivering new functional genes to dystrophic muscle in mouse models using gene therapy and stem cell transplantation. However, significant barriers have thwarted translation of these approaches to true therapies, including insufficient therapeutic material (eg, cells and viral vectors), challenge...

  9. Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

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    Han Renzhi

    2011-12-01

    Full Text Available Abstract Background Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. Methods To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. Results The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin

  10. Dystrophin hydrophobic regions in the pathogenesis of Duchenne and Becker muscular dystrophies

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    Yingyin Liang

    2015-05-01

    Full Text Available The aim of our study was to determine the role of dystrophin hydrophobic regions in the pathogenesis of Duchenne (DMD and Becker (BMD muscular dystrophies, by the Kyte-Doolittle scale mean hydrophobicity profile and 3D molecular models. A total of 1038 cases diagnosed with DMD or BMD with the in-frame mutation were collected in our hospital and the Leiden DMD information database in the period 2002-2013. Correlation between clinical types and genotypes were determined on the basis of these two sources. In addition, the Kyte-Doolittle scale mean hydrophobicity of dystrophin was analyzed using BioEdit software and the models of the hydrophobic domains of dystrophin were constructed. The presence of four hydrophobic regions is confirmed. They include the calponin homology CH2 domain on the actin-binding domain (ABD, spectrin-type repeat 16, hinge III and the EF Hand domain. The severe symptoms of DMD usually develop as a result of the mutational disruption in the hydrophobic regions I, II and IV of dystrophin – those that bind associated proteins of the dystrophin-glycoprotein complex (DGC. On the other hand, when the hydrophobic region III is deleted, the connection of the ordered repeat domains of the central rod domain remains intact, resulting in the less severe clinical presentation. We conclude that mutational changes in the structure of hydrophobic regions of dystrophin play an important role in the pathogenesis of DMD.

  11. Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

    Science.gov (United States)

    Kornegay, Joe N; Bogan, Daniel J; Bogan, Janet R; Dow, Jennifer L; Wang, Jiahui; Fan, Zheng; Liu, Naili; Warsing, Leigh C; Grange, Robert W; Ahn, Mihye; Balog-Alvarez, Cynthia J; Cotten, Steven W; Willis, Monte S; Brinkmeyer-Langford, Candice; Zhu, Hongtu; Palandra, Joe; Morris, Carl A; Styner, Martin A; Wagner, Kathryn R

    2016-01-01

    Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no

  12. An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

    Science.gov (United States)

    Collins, Katherine M; Kainov, Yaroslav A; Christodolou, Evangelos; Ray, Debashish; Morris, Quaid; Hughes, Timothy; Taylor, Ian A; Makeyev, Eugene V; Ramos, Andres

    2017-04-04

    RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping.

  13. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  14. Intron Retention and TE Exonization Events in ZRANB2

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    Sang-Je Park

    2012-01-01

    Full Text Available The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2, contains arginine/serine-rich (RS domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3 between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3. Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species. Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3. RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys and were expressed via intron retention (IR. Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

  15. Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene

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    Shomron Noam

    2007-11-01

    Full Text Available Abstract Background Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. Results Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. Conclusion The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.

  16. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.

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    Mariya M Kucherenko

    Full Text Available The Dystroglycan-Dystrophin (Dg-Dys complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.

  17. Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein

    OpenAIRE

    2008-01-01

    The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx...

  18. Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Nik-Ahd, Farnoosh; Bertoni, Carmen

    2014-07-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients.

  19. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.

    Science.gov (United States)

    Dumont, Nicolas A; Wang, Yu Xin; von Maltzahn, Julia; Pasut, Alessandra; Bentzinger, C Florian; Brun, Caroline E; Rudnicki, Michael A

    2015-12-01

    Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

  20. Deficiency in Cardiac Dystrophin Affects the Abundance of the α-/β-Dystroglycan Complex

    Directory of Open Access Journals (Sweden)

    James Lohan

    2005-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily categorised as a skeletal muscle disease, deficiency in the membrane cytoskeletal protein dystrophin also affects the heart. The central transsarcolemmal linker between the actin membrane cytoskeleton and the extracellular matrix is represented by the dystrophin-associated dystroglycans. Chemical cross-linking analysis revealed no significant differences in the dimeric status of the α-/β-dystroglycan subcomplex in the dystrophic mdx heart as compared to normal cardiac tissue. In analogy to skeletal muscle fibres, heart muscle also exhibited a greatly reduced abundance of both dystroglycans in dystrophin-deficient cells. Immunoblotting demonstrated that the degree of reduction in α-dystroglycan is more pronounced in matured mdx skeletal muscle as contrasted to the mdx heart. The fact that the deficiency in dystrophin triggers a similar pathobiochemical response in both types of muscle suggests that the cardiomyopathic complications observed in x-linked muscular dystrophy might be initiated by the loss of the dystrophin-associated surface glycoprotein complex.

  1. First Exon Length Controls Active Chromatin Signatures and Transcription

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    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  2. The complete exon-intron structure of the 156-kb human gene NFKB1, which encodes the p105 and p50 proteins of transcription factors NF-{kappa}B and I{kappa}B-{gamma}: Implications for NF-{kappa}B-mediated signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Heron, E.; Deloukas, P.; van Loon, A.P.G.M. [F. Hoffmann-La Roche Limited, Basel (Switzerland)

    1995-12-10

    The NFKB1 gene encodes three proteins of the NF-{kappa}/Rel and I{kappa}B families: p105, p50, and (in mouse) I{kappa}B-{gamma}. We determined the complete genomic structure of human NFKB1. NFKB1 spans 156 kb and has 24 exons with introns varying between 40,000 and 323 bp in length. Although NFKB2, which encodes p100 and p52, also has 24 exons and has a comparable exon-intron structure, it is 20 times shorter than NFKB1. We propose that the long size of NFKB1 is important for transient activation of NF-{kappa}B complexes containing p50. I{kappa}B-{gamma} corresponds to the carboxyl-terminal half of p105. DNA sequence analysis showed that the 3{prime}-end of human intron 11 and the 5{prime}-end of exon 12 of NFKB1 are colinear with the 5{prime}-untranslated region of mouse I{kappa}B-{gamma} cDNA. I{kappa}B-{gamma} is thus likely to be generated by transcription starting within intron 11 and not by alternative splicing of the mouse mRNA encoding p105 and p50. 71 refs., 5 figs., 1 tab.

  3. Automatic detection of exonic splicing enhancers (ESEs using SVMs

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    Suhai Sándor

    2008-09-01

    Full Text Available Abstract Background Exonic splicing enhancers (ESEs activate nearby splice sites and promote the inclusion (vs. exclusion of exons in which they reside, while being a binding site for SR proteins. To study the impact of ESEs on alternative splicing it would be useful to have a possibility to detect them in exons. Identifying SR protein-binding sites in human DNA sequences by machine learning techniques is a formidable task, since the exon sequences are also constrained by their functional role in coding for proteins. Results The choice of training examples needed for machine learning approaches is difficult since there are only few exact locations of human ESEs described in the literature which could be considered as positive examples. Additionally, it is unclear which sequences are suitable as negative examples. Therefore, we developed a motif-oriented data-extraction method that extracts exon sequences around experimentally or theoretically determined ESE patterns. Positive examples are restricted by heuristics based on known properties of ESEs, e.g. location in the vicinity of a splice site, whereas negative examples are taken in the same way from the middle of long exons. We show that a suitably chosen SVM using optimized sequence kernels (e.g., combined oligo kernel can extract meaningful properties from these training examples. Once the classifier is trained, every potential ESE sequence can be passed to the SVM for verification. Using SVMs with the combined oligo kernel yields a high accuracy of about 90 percent and well interpretable parameters. Conclusion The motif-oriented data-extraction method seems to produce consistent training and test data leading to good classification rates and thus allows verification of potential ESE motifs. The best results were obtained using an SVM with the combined oligo kernel, while oligo kernels with oligomers of a certain length could be used to extract relevant features.

  4. Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes.

    Science.gov (United States)

    Rubi, Lena; Koenig, Xaver; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz

    2017-03-04

    Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK1, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

  5. The sarcoglycan-sarcospan complex localization in mouse retina is independent from dystrophins

    Science.gov (United States)

    Fort, Patrice; Estrada, Francisco-Javier; Bordais, Agnès; Mornet, Dominique; Sahel, José-Alain; Picaud, Serge; Vargas, Haydeé Rosas; Coral-Vázquez, Ramón M.; Rendon, Alvaro

    2005-01-01

    The sarcoglycan–sarcospan (SG–SSPN) complex is part of the dystrophin-glycoprotein complex that has been extensively characterized in muscle. To establish the framework for functional studies of sarcoglycans in retina here, we quantified sarcoglycans mRNA levels with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and performed immunohistochemistry to determine their cellular and subcellular distribution. We showed that the β-, δ-, γ-, ε-sarcoglycans and sarcospan are expressed in mouse retina. They are localized predominantly in the outer and the inner limiting membranes, probably in the Müller cells and also in the ganglion cells axons where the expression of dystrophins have never been reported. We also investigated the status of the sarcoglycans in the retina of mdx3cv mutant mice for all Duchene Muscular Dystrophy (DMD) gene products. The absence of dystrophin did not produce any change in the sarcoglycan–sarcospan components expression and distribution. PMID:15993965

  6. The structure of a human neurofilament gene (NF-L): A unique exon-intron organization in the intermediate filament gene family.

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); F.G. Grosveld (Frank); K. Yazdanbakhsh; D. Flavell (David); D.N. Meijer (Dies); W.E. Mushynski

    1987-01-01

    textabstractWe have cloned and determined the nucleotide sequence of the human gene for the neurofilament subunit NF-L. The cloned DNA contains the entire transcriptional unit and generates two mRNAs of approx. 2.6 and 4.3 kb after transfection into mouse L-cells. The NF-L gene has an unexpected

  7. Regional genomic instability predisposes to complex dystrophin gene rearrangements.

    Science.gov (United States)

    Oshima, Junko; Magner, Daniel B; Lee, Jennifer A; Breman, Amy M; Schmitt, Eric S; White, Lisa D; Crowe, Carol A; Merrill, Michelle; Jayakar, Parul; Rajadhyaksha, Aparna; Eng, Christine M; del Gaudio, Daniela

    2009-09-01

    Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.

  8. The emergence of alternative 3' and 5' splice site exons from constitutive exons.

    Directory of Open Access Journals (Sweden)

    Eli Koren

    2007-05-01

    Full Text Available Alternative 3' and 5' splice site (ss events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving, similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.

  9. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    Science.gov (United States)

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  10. Variants Within TSC2 Exons 25 and 31 Are Very Unlikely to Cause Clinically Diagnosable Tuberous Sclerosis.

    Science.gov (United States)

    Ekong, Rosemary; Nellist, Mark; Hoogeveen-Westerveld, Marianne; Wentink, Marjolein; Panzer, Jessica; Sparagana, Steven; Emmett, Warren; Dawson, Natalie L; Malinge, Marie Claire; Nabbout, Rima; Carbonara, Caterina; Barberis, Marco; Padovan, Sergio; Futema, Marta; Plagnol, Vincent; Humphries, Steve E; Migone, Nicola; Povey, Sue

    2016-04-01

    Inactivating mutations in TSC1 and TSC2 cause tuberous sclerosis complex (TSC). The 2012 international consensus meeting on TSC diagnosis and management agreed that the identification of a pathogenic TSC1 or TSC2 variant establishes a diagnosis of TSC, even in the absence of clinical signs. However, exons 25 and 31 of TSC2 are subject to alternative splicing. No variants causing clinically diagnosed TSC have been reported in these exons, raising the possibility that such variants would not cause TSC. We present truncating and in-frame variants in exons 25 and 31 in three individuals unlikely to fulfil TSC diagnostic criteria and examine the importance of these exons in TSC using different approaches. Amino acid conservation analysis suggests significantly less conservation in these exons compared with the majority of TSC2 exons, and TSC2 expression data demonstrates that the majority of TSC2 transcripts lack exons 25 and/or 31 in many human adult tissues. In vitro assay of both exons shows that neither exon is essential for TSC complex function. Our evidence suggests that variants in TSC2 exons 25 or 31 are very unlikely to cause classical TSC, although a role for these exons in tissue/stage specific development cannot be excluded.

  11. Motor physical therapy affects muscle collagen type I and decreases gait speed in dystrophin-deficient dogs.

    Directory of Open Access Journals (Sweden)

    Thaís P Gaiad

    Full Text Available Golden Retriever Muscular Dystrophy (GRMD is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD maintained their routine of activities of daily living. At t0 (pre and t1 (post-physical therapy, collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy, mediolateral (Fz and craniocaudal (Fx ground reaction forces (GRF were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000. The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.

  12. Predicting mutually exclusive spliced exons based on exon length, splice site and reading frame conservation, and exon sequence homology

    Directory of Open Access Journals (Sweden)

    Hammesfahr Björn

    2011-06-01

    Full Text Available Abstract Background Alternative splicing of pre-mature RNA is an important process eukaryotes utilize to increase their repertoire of different protein products. Several types of different alternative splice forms exist including exon skipping, differential splicing of exons at their 3'- or 5'-end, intron retention, and mutually exclusive splicing. The latter term is used for clusters of internal exons that are spliced in a mutually exclusive manner. Results We have implemented an extension to the WebScipio software to search for mutually exclusive exons. Here, the search is based on the precondition that mutually exclusive exons encode regions of the same structural part of the protein product. This precondition provides restrictions to the search for candidate exons concerning their length, splice site conservation and reading frame preservation, and overall homology. Mutually exclusive exons that are not homologous and not of about the same length will not be found. Using the new algorithm, mutually exclusive exons in several example genes, a dynein heavy chain, a muscle myosin heavy chain, and Dscam were correctly identified. In addition, the algorithm was applied to the whole Drosophila melanogaster X chromosome and the results were compared to the Flybase annotation and an ab initio prediction. Clusters of mutually exclusive exons might be subsequent to each other and might encode dozens of exons. Conclusions This is the first implementation of an automatic search for mutually exclusive exons in eukaryotes. Exons are predicted and reconstructed in the same run providing the complete gene structure for the protein query of interest. WebScipio offers high quality gene structure figures with the clusters of mutually exclusive exons colour-coded, and several analysis tools for further manual inspection. The genome scale analysis of all genes of the Drosophila melanogaster X chromosome showed that WebScipio is able to find all but two of the 28

  13. A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.

    OpenAIRE

    Takacs, A M; Denker, J A; Perrine, K G; Maroney, P A; Nilsen, T W

    1988-01-01

    The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt se...

  14. Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

    Science.gov (United States)

    Lehto, Taavi; Castillo Alvarez, Alejandra; Gauck, Sarah; Gait, Michael J; Coursindel, Thibault; Wood, Matthew J A; Lebleu, Bernard; Boisguerin, Prisca

    2014-03-01

    Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

  15. Polyquaternium-mediated delivery of morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    Science.gov (United States)

    Wang, Mingxing; Wu, Bo; Shah, Sapana N; Lu, Peijuan; Lu, Qilong

    2017-11-01

    Antisense oligonucleotide therapy for Duchenne muscular dystrophy has shown great potential in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few polyquaterniums (PQs) with different size and composition for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that Luviquat(TM) series, especially PQ-1 and PQ-3, promoted the exon-skipping efficiency comparable to Endoporter-mediated PMO delivery in vitro. Significant enhancement in skipping dystrophin exon 23 has also been achieved with PQ-3 up to seven-fold when compared to PMO alone in mdx mice. Cytotoxicity of the PQs was lower than Endoporter and PEI 25 K in vitro and muscle damage not clearly detected in vivo under the tested concentrations. These results together demonstrate that the optimization of PQ in molecular size, composition and distribution of positive charges is the key factor to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow polyquaternium series as AO delivery enhancing agents for treating muscular dystrophy and other diseases.

  16. Identification of protein features encoded by alternative exons using Exon Ontology.

    Science.gov (United States)

    Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

    2017-06-01

    Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Fourier Power Spectrum Analysis of Exons for the Period-3 Behavior

    Institute of Scientific and Technical Information of China (English)

    Yuan Xin TIAN; Chao CHEN; Xiao Yong ZOU; Jian Ding QIU; Pei Xiang CAI; Jin Yuan MO

    2005-01-01

    The period-3 behaviors of 105 exons from 20 genes in human were studied by Fourier power spectrum. The results indicated that not all exons show the period-3 behavior. The exons were adjusted in order to make them accord with the order of the protein translated, and we found that the period-3 character is relation to the length of exons and the bases distribution in the three codon position. Furthermore, as long as the exons with period-3 behavior accord with the order of protein translated, they would exhibit the synonymous codons usage preference, and the codons with g/c at the third position are used in higher frequency. The results are significant to the gene prediction and the research on the introns.

  18. Exon circularization in mammalian nuclear extracts.

    Science.gov (United States)

    Pasman, Z; Been, M D; Garcia-Blanco, M A

    1996-06-01

    Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

  19. Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion

    DEFF Research Database (Denmark)

    Duguez, S.; Duddy, W.; Johnston, H.

    2013-01-01

    Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved...... of new potential therapeutic targets....

  20. Proteomic Profiling of the Dystrophin-Deficient mdx Phenocopy of Dystrophinopathy-Associated Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ashling Holland

    2014-01-01

    Full Text Available Cardiorespiratory complications are frequent symptoms of Duchenne muscular dystrophy, a neuromuscular disorder caused by primary abnormalities in the dystrophin gene. Loss of cardiac dystrophin initially leads to changes in dystrophin-associated glycoproteins and subsequently triggers secondarily sarcolemmal disintegration, fibre necrosis, fibrosis, fatty tissue replacement, and interstitial inflammation. This results in progressive cardiac disease, which is the cause of death in a considerable number of patients afflicted with X-linked muscular dystrophy. In order to better define the molecular pathogenesis of this type of cardiomyopathy, several studies have applied mass spectrometry-based proteomics to determine proteome-wide alterations in dystrophinopathy-associated cardiomyopathy. Proteomic studies included both gel-based and label-free mass spectrometric surveys of dystrophin-deficient heart muscle from the established mdx animal model of dystrophinopathy. Comparative cardiac proteomics revealed novel changes in proteins associated with mitochondrial energy metabolism, glycolysis, signaling, iron binding, antibody response, fibre contraction, basal lamina stabilisation, and cytoskeletal organisation. This review summarizes the importance of studying cardiomyopathy within the field of muscular dystrophy research, outlines key features of the mdx heart and its suitability as a model system for studying cardiac pathogenesis, and discusses the impact of recent proteomic findings for exploring molecular and cellular aspects of cardiac abnormalities in inherited muscular dystrophies.

  1. The polyproline site in hinge 2 influences the functional capacity of truncated dystrophins.

    Directory of Open Access Journals (Sweden)

    Glen B Banks

    2010-05-01

    Full Text Available Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophin(DeltaR4-R23/DeltaCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophin(DeltaR4-R23/DeltaCT led to small myofibers (12% smaller than wild-type, Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophin(DeltaR4-R23/DeltaCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid alpha-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.

  2. Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity.

    Science.gov (United States)

    van den Bergen, J C; Wokke, B H A; Hulsker, M A; Verschuuren, J J G M; Aartsma-Rus, A M

    2015-03-01

    Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

  3. Human fructose-1,6-bisphosphatase gene (FBP1): Exon-intron organization, localization to chromosome bands 9q22.2-q22.3, and mutation screening in subjects with fructose-1,6-bisphosphatase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    El-Maghrabi, M.R.; Jiang, W. [State Univ. of New York, Stony Brook, NY (United States)] [and others

    1995-06-10

    Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6-bisphosphatase deficiency. Four nucleotide substitutions were identified, two of which were silent mutations in the codons for Ala-216 (GCT {yields} GCC) and Gly-319 (GGG {yields} GGA). The other substitutions were in intron 3, a C {yields} T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A {yields} T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity. 18 refs., 3 figs., 3 tabs.

  4. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy

    National Research Council Canada - National Science Library

    Finkel, Richard S; Flanigan, Kevin M; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H Lee; Reha, Allen; Northcutt, Valerie J; Elfring, Gary; Barth, Jay; Peltz, Stuart W

    2013-01-01

    Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124...

  5. Variants affecting exon skipping contribute to complex traits.

    Directory of Open Access Journals (Sweden)

    Younghee Lee

    Full Text Available DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1 experimentally validate our in silico predictions of skipped exons and 2 characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits.

  6. Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient

    DEFF Research Database (Denmark)

    Zhou, J; Hertz, Jens Michael; Leinonen, A;

    1992-01-01

    alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin...

  7. Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas.

    Science.gov (United States)

    Murakami, A; Mori, T; Sakai, H; Murakami, M; Yanai, T; Hoshino, Y; Maruo, K

    2011-09-01

    KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.

  8. How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

    Science.gov (United States)

    Sawada, Ryusuke; Mitaku, Shigeki

    2011-01-01

    The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.

  9. The evolutionary fate of alternatively spliced homologous exons after gene duplication.

    Science.gov (United States)

    Abascal, Federico; Tress, Michael L; Valencia, Alfonso

    2015-04-29

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Bartolo, C; Sedra, M S; Western, L M; Mendell, J R

    1993-08-01

    About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

  11. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure.

    Science.gov (United States)

    Noutsios, Georgios T; Silveyra, Patricia; Bhatti, Faizah; Floros, Joanna

    2013-06-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5' untranslated (5'UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5'UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA.

  12. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Science.gov (United States)

    Barnabei, Matthew S; Metzger, Joseph M

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  13. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

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    Matthew S Barnabei

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  14. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    OpenAIRE

    2011-01-01

    Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle ...

  15. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

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    Lei ZHAO

    2015-07-01

    Full Text Available Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles of DMD patients after birth are in the natural state of actual path of force transmission which carried high tension from eccentric exercise. This paper investigated systematically whether expression of DGC is associated with progressive muscle weakness in natural history of DMD, and analyzed the expression of DGC at the sarcolemma of 197 confirmed DMD cases (9 days-12 years old.  Methods  The expression of α- and β-dystroglycan (DG, α-, β-, γ- and δ-sarcoglycan (SG and syntrophin at the sarcolemma of DMD patients was analyzed by immunofluorescent staining.  Results  The study showed that there was no relationship between lack of proteins and progressive muscle weakness with increasing age, although expression of α- and β-DG, α-, β-, γ- and δ-SG and syntrophin at the sarcolemma at different stages of 197 DMD patients (9 days-12 years old had different degrees of deficiency.  Conclusions  Deficiency of DGC may occur before birth and DMD patients were recommended to avoid further damage to skeletal muscles from eccentric exercise and high-resistance movement in activities of daily life and rehabilitation training. DOI: 10.3969/j.issn.1672-6731.2015.06.006

  16. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling

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    Alejandro Martínez-Herrera

    2015-09-01

    Full Text Available Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015 115–120 [1].

  17. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    Science.gov (United States)

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

  18. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Cação-Benedini, L.O.; Ribeiro, P.G. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Prado, C.M.; Chesca, D.L. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Mattiello-Sverzut, A.C. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-05-09

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

  19. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

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    Guangrong Xie

    2016-05-01

    Full Text Available The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G with increased homology (91.45% with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C.

  20. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons

    Institute of Scientific and Technical Information of China (English)

    Zhenxun Wang; Deblina Chatterjee; Hyun Yong Jeon; Martin Akerman; Matthew G. Vander Heiden; Lewis C. Cantley; Adrian R. Krainer

    2012-01-01

    Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth.However,the cancer-specific alternative splicing regulation of PK-M is not completely understood.Here,we demonstrate that PK-M is regulated by reciprocal affects on the mutually exclusive exons 9 and 10,such that exon 9 is repressed and exon 10 is activated in cancer cells.Strikingly,exonic,rather than intronic,cis-elements are key determinants ef PK-M splicing isoform ratios.Using a systematic sub-exonic duplication approach,we identify a potent exonlc splicing enhancer in exon 10,which differs from its homologous counterpart in exon 9 by only two nucleotides.We identify SRSF3 as one of the cognate factors,and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism.These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect,and also have implications for other genes with a similar pattern of alternative splicing.

  1. Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

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    Nyiraneza Christine

    2012-06-01

    Full Text Available Abstract Background It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI, low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs. In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. Results Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%, whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%. Only seven of ninety-eight tumors (7% had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13 versus 0 to 17 (95% CI 0 to 2 in adjacent colon mucosa (P = 0.004. Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03, and MDM2 overexpression (P = 0.02, independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational

  2. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    Science.gov (United States)

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  3. Faster exon assembly by sparse spliced alignment

    CERN Document Server

    Tiskin, Alexander

    2007-01-01

    Assembling a gene from candidate exons is an important problem in computational biology. Among the most successful approaches to this problem is \\emph{spliced alignment}, proposed by Gelfand et al., which scores different candidate exon chains within a DNA sequence of length $m$ by comparing them to a known related gene sequence of length n, $m = \\Theta(n)$. Gelfand et al.\\ gave an algorithm for spliced alignment running in time O(n^3). Kent et al.\\ considered sparse spliced alignment, where the number of candidate exons is O(n), and proposed an algorithm for this problem running in time O(n^{2.5}). We improve on this result, by proposing an algorithm for sparse spliced alignment running in time O(n^{2.25}). Our approach is based on a new framework of \\emph{quasi-local string comparison}.

  4. Cryptic exon incorporation occurs in Alzheimer's brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43.

    Science.gov (United States)

    Sun, Mingkuan; Bell, William; LaClair, Katherine D; Ling, Jonathan P; Han, Heather; Kageyama, Yusuke; Pletnikova, Olga; Troncoso, Juan C; Wong, Philip C; Chen, Liam L

    2017-06-01

    Abnormal accumulation of TDP-43 into cytoplasmic or nuclear inclusions with accompanying nuclear clearance, a common pathology initially identified in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), has also been found in Alzheimer' disease (AD). TDP-43 serves as a splicing repressor of nonconserved cryptic exons and that such function is compromised in brains of ALS and FTD patients, suggesting that nuclear clearance of TDP-43 underlies its inability to repress cryptic exons. However, whether TDP-43 cytoplasmic aggregates are a prerequisite for the incorporation of cryptic exons is not known. Here, we assessed hippocampal tissues from 34 human postmortem brains including cases with confirmed diagnosis of AD neuropathologic changes along with age-matched controls. We found that cryptic exon incorporation occurred in all AD cases exhibiting TDP-43 pathology. Furthermore, incorporation of cryptic exons was observed in the hippocampus when TDP-43 inclusions was restricted only to the amygdala, the earliest stage of TDP-43 progression. Importantly, cryptic exon incorporation could be detected in AD brains lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. These data supports the notion that the functional consequence of nuclear depletion of TDP-43 as determined by cryptic exon incorporation likely occurs as an early event of TDP-43 proteinopathy and may have greater contribution to the pathogenesis of AD than currently appreciated. Early detection and effective repression of cryptic exons in AD patients may offer important diagnostic and therapeutic implications for this devastating illness of the elderly.

  5. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

    Science.gov (United States)

    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

  6. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Chantal Beekman

    Full Text Available Duchenne muscular dystrophy (DMD is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2-17% and intra-assay precision, CV 2-10%. Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

  7. Effect of exonic splicing regulation on synonymous codon usage in alternatively spliced exons of Dscam

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    Takahashi Aya

    2009-08-01

    Full Text Available Abstract Background Synonymous codon usage is typically biased towards translationally superior codons in many organisms. In Drosophila, genomic data indicates that translationally optimal codons and splice optimal codons are mostly mutually exclusive, and adaptation to translational efficiency is reduced in the intron-exon boundary regions where potential exonic splicing enhancers (ESEs reside. In contrast to genomic scale analyses on large datasets, a refined study on a well-controlled set of samples can be effective in demonstrating the effects of particular splice-related factors. Down syndrome cell adhesion molecule (Dscam has the largest number of alternatively spliced exons (ASEs known to date, and the splicing frequency of each ASE is accessible from the relative abundance of the transcript. Thus, these ASEs comprise a unique model system for studying the effect of splicing regulation on synonymous codon usage. Results Codon Bias Indices (CBI in the 3' boundary regions were reduced compared to the rest of the exonic regions among 48 and 33 ASEs of exon 6 and 9 clusters, respectively. These regional differences in CBI were affected by splicing frequency and distance from adjacent exons. Synonymous divergence levels between the 3' boundary region and the remaining exonic region of exon 6 ASEs were similar. Additionally, another sensitive comparison of paralogous exonic regions in recently retrotransposed processed genes and their parental genes revealed that, in the former, the differences in CBI between what were formerly the central regions and the boundary regions gradually became smaller over time. Conclusion Analyses of the multiple ASEs of Dscam allowed direct tests of the effect of splice-related factors on synonymous codon usage and provided clear evidence that synonymous codon usage bias is restricted by exonic splicing signals near the intron-exon boundary. A similar synonymous divergence level between the different exonic

  8. Relation between p53 (exon 7) mutation and p53 overexpression in human cervical cancers%宫颈癌p53外显子7突变与p53蛋白高表达的关系

    Institute of Scientific and Technical Information of China (English)

    张娜; 李惠芳; 常艳丽; 梁莎

    2001-01-01

    目的探讨宫颈癌p53外显子7突变与p53蛋白高表达的关系。方法采用免疫组织化学、聚合酶链反应(PCR)、限制性酶解片段长度多态性(RFLP)分析等方法对49例宫颈癌组织石蜡包埋标本中p53外显子7的突变与p53蛋白表达进行了检测。结果 p53外显子7的突变率8.2%(4/49)显著低于p53蛋白阳性率49.0%(24/49)(χ2=18.05,P<0.001);p53外显子7突变不一定p53蛋白阳性。结论 p53外显子7突变可能是部分宫颈癌变的一个重要因素;大部分宫颈癌可能主要由于高危人乳头状瘤病毒(HPV)感染后,通过E6/p53蛋白复合物的形成使p53蛋白失活所致。%Objective To investigate the relation between p53 (exon 7) mutations and p53 overexpression in human cervical cancer.Methods p53 (exon 7) mutation and p53 overexpression were examined by immunohistochemistry,polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in 49 cases of cervical cancers on their paraffin-embedded tissue specimens.Results There was significant difference between p53 (exon 7) mutation 4/49 (8.2%) and p53 overexpression 24/49 (49.0%) in cervical cancer (χ2=18.05,P<0.001);not all cases of p53 mutation had p53 protein positive.Conclusion The p53 (exon 7) mutation is an important factor in part of cervical cancers,but anomalous structure and inactivation of p53 proteins caused by E6/p53 protein complex formed in high risk HPV infection are the significant cause of the greater part of cervical cancers.

  9. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    Directory of Open Access Journals (Sweden)

    Bailey Nichols

    2015-07-01

    Full Text Available Dystrophin-glycoprotein complex (DGC is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin of a muscle fiber to the extracellular matrix (ECM. Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies, and limb-girdle muscular dystrophies (sarcoglycanopathies, are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS, which is localized at the muscle membrane by DGC members (dystrophin and syntrophins, plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain.

  10. Current understanding of dystrophin-related muscular dystrophy and therapeutic challenges ahead

    Institute of Scientific and Technical Information of China (English)

    ZHOU Guang-qian; XIE Hui-qi; ZHANG Su-zhen; YANG Zhi-ming

    2006-01-01

    Objective To review the recent research progress in dystrophin-related muscular dystrophy includes X-linked hereditary Duchenne and Becker muscular dystrophies (DMD and BMD).Data sources Information included in this article was identified by searches of PUBMED and other online resources using the key terms DMD, dystrophin, mutations, animal models, pathophysiology, gene expression, stem cells, gene therapy, cell therapy, and pharmacological.Study selection Mainly original milestone articles and timely reviews written by major pioneer investigators of the field were selected.Results The key issues related to the genetic basis and pathophysiological factors of the diseases were critically addressed. The availabilities and advantages of various animal models for the diseases were described. Major molecular and cellular therapeutic approaches were also discussed, many of which have indeed exhibited some success in pre-clinical studies but at the same time encountered a number of technical hurdles, including the efficient and systemic delivery of a functional gene and myogenic precursor/stem cells to repair genetic defects.Conclusions Further understanding of pathophysiological mechanisms at molecular levels and regenerative properites of myogenic precursor/stem cells will promote the development of multiple therapeutic strategies. The combined use of multiple strategies may represent the major challenge as well as the greatest hope for the therapy of these diseases in coming years.

  11. Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

    Directory of Open Access Journals (Sweden)

    Hiroshi Sakai

    Full Text Available Muscle satellite cells (SCs are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs isolated from Pax3(GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease.

  12. Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice

    Directory of Open Access Journals (Sweden)

    Sali Arpana

    2011-05-01

    Full Text Available Abstract Background Cardiomyopathy in Duchenne muscular dystrophy (DMD is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy. Methods Three month old female mdx mice were exposed to the β1 receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188 (460 mg/kg/dose i.p. daily. Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD and picrosirius red staining. Results BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p Conclusions This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice.

  13. Single nucleotide polymorphism-based validation of exonic splicing enhancers.

    Directory of Open Access Journals (Sweden)

    William G Fairbrother

    2004-09-01

    Full Text Available Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality, which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE-ESE method. Aligning the single nucleotide polymorphisms (SNPs from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05. This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.

  14. Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta.

    Science.gov (United States)

    Poulter, James A; Murillo, Gina; Brookes, Steven J; Smith, Claire E L; Parry, David A; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

    2014-10-15

    Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.

  15. Modulating Calcium Signals to Boost AON Exon Skipping for DMD

    Science.gov (United States)

    2016-10-01

    RyR antags for RNASeQ (18 months; 70% complete) We are in the process of prioritizing based on initial findings. As of now CDMD1003 exon 45...sequence analysis Subtask 1 - Optimize alternate splicing assay using exon capture and RNASeq (12 months, 80% complete). Additionally, we have...begun optimizing the exon capture and performed preliminary RNASeq experiments as described using exon capture. Subtask 2 - High depth RNASeQ on

  16. Exon - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ontents Exons in variants Data file File name: astra_exon.zip File URL: ftp://ftp.biosciencedbc.jp/archive/a... About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Exon - ASTRA | LSDB Archive ...

  17. Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers.

    Science.gov (United States)

    Miyazaki, Daigo; Nakamura, Akinori; Fukushima, Kazuhiro; Yoshida, Kunihiro; Takeda, Shin'ichi; Ikeda, Shu-ichi

    2011-05-01

    Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.

  18. A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.

    NARCIS (Netherlands)

    Tarpey, P.S.; Smith, R.; Pleasance, E.; Whibley, A.; Edkins, S.; Hardy, C.; O'Meara, S.; Latimer, C.; Dicks, E.; Menzies, A.; Stephens, P.; Blow, M.; Greenman, C.; Xue, Y.; Tyler-Smith, C.; Thompson, D.; Gray, K.; Andrews, J.; Barthorpe, S.; Buck, G.; Cole, J.; Dunmore, R.; Jones, D.; Maddison, M.; Mironenko, T.; Turner, R.; Turrell, K.; Varian, J.; West, S.; Widaa, S.; Wray, P.; Teague, J.; Butler, A.; Jenkinson, A.; Jia, M.; Richardson, D.; Shepherd, R.; Wooster, R.; Tejada, M.I.; Martinez, F.; Carvill, G.; Goliath, R.; Brouwer, A.P.M. de; Bokhoven, H. van; Esch, H. van; Chelly, J.; Raynaud, M.; Ropers, H.H.; Abidi, F.E.; Srivastava, A.K.; Cox, J.; Luo, Y.; Mallya, U.; Moon, J.; Parnau, J.; Mohammed, S.; Tolmie, J.L.; Shoubridge, C.; Corbett, M.; Gardner, A.; Haan, E.; Rujirabanjerd, S.; Shaw, M.A.; Vandeleur, L.; Fullston, T.; Easton, D.F.; Boyle, J.; Partington, M.; Hackett, A.; Field, M.; Skinner, C.; Stevenson, R.E.; Bobrow, M.; Turner, G.; Schwartz, C.E.; Gecz, J.; Raymond, F.L.; Futreal, P.A.; Stratton, M.R.

    2009-01-01

    Large-scale systematic resequencing has been proposed as the key future strategy for the discovery of rare, disease-causing sequence variants across the spectrum of human complex disease. We have sequenced the coding exons of the X chromosome in 208 families with X-linked mental retardation (XLMR),

  19. Evidence for association of multi-exon skipping events with tumors

    Institute of Scientific and Technical Information of China (English)

    Jianning BI; Tao PENG; Yanda LI

    2008-01-01

    Alternative splicing(AS)has been shown to be frequently present in human tumors.Specifically,it has been observed in some experimental studies that multi-exon skipping(MES)events often appear in tumorous tissues.Prompted by this observation,we conducted a genomewide analysis of MES events to investigate their association with tumors.The results show that MES events are more likely associated with tumors than single-exon skipping (SES) and the degree of association increases with the number of skipped exons.Furthermore,MES events are found to be less conserved than their SES counterparts,which provides additional evidence for our results because disease-associated AS events should be eliminated during evolution.Interestingly,these differences still existed even after comparison Of MES and SES events with similarlength skipped regions.These results demonstrate that MES events mav be associated with tumors and suggest that MES isoforms might be useful in cancer diagnosis.

  20. Computational analysis and prediction for exons of PAC579 genomic sequence

    Institute of Scientific and Technical Information of China (English)

    黄弋; 覃文新; 万大方; 赵新泰; 顾健人

    2001-01-01

    To isolate the novel genes related to human hepatocellular carcinoma (HCC), we sequenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC, Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0-60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.

  1. Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing.

    Directory of Open Access Journals (Sweden)

    Elisabete Hernández-Imaz

    Full Text Available Neurofibromatosis type 1 (NF1 is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.

  2. Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding - explains hyperglycemia in type 2 diabetic patients.

    Science.gov (United States)

    Yellapu, Nandakumar; Mahto, Manoj Kumar; Valasani, Koteswara Rao; Sarma, P V G K; Matcha, Bhaskar

    2015-01-01

    Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix-helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

  3. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    Directory of Open Access Journals (Sweden)

    Jessica D. Gumerson

    2011-01-01

    Full Text Available Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle may contribute to a higher degree of muscle damage which eventually overwhelms muscle regeneration capacity. While increased susceptibility of muscle to mechanical injury is thought to be an important contributor to disease pathology, it is becoming clear that not all DGC-associated diseases share this supposed hallmark feature. This paper outlines experimental support for a function of the DGC in preventing muscle damage and examines the evidence that supports novel functions for this complex in muscle that when impaired, may contribute to the pathogenesis of muscular dystrophy.

  4. Proteasome inhibitor (MG132 rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx5cv mice

    Directory of Open Access Journals (Sweden)

    Jean-Sebastien eRougier

    2013-03-01

    Full Text Available The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex (DMC at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (INa were both decreased in cardiomyocytes of dystrophin-deficient mdx5cv mice. In this study, wild-type (WT and mdx5cv mice were treated for 7 days with the proteasome inhibitor MG132 (10 µg/Kg/24 h using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and INa but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

  5. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Takahiro; Itoh, Kyoko, E-mail: kxi14@koto.kpu-m.ac.jp; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  6. New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye

    Directory of Open Access Journals (Sweden)

    Rishko Valentyna M

    2011-09-01

    Full Text Available Abstract Background The Dystrophin Glycoprotein Complex (DGC is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys and Dystroglycan (Dg are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye. Results Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, we have determined the function of previously screened components and their genetic interaction with the DGC in this tissue. Our study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. We determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. Conclusions Our data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton.

  7. The proton pump inhibitor lansoprazole improves the skeletal phenotype in dystrophin deficient mdx mice.

    Directory of Open Access Journals (Sweden)

    Arpana Sali

    Full Text Available BACKGROUND: In Duchenne muscular dystrophy (DMD, loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology. METHODOLOGY/PRINCIPAL FINDINGS: We designed a preclinical trial to investigate the effects of lansoprazole (LANZO administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group: (1 vehicle control; (2 5 mg/kg/day LANZO; (3 5 mg/kg/day prednisolone; and (4 combined treatment of 5 mg/kg/day prednisolone (PRED and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan and functional outcomes (grip strength and Rotarod were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions. CONCLUSIONS/SIGNIFICANCE: Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings

  8. First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer.

    Science.gov (United States)

    Bianchini, D; Omlin, A; Pezaro, C; Lorente, D; Ferraldeschi, R; Mukherji, D; Crespo, M; Figueiredo, I; Miranda, S; Riisnaes, R; Zivi, A; Buchbinder, A; Rathkopf, D E; Attard, G; Scher, H I; de Bono, J; Danila, D C

    2013-11-12

    Prostate cancer remains dependent of androgen receptor (AR) signalling, even after emergence of castration resistance. EZN-4176 is a third-generation antisense oligonucleotide that binds to the hinge region (exon 4) of AR mRNA resulting in full-length AR mRNA degradation and decreased AR protein expression. This Phase I study aimed to evaluate EZN-4176 in men with castration-resistant prostate cancer (CRPC). Patients with progressing CRPC were eligible; prior abiraterone and enzalutamide treatment were allowed. EZN-4176 was administered as a weekly (QW) 1-h intravenous infusion. The starting dose was 0.5 mg kg(-1) with a 4-week dose-limiting toxicity (DLT) period and a 3+3 modified Fibonacci dose escalation design. After determination of the DLT for weekly administration, an every 2 weeks schedule was initiated. A total of 22 patients were treated with EZN-4176. At 10 mg kg(-1) QW, two DLTs were observed due to grade 3-4 ALT or AST elevation. No confirmed biochemical or soft tissue responses were observed. Of eight patients with <5 circulating tumour cells at baseline, a conversion to <5 was observed in three (38%) patients. The most common EZN-4176-related toxicities (all grades) were fatigue (59%), reversible abnormalities in liver function tests ALT (41%) and AST (41%) and infusion-related reactions including chills (36%) and pyrexia (14%). Activity of EZN-4176 at the doses and schedules explored was minimal. The highest dose of 10 mg kg(-1) QW was associated with significant but reversible transaminase elevation.

  9. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Beggs, A.H.; Neumann, P.E.; Anderson, M.S.; Kunkel, L.M. (Harvard Medical School, Boston, MA (United States)); Arahata, Kiichi; Arikawa, Eri; Nonaka, Ikuya (National Inst. of Neuroscience, Tokyo (Japan))

    1992-01-15

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3,500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, the authors propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.

  10. Exonic splicing regulatory elements skew synonymous codon usage near intron-exon boundaries in mammals.

    NARCIS (Netherlands)

    Parmley, J.L.; Hurst, L.D.

    2007-01-01

    In mammals there is a bias in amino acid usage near splice sites that is explained, in large part, by the high density of exonic splicing enhancers (ESEs) in these regions. Is there a similar bias for the relative use of synonymous codons, and can any such bias be predicted by their abundance in ESE

  11. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance...... of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  12. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Fumio; Awata, Hisataka; Matsuda, Ichiro [Kumamoto Univ. (Japan)

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this train together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  13. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

    Science.gov (United States)

    Endo, F; Awata, H; Katoh, H; Matsuda, I

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  14. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy

    OpenAIRE

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of exten...

  15. Exon Deletions of Parkin Gene in Patients with Parkinson Disease

    Institute of Scientific and Technical Information of China (English)

    王涛; 梁直厚; 孙圣刚; 曹学兵; 彭海; 刘红进; 童萼塘

    2004-01-01

    Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

  16. Muscle dysfunction and structural defects of dystrophin-null sapje mutant zebrafish larvae are rescued by ataluren treatment.

    Science.gov (United States)

    Li, Mei; Andersson-Lendahl, Monika; Sejersen, Thomas; Arner, Anders

    2014-04-01

    Sapje zebrafish carry a mutation in the dystrophin gene, which results in a premature stop codon, and a severe muscle phenotype. They display several of the structural characteristics of Duchenne muscular dystrophy (DMD). Ataluren (PTC124) is proposed to cause readthrough of premature stop codons and has been introduced as a potential treatment of genetic disorders. Clinical trials in DMD have shown promise, although with complex dose dependency. We have established physiology techniques, enabling high resolution of contractile function in skeletal muscle of zebrafish larvae. We aimed to provide a mechanical analysis of sapje larval muscle and examine effects of ataluren. Homozygous 5 d postfertilization (dpf) sapje larvae exhibited structural defects with 50% decrease in active tension. Ataluren (0.1-1 μM, 3-5 dpf) improved contractile function (~60% improvement of force at 0.5 μM) and dystrophin expression. Controls were not affected. Higher doses (5 μM, 35 μM) impaired contractile function, an effect also observed in controls, suggesting unspecific negative effects at high concentrations. In summary, Sapje larvae exhibit impaired contractile performance and provide a relevant DMD model for functional studies. Ataluren significantly improves skeletal muscle function in the sapje larvae, most likely reflecting an observed increase in dystrophin expression. The bell-shaped dose dependence in sapje resembles that previously reported in clinical DMD studies.

  17. An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

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    Linu S Abraham

    2010-08-01

    Full Text Available The large conductance, voltage- and calcium-dependent potassium (BK channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

  18. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

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    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  19. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

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    Federico Abascal

    2015-06-01

    Full Text Available Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

  20. Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice.

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    Gray, Sarah K; McGee-Lawrence, Meghan E; Sanders, Jennifer L; Condon, Keith W; Tsai, Chung-Jui; Donahue, Seth W

    2012-09-01

    Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that has deleterious consequences in muscle and bone, leading to decreased mobility, progressive osteoporosis, and premature death. Patients with DMD experience a higher-than-average fracture rate, particularly in the proximal and distal femur and proximal tibia. The dystrophin-deficient mdx mouse is a model of DMD that demonstrates muscle degeneration and fibrosis and osteoporosis. Parathyroid hormone, an effective anabolic agent for post-menopausal and glucocorticoid-induced osteoporosis, has not been explored for DMD. Black bear parathyroid hormone (bbPTH) has been implicated in the maintenance of bone properties during extended periods of disuse (hibernation). We cloned bbPTH and found 9 amino acid residue differences from human PTH. Apoptosis was mitigated and cAMP was activated by bbPTH in osteoblast cultures. We administered 28nmol/kg of bbPTH 1-84 to 4-week old male mdx and wild type mice via daily (5×/week) subcutaneous injection for 6 weeks. Vehicle-treated mdx mice had 44% lower trabecular bone volume fraction than wild type mice. No changes were found in femoral cortical bone geometry or mechanical properties with bbPTH treatment in wild type mice, and only medio-lateral moment of inertia changed with bbPTH treatment in mdx femurs. However, μCT analyses of the trabecular regions of the distal femur and proximal tibia showed marked increases in bone volume fraction with bbPTH treatment, with a greater anabolic response (7-fold increase) in mdx mice than wild type mice (2-fold increase). Trabecular number increased in mdx long bone, but not wild type bone. Additionally, greater osteoblast area and decreased osteoclast area were observed with bbPTH treatment in mdx mice. The heightened response to PTH in mdx bone compared to wild type suggests a link between dystrophin deficiency, altered calcium signaling, and bone. These findings support further investigation of PTH as an anabolic

  1. Novel exon of mammalian ADAR2 extends open reading frame.

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    Stefan Maas

    Full Text Available BACKGROUND: The post-transcriptional processing of pre-mRNAs by RNA editing contributes significantly to the complexity of the mammalian transcriptome. RNA editing by site-selective A-to-I modification also regulates protein function through recoding of genomically specified sequences. The adenosine deaminase ADAR2 is the main enzyme responsible for recoding editing and loss of ADAR2 function in mice leads to a phenotype of epilepsy and premature death. Although A-to-I RNA editing is known to be subject to developmental and cell-type specific regulation, there is little knowledge regarding the mechanisms that regulate RNA editing in vivo. Therefore, the characterization of ADAR expression and identification of alternative ADAR variants is an important prerequisite for understanding the mechanisms for regulation of RNA editing and the causes for deregulation in disease. METHODOLOGY/PRINCIPAL FINDINGS: Here we present evidence for a new ADAR2 splice variant that extends the open reading frame of ADAR2 by 49 amino acids through the utilization of an exon located 18 kilobases upstream of the previously annotated first coding exon and driven by a candidate alternative promoter. Interestingly, the 49 amino acid extension harbors a sequence motif that is closely related to the R-domain of ADAR3 where it has been shown to function as a basic, single-stranded RNA binding domain. Quantitative expression analysis shows that expression of the novel ADAR2 splice variant is tissue specific being highest in the cerebellum. CONCLUSIONS/SIGNIFICANCE: The strong sequence conservation of the ADAR2 R-domain between human, mouse and rat ADAR2 genes suggests a conserved function for this isoform of the RNA editing enzyme.

  2. Aberrant location of inhibitory synaptic marker proteins in the hippocampus of dystrophin-deficient mice: implications for cognitive impairment in duchenne muscular dystrophy.

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    Elżbieta Krasowska

    Full Text Available Duchenne muscular dystrophy (DMD is a neuromuscular disease that arises from mutations in the dystrophin-encoding gene. Apart from muscle pathology, cognitive impairment, primarily of developmental origin, is also a significant component of the disorder. Convergent lines of evidence point to an important role for dystrophin in regulating the molecular machinery of central synapses. The clustering of neurotransmitter receptors at inhibitory synapses, thus impacting on synaptic transmission, is of particular significance. However, less is known about the role of dystrophin in influencing the precise expression patterns of proteins located within the pre- and postsynaptic elements of inhibitory synapses. To this end, we exploited molecular markers of inhibitory synapses, interneurons and dystrophin-deficient mouse models to explore the role of dystrophin in determining the stereotypical patterning of inhibitory connectivity within the cellular networks of the hippocampus CA1 region. In tissue from wild-type (WT mice, immunoreactivity of neuroligin2 (NL2, an adhesion molecule expressed exclusively in postsynaptic elements of inhibitory synapses, and the vesicular GABA transporter (VGAT, a marker of GABAergic presynaptic elements, were predictably enriched in strata pyramidale and lacunosum moleculare. In acute contrast, NL2 and VGAT immunoreactivity was relatively evenly distributed across all CA1 layers in dystrophin-deficient mice. Similar changes were evident with the cannabinoid receptor 1, vesicular glutamate transporter 3, parvalbumin, somatostatin and the GABAA receptor alpha1 subunit. The data show that in the absence of dystrophin, there is a rearrangement of the molecular machinery, which underlies the precise spatio-temporal pattern of GABAergic synaptic transmission within the CA1 sub-field of the hippocampus.

  3. Characterization of major histocompatibility complex (MHC DRB exon 2 and DRA exon 3 fragments in a primary terrestrial rabies vector (Procyon lotor.

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    Sarrah Castillo

    Full Text Available The major histocompatibility complex (MHC presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor. Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250 bp and DRB exon 2 (228 bp. MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4-15.8% divergence and translated into 1 to 21 (1.3-27.6% divergence amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005, indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host.

  4. Comprehensive survey of SNPs in the Affymetrix exon array using the 1000 Genomes dataset.

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    Eric R Gamazon

    Full Text Available Microarray gene expression data has been used in genome-wide association studies to allow researchers to study gene regulation as well as other complex phenotypes including disease risks and drug response. To reach scientifically sound conclusions from these studies, however, it is necessary to get reliable summarization of gene expression intensities. Among various factors that could affect expression profiling using a microarray platform, single nucleotide polymorphisms (SNPs in target mRNA may lead to reduced signal intensity measurements and result in spurious results. The recently released 1000 Genomes Project dataset provides an opportunity to evaluate the distribution of both known and novel SNPs in the International HapMap Project lymphoblastoid cell lines (LCLs. We mapped the 1000 Genomes Project genotypic data to the Affymetrix GeneChip Human Exon 1.0ST array (exon array, which had been used in our previous studies and for which gene expression data had been made publicly available. We also evaluated the potential impact of these SNPs on the differentially spliced probesets we had identified previously. Though the 1000 Genomes Project data allowed a comprehensive survey of the SNPs in this particular array, the same approach can certainly be applied to other microarray platforms. Furthermore, we present a detailed catalogue of SNP-containing probesets (exon-level and transcript clusters (gene-level, which can be considered in evaluating findings using the exon array as well as benefit the design of follow-up experiments and data re-analysis.

  5. Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line.

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    Frederique Ruf-Zamojski

    Full Text Available Dystrophin (Dmd is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine ct90a. We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.

  6. Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs

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    Voit Thomas

    2011-04-01

    Full Text Available Abstract Background This study aimed to measure the gait abnormalities in GRMD (Golden retriever muscular dystrophy dogs during growth and disease progression using an ambulatory gait analyzer (3D-accelerometers as a possible tool to assess the effects of a therapeutic intervention. Methods Six healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status with repeated measurements. A principal component analysis (PCA was applied to perfect multivariate data interpretation. Results Speed, stride length, total power and force significantly already decreased (p Conclusion The gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.

  7. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

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    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  8. Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model

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    Jahnke Vanessa E

    2012-08-01

    Full Text Available Abstract Background Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR, separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s' effects. Results We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.

  9. Detection of deletion in the dystrophin gene of a patient with quadriceps myopathy.

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    Kumari D

    2000-01-01

    Full Text Available A 43 year old male presented with slowly progressive weakness of limbs and hypertrophy of triceps, brachioradialis and calf muscles for four years. There was thinning of quadriceps muscles in both thighs. Histological study was compatible with Becker muscular dystrophy (BMD. Genomic DNA analysis showed a deletion of the Hind III fragments, spanning exons 45-47. A junction fragment of 11.0 kb was observed along with a deletion of a 3.4 kb PstI fragment containing exon 51 in the patient, and in one of his two sisters. The clinical and laboratory characteristics in this patient are in keeping with what has been described ′quadriceps myopathy′ and fall within the phenotypic variants of BMD as has been shown by others.

  10. Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

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    Sandra Murphy

    2015-06-01

    Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

  11. Performance of genotype imputation for rare variants identified in exons and flanking regions of genes.

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    Li Li

    Full Text Available Genotype imputation has the potential to assess human genetic variation at a lower cost than assaying the variants using laboratory techniques. The performance of imputation for rare variants has not been comprehensively studied. We utilized 8865 human samples with high depth resequencing data for the exons and flanking regions of 202 genes and Genome-Wide Association Study (GWAS data to characterize the performance of genotype imputation for rare variants. We evaluated reference sets ranging from 100 to 3713 subjects for imputing into samples typed for the Affymetrix (500K and 6.0 and Illumina 550K GWAS panels. The proportion of variants that could be well imputed (true r(2>0.7 with a reference panel of 3713 individuals was: 31% (Illumina 550K or 25% (Affymetrix 500K with MAF (Minor Allele Frequency less than or equal 0.001, 48% or 35% with 0.0010.05. The performance for common SNPs (MAF>0.05 within exons and flanking regions is comparable to imputation of more uniformly distributed SNPs. The performance for rare SNPs (0.01humans via targeted exon resequencing into additional samples with GWAS data, but imputation of very rare variants (MAF< = 0.005 will require reference panels with thousands of subjects.

  12. Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

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    Mameli, Eva; Lepori, Maria Barbara; Chiappe, Francesca; Ranucci, Giusy; Di Dato, Fabiola; Iorio, Raffaele; Loudianos, Georgios

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease.

  13. Nestin expression in end-stage disease in dystrophin-deficient heart: implications for regeneration from endogenous cardiac stem cells.

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    Berry, Suzanne E; Andruszkiewicz, Peter; Chun, Ju Lan; Hong, Jun

    2013-11-01

    Nestin(+) cardiac stem cells differentiate into striated cells following myocardial infarct. Transplantation of exogenous stem cells into myocardium of a murine model for Duchenne muscular dystrophy (DMD) increased proliferation of endogenous nestin(+) stem cells and resulted in the appearance of nestin(+) striated cells. This correlated with, and may be responsible for, prevention of dilated cardiomyopathy. We examined nestin(+) stem cells in the myocardium of dystrophin/utrophin-deficient (mdx/utrn(-/-)) mice, a model for DMD. We found that 92% of nestin(+) interstitial cells expressed Flk-1, a marker present on cardiac progenitor cells that differentiate into the cardiac lineage, and that a subset expressed Sca-1, present on adult cardiac cells that become cardiomyocytes. Nestin(+) interstitial cells maintained expression of Flk-1 but lost Sca-1 expression with age and were present in lower numbers in dystrophin-deficient heart than in wild-type heart. Unexpectedly, large clusters of nestin(+) striated cells ranging in size from 20 to 250 cells and extending up to 500 μm were present in mdx/utrn(-/-) heart near the end stage of disease. These cells were also present in dystrophin-deficient mdx/utrn(+/-) and mdx heart but not wild-type heart. Nestin(+) striated cells expressed cardiac troponin I, desmin, and Connexin 43 and correlated with proinflammatory CD68(+) macrophages. Elongated nestin(+) interstitial cells with striations were observed that did not express Flk-1 or the late cardiac marker cardiac troponin I but strongly expressed the early cardiac marker desmin. Nestin was also detected in endothelial and smooth muscle cells. These data indicate that new cardiomyocytes form in dystrophic heart, and nestin(+) interstitial cells may generate them in addition to other cells of the cardiac lineage.

  14. Ultrastructural changes in the interstitial cells of Cajal and gastric dysrhythmias in mice lacking full-length dystrophin (mdx mice).

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    Vannucchi, Maria-Giuliana; Zizzo, Maria-Grazia; Zardo, Claudio; Pieri, Laura; Serio, Rosa; Mulè, Flavia; Faussone-Pellegrini, Maria-Simonetta

    2004-05-01

    At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca(2+) handling, and with defective generation and regeneration of slow wave activity.

  15. GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

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    Erik van der Wal

    2017-06-01

    Full Text Available Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1 GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs.

  16. Disruption of action potential and calcium signaling properties in malformed myofibers from dystrophin-deficient mice.

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    Hernández-Ochoa, Erick O; Pratt, Stephen J P; Garcia-Pelagio, Karla P; Schneider, Martin F; Lovering, Richard M

    2015-04-01

    Duchenne muscular dystrophy (DMD), the most common and severe muscular dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e., increased susceptibility to damage) are presumably due to structural instability of the myofiber cytoskeleton, but recent studies suggest that the increased presence of malformed/branched myofibers in dystrophic muscle may also play a role. We have previously studied myofiber morphology in healthy wild-type (WT) and dystrophic (MDX) skeletal muscle. Here, we examined myofiber excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) to assess the action potential (AP) properties. We also examined AP-induced Ca(2+) transients using high-speed confocal microscopy with rhod-2, and assessed sarcolemma fragility using elastimetry. AP recordings showed an increased width and time to peak in malformed MDX myofibers compared to normal myofibers from both WT and MDX, but no significant change in AP amplitude. Malformed MDX myofibers also exhibited reduced AP-induced Ca(2+) transients, with a further Ca(2+) transient reduction in the branches of malformed MDX myofibers. Mechanical studies indicated an increased sarcolemma deformability and instability in malformed MDX myofibers. The data suggest that malformed myofibers are functionally different from myofibers with normal morphology. The differences seen in AP properties and Ca(2+) signals suggest changes in excitability and remodeling of the global Ca(2+) signal, both of which could underlie reported weakness in dystrophic muscle. The biomechanical changes in the sarcolemma support the notion that malformed myofibers are more susceptible to damage. The high prevalence of malformed myofibers in dystrophic muscle may contribute to the progressive strength loss and fragility seen in dystrophic muscles. © 2015 The Authors. Physiological Reports published by Wiley

  17. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

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    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P muscle (65.7 versus 73.8 per cent; P muscle was lower than in the controls (25.9 versus 30.8 per cent; P muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  18. Alternative splicing in colon, bladder, and prostate cancer identified by exon-array analysis

    DEFF Research Database (Denmark)

    Thorsen, Kasper; Sørensen, Karina D.; Brems-Eskildsen, Anne Sofie;

    2008-01-01

    Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages......, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2...... from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19...

  19. Polymorphism of the second exon of human leukocyte antigen-DQA1, -DQB1 gene and genetic susceptibility to idiopathic dilated cardiomyopathy in people of the Han nationality in northern China

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LI Wei-min; SUN Ning-ling

    2005-01-01

    @@ Idiopathic dilated cardiomyopathy (IDC) is characterized by dilation and impaired contraction of the left ventricle or both, and it is a relevant cause of heart failure and a common indication for heart transplantation. The major pathogenetic hypothesis in IDC involves autoimmune mediated damage to myocytes. The development of autoimmune inflammatory damage occurs only in patients with a predisposing genetic background. Changes in the immune system concerning cell-mediated and humoral immunity have been detected. The immune system is strictly related to human leukocyte antigen (HLA), which is located on the surface of antigen presenting cells. Its primary function is to restrict T-cell receptors in the process of recognizing auto- or exterior antigen, and thus participates in or mediates immunological recognition, immunological response and immune regulation at various levels. HLA is a genetic marker of susceptibility to autoimmune myocardial damage.1 In the present study, the HLA-DQA1 and -DQB1 alleles in IDC patients were detected with the techniques of polymerase chain reaction-sequence specific primers (PCR-SSP) to explore the immunogenetic mechanisms involved in the pathogenesis of IDC.

  20. Origin of introns by 'intronization' of exonic sequences

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David;

    2008-01-01

    The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting...

  1. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern...

  2. Exon silencing by UAGG motifs in response to neuronal excitation.

    Directory of Open Access Journals (Sweden)

    Ping An

    2007-02-01

    Full Text Available Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

  3. The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

    Science.gov (United States)

    Ouafik, L H; Stoffers, D A; Campbell, T A; Johnson, R C; Bloomquist, B T; Mains, R E; Eipper, B A

    1992-10-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along

  4. Comparison of Affymetrix Gene Array with the Exon Array shows potential application for detection of transcript isoform variation

    Directory of Open Access Journals (Sweden)

    Coulombe-Huntington Jasmin

    2009-11-01

    Full Text Available Abstract Background The emergence of isoform-sensitive microarrays has helped fuel in-depth studies of the human transcriptome. The Affymetrix GeneChip Human Exon 1.0 ST Array (Exon Array has been previously shown to be effective in profiling gene expression at the isoform level. More recently, the Affymetrix GeneChip Human Gene 1.0 ST Array (Gene Array has been released for measuring gene expression and interestingly contains a large subset of probes from the Exon Array. Here, we explore the potential of using Gene Array probes to assess expression variation at the sub-transcript level. Utilizing datasets of the high quality Microarray Quality Control (MAQC RNA samples previously assayed on the Exon Array and Gene Array, we compare the expression measurements of the two platforms to determine the performance of the Gene Array in detecting isoform variations. Results Overall, we show that the Gene Array is comparable to the Exon Array in making gene expression calls. Moreover, to examine expression of different isoforms, we modify the Gene Array probe set definition file to enable summarization of probe intensity values at the exon level and show that the expression profiles between the two platforms are also highly correlated. Next, expression calls of previously known differentially spliced genes were compared and also show concordant results. Splicing index analysis, representing estimates of exon inclusion levels, shows a lower but good correlation between platforms. As the Gene Array contains a significant subset of probes from the Exon Array, we note that, in comparison, the Gene Array overlaps with fewer but still a high proportion of splicing events annotated in the Known Alt Events UCSC track, with abundant coverage of cassette exons. We discuss the ability of the Gene Array to detect alternative splicing and isoform variation and address its limitations. Conclusion The Gene Array is an effective expression profiling tool at gene and

  5. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

    Directory of Open Access Journals (Sweden)

    Sugnet Charles

    2006-12-01

    Full Text Available Abstract Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic

  6. The silent mutation nucleotide 744 G --> A, Lys172Lys, in exon 6 of BRCA2 results in exon skipping

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Steffensen, Ane Y; Jønson, Lars

    2009-01-01

    Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent...... and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G --> A/c.516 G --> A, Lys172Lys) in exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon trapping analysis showed that the mutation results in skipping...... of exon 6 and/or both exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation....

  7. Occipital horn syndrome and classical Menkes syndrome caused by deep intronic mutations, leading to the activation of ATP7A pseudo-exon

    DEFF Research Database (Denmark)

    Yasmeen, Saiqa; Lund, Katrine; De Paepe, Anne

    2014-01-01

    Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations...... patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo...... mechanism, which has hitherto been overlooked.European Journal of Human Genetics advance online publication, 4 September 2013; doi:10.1038/ejhg.2013.191....

  8. Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Lewis

    2010-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

  9. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    Science.gov (United States)

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

  10. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    Science.gov (United States)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  11. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    Directory of Open Access Journals (Sweden)

    Mei Li

    Full Text Available Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM. This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

  12. 免疫组织化学dystrophin染色诊断Duchenne型肌营养不良症的研究%Diagnosis of Duchenne muscular dystrophy through dystrophin expression detection by immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    刘鹏; 沈定国

    2008-01-01

    目的 探讨Duchenne型肌营养不良症(DMD)肌萎缩蛋白(dystrophin)表达规律和临床意义.方法 收集我院7例DMD患者作为试验组,7例非DMD患者为对照组.使用抗dystrophin杆状结构域单抗、免疫组织化学染色,观察肌膜dystrophin表达.结果 7例DMD患者肌细胞膜dystrophin阴性,7例非DMD患者dystrophin染色阳性.结论 证实DMD患者肌细胞膜dystrophin表达阴性,揭示dystrophin缺失是其发病机制,可以作为确诊DMD手段,对临床诊断DMD有实际意义.%Objective To study dystrophin expression in Duchenne muscular dystrophy (DMD) and non-DMD patients. Methods With immunohistochemistry method, using monoclonal antibody of dystrophin, expression of dystrophin was analyzed in 7 DMD patients (experimental group)and 7 non-DMD patients (control group). Results In 7 non-DMD patients, uniform and continuous dystrophin expression was found along the sarolemma, while not in 7 DMD patients. Conclusions Dystrophin expression in myocyte membrane is negative in DMD patients, which indicates that dystrophin loss may be involved in the pathogenesis of DMD. It can be used as a "gold standard" in diagnosing DMD.

  13. Intron phase correlations and the evolution of the intron/exon structure of genes.

    Science.gov (United States)

    Long, M; Rosenberg, C; Gilbert, W

    1995-01-01

    Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns. Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets. We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution. Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions. This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes. PMID:8618928

  14. Fetal microchimeric cells in a fetus-treats-its-mother paradigm do not contribute to dystrophin production in serially parous mdx females.

    Science.gov (United States)

    Seppanen, Elke Jane; Hodgson, Samantha Susan; Khosrotehrani, Kiarash; Bou-Gharios, George; Fisk, Nicholas M

    2012-10-10

    Throughout every pregnancy, genetically distinct fetal microchimeric stem/progenitor cells (FMCs) engraft in the mother, persist long after delivery, and may home to damaged maternal tissues. Phenotypically normal fetal lymphoid progenitors have been described to develop in immunodeficient mothers in a fetus-treats-its-mother paradigm. Since stem cells contribute to muscle repair, we assessed this paradigm in the mdx mouse model of Duchenne muscular dystrophy. mdx females were bred serially to either ROSAeGFP males or mdx males to obtain postpartum microchimeras that received either wild-type FMCs or dystrophin-deficient FMCs through serial gestations. To enhance regeneration, notexin was injected into the tibialis anterior of postpartum mice. FMCs were detected by qPCR at a higher frequency in injected compared to noninjected side muscle (P=0.02). However, the number of dystrophin-positive fibers was similar in mothers delivering wild-type compared to mdx pups. In addition, there was no correlation between FMC detection and percentage dystrophin, and no GFP+ve FMCs were identified that expressed dystrophin. In 10/11 animals, GFP+ve FMCs were detected by immunohistochemistry, of which 60% expressed CD45 with 96% outside the basal lamina defining myofiber contours. Finally we confirmed lack of FMC contribution to statellite cells in postpartum mdx females mated with Myf5-LacZ males. We conclude that the FMC contribution to regenerating muscles is insufficient to have a functional impact.

  15. Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-09-01

    Full Text Available The full-length dystrophin protein isoform of 427 kDa (Dp427, the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.

  16. Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

    Science.gov (United States)

    De Luca, Annamaria; Nico, Beatrice; Rolland, Jean-François; Cozzoli, Anna; Burdi, Rosa; Mangieri, Domenica; Giannuzzi, Viviana; Liantonio, Antonella; Cippone, Valentina; De Bellis, Michela; Nicchia, Grazia Paola; Camerino, Giulia Maria; Frigeri, Antonio; Svelto, Maria; Camerino, Diana Conte

    2008-11-01

    Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

  17. More deletions in the 5{prime} region than in the central region of the dystrophin gene were identified among Filipino Duchenne and Becker muscular dystrophy patients

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-11-06

    This report describes mutations in the dystrophin gene and the frequency of these mutations in Filipino pedigrees with Duchenne and Becker muscular dystrophy (DMD/BMD). The findings suggest the presence of genetic variability among DMD/BMD patients in different populations. 13 refs., 1 tab.

  18. Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

    Science.gov (United States)

    Allen, David G; Whitehead, Nicholas P; Froehner, Stanley C

    2016-01-01

    Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease.

  19. Transcriptional enhancers in protein-coding exons of vertebrate developmental genes.

    Directory of Open Access Journals (Sweden)

    Deborah I Ritter

    Full Text Available Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77% exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less. GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%, suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147 or tissue-specificity (coding: 14/24 vs. noncoding: 50/105. Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81. Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

  20. microRNA-340-5p Functions Downstream of Cardiotrophin-1 to Regulate Cardiac Eccentric Hypertrophy and Heart Failure via Target Gene Dystrophin.

    Science.gov (United States)

    Zhou, Jian; Gao, Jie; Zhang, Xiaoya; Liu, Yan; Gu, Song; Zhang, Xitao; An, Xiangguang; Yan, Jun; Xin, Yue; Su, Pixiong

    2015-01-01

    Pathological cardiac hypertrophy inevitably leads to the unfavorable outcomes of heart failure (HF) or even sudden death. microRNAs are key regulation factors participating in many pathophysiological processes. Recently, we observed upregulation of microRNA-340-5p (miR-340) in failing human hearts because of dilated cardiomyopathy, but the functional consequence of miR-340 remains to be clarified.We transfected neonatal cardiomyocytes with miR-340 and found fetal gene expression including Nppa, Nppb and Myh7. We also observed eccentric hypertrophy development upon treatment which was analogous to the phenotype after cardiotrophin-1 (CT-1) stimulation. As a potent inducer of cardiac eccentric hypertrophy, treatment by IL-6 family members CT-1 and leukemia inhibitory factor (LIF) led to the elevation of miR-340. Knockdown of miR-340 using antagomir attenuated fetal gene expression and hypertrophy formation, which means miR-340 could convey the hypertrophic signal of CT-1. To demonstrate the initial factor of miR-340 activation, we constructed a volume overloaded abdominal aorta-inferior vena cava fistula rat HF model. miR-340 and CT-1 were found to be up-regulated in the left ventricle. Dystrophin (DMD), a putative target gene of miR-340 which is eccentric hypertrophy-susceptible, was decreased in this HF model upon Western blotting and immunohistochemistry tests. Luciferase assay constructed in two seed sequence of DMD gene 3'UTR showed decreased luciferase activities, and miR-340 transfected cells resulted in the degradation of DMD.miR-340 is a pro-eccentric hypertrophy miRNA, and its expression is dependent on volume overload and cytokine CT-1 activation. Cardiomyocyte structure protein DMD is a target of miR-340.

  1. Distribution of components of basal lamina and dystrophin-dystroglycan complex in the rat pineal gland: differences from the brain tissue and between the subdivisions of the gland.

    Science.gov (United States)

    Bagyura, Zsolt; Pócsai, Károly; Kálmán, Mihály

    2010-01-01

    The pineal gland is an evagination of the brain tissue, a circumventricular neuroendocrine organ. Our immunohistochemical study investigates basal lamina components (laminin, agrin, perlecan, fibronectin), their receptor, the dystrophin-dystroglycan complex (beta-dystroglycan, dystrophin utrophin), aquaporins (-4,-9) and cellular markers (S100, neurofilament, GFAP, glutamine synthetase) in the adult rat corpus pineale. The aim was to compare the immunohistochemical features of the cerebral and pineal vessels and their environment, and to compare their features in the distal and proximal subdivisions of the so-called 'superficial pineal gland'. In contrast to the cerebral vessels, pineal vessels proved to be immunonegative to alpha1-dystrobrevin, but immunoreactive to laminin. An inner, dense, and an outer, loose layer of laminin as two basal laminae were present. The gap between them contained agrin and perlecan. Basal lamina components enmeshed the pinealocytes, too. Components of dystrophin-dystroglycan complex were also distributed along the vessels. Dystrophin, utrophin and agrin gave a 'patchy' distribution rather than a continuous one. The vessels were interconnected by wing-like structures, composed of basal lamina-components: a delicate network forming nests for cells. Cells immunostained with glutamine synthetase, S100-protein or neurofilament protein contacted the vessels, as well as GFAP- or aquaporin-immunostained astrocytes. Within the body a smaller, proximal, GFAP-and aquaporin-containing subdivision, and a larger, distal, GFAP-and aquaporin-free subdivision could be distinguished. The vascular localization of agrin and utrophin, as well as dystrophin, delineated vessels unequally, preferring the proximal or distal end of the body, respectively.

  2. Antisense-mediated exon skipping to reframe transcripts.

    Science.gov (United States)

    Turczynski, Sandrina; Titeux, Matthias; Pironon, Nathalie; Hovnanian, Alain

    2012-01-01

    Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.

  3. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    Science.gov (United States)

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  4. Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.

    Directory of Open Access Journals (Sweden)

    Sushma Grellscheid

    2011-12-01

    Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

  5. Increased constitutive nitric oxide production by whole body periodic acceleration ameliorates alterations in cardiomyocytes associated with utrophin/dystrophin deficiency.

    Science.gov (United States)

    Lopez, Jose R; Kolster, Juan; Zhang, Rui; Adams, Jose

    2017-07-01

    Duchenne Muscular Dystrophy (DMD) cardiomyopathy is a progressive lethal disease caused by the lack of the dystrophin protein in the heart. The most widely used animal model of DMD is the dystrophin-deficient mdx mouse; however, these mice exhibit a mild dystrophic phenotype with heart failure only late in life. In contrast, mice deficient for both dystrophin and utrophin (mdx/utrn(-/-), or dKO) can be used to model severe DMD cardiomyopathy where pathophysiological indicators of heart failure are detectable by 8-10weeks of age. Nitric oxide (NO) is an important signaling molecule involved in vital functions of regulating rhythm, contractility, and microcirculation of the heart, and constitutive NO production affects the function of proteins involved in excitation-contraction coupling. In this study, we explored the efficacy of enhancing NO production as a therapeutic strategy for treating DMD cardiomyopathy using the dKO mouse model of DMD. Specifically, NO production was induced via whole body periodic acceleration (pGz), a novel non-pharmacologic intervention which enhances NO synthase (NOS) activity through sinusoidal motion of the body in a headward-footward direction, introducing pulsatile shear stress to the vascular endothelium and cardiomyocyte plasma membrane. Male dKO mice were randomized at 8weeks of age to receive daily pGz (480cpm, Gz±3.0m/s(2), 1h/d) for 4weeks or no treatment, and a separate age-matched group of WT animals (pGz-treated and untreated) served as non-diseased controls. At the conclusion of the protocol, cardiomyocytes from untreated dKO animals had, respectively, 4.3-fold and 3.5-fold higher diastolic resting concentration of Ca(2+) ([Ca(2+)]d) and Na(+) ([Na(+)]d) compared to WT, while pGz treatment significantly reduced these levels. For dKO cardiomyocytes, pGz treatment also improved the depressed contractile function, decreased oxidative stress, blunted the elevation in calpain activity, and mitigated the abnormal increase in [Ca

  6. The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

    OpenAIRE

    Chiba, H; Komatsu, K.; Lee, Y.C.; Tomizuka, T; Strott, C A

    1995-01-01

    The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal...

  7. Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing.

    Science.gov (United States)

    Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

    2016-11-24

    Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.

  8. Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Andresen, B S; Jensen, T G; Bross, P

    1994-01-01

    spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11......Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985......), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot...

  9. Defects in mitochondrial ATP synthesis in dystrophin-deficient mdx skeletal muscles may be caused by complex I insufficiency.

    Directory of Open Access Journals (Sweden)

    Emma Rybalka

    Full Text Available Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca2+ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca2+ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca2+ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb's cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca2+ load in comparison to controls, and 400 nM extramitochondrial Ca2+ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.

  10. Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse.

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    Christopher F Spurney

    Full Text Available Thymosin beta-4 (Tbeta4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

  11. Early manifestation of alteration in cardiac function in dystrophin deficient mdx mouse using 3D CMR tagging

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    Zhong Jia

    2009-10-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is caused by the absence of the cytoskeletal protein, dystrophin. In DMD patients, dilated cardiomyopathy leading to heart failure may occur during adolescence. However, early cardiac dysfunction is frequently undetected due to physical inactivity and generalized debilitation. The objective of this study is to determine the time course of cardiac functional alterations in mdx mouse, a mouse model of DMD, by evaluating regional ventricular function with CMR tagging. Methods In vivo myocardial function was evaluated by 3D CMR tagging in mdx mice at early (2 months, middle (7 months and late (10 months stages of disease development. Global cardiac function, regional myocardial wall strains, and ventricular torsion were quantified. Myocardial lesions were assessed with Masson's trichrome staining. Results Global contractile indexes were similar between mdx and C57BL/6 mice in each age group. Histology analysis showed that young mdx mice were free of myocardial lesions. Interstitial fibrosis was present in 7 month mdx mice, with further development into patches or transmural lesions at 10 months of age. As a result, 10 month mdx mice showed significantly reduced regional strain and torsion. However, young mdx mice showed an unexpected increase in regional strain and torsion, while 7 month mdx mice displayed similar regional ventricular function as the controls. Conclusion Despite normal global ventricular function, CMR tagging detected a biphasic change in myocardial wall strain and torsion, with an initial increase at young age followed by progressive decrease at older ages. These results suggest that CMR tagging can provide more sensitive measures of functional alterations than global functional indexes in dystrophin-related cardiomyopathies.

  12. Sparing of the dystrophin-deficient cranial sartorius muscle is associated with classical and novel hypertrophy pathways in GRMD dogs.

    Science.gov (United States)

    Nghiem, Peter P; Hoffman, Eric P; Mittal, Priya; Brown, Kristy J; Schatzberg, Scott J; Ghimbovschi, Svetlana; Wang, Zuyi; Kornegay, Joe N

    2013-11-01

    Both Duchenne and golden retriever muscular dystrophy (GRMD) are caused by dystrophin deficiency. The Duchenne muscular dystrophy sartorius muscle and orthologous GRMD cranial sartorius (CS) are relatively spared/hypertrophied. We completed hierarchical clustering studies to define molecular mechanisms contributing to this differential involvement and their role in the GRMD phenotype. GRMD dogs with larger CS muscles had more severe deficits, suggesting that selective hypertrophy could be detrimental. Serial biopsies from the hypertrophied CS and other atrophied muscles were studied in a subset of these dogs. Myostatin showed an age-dependent decrease and an inverse correlation with the degree of GRMD CS hypertrophy. Regulators of myostatin at the protein (AKT1) and miRNA (miR-539 and miR-208b targeting myostatin mRNA) levels were altered in GRMD CS, consistent with down-regulation of myostatin signaling, CS hypertrophy, and functional rescue of this muscle. mRNA and proteomic profiling was used to identify additional candidate genes associated with CS hypertrophy. The top-ranked network included α-dystroglycan and like-acetylglucosaminyltransferase. Proteomics demonstrated increases in myotrophin and spectrin that could promote hypertrophy and cytoskeletal stability, respectively. Our results suggest that multiple pathways, including decreased myostatin and up-regulated miRNAs, α-dystroglycan/like-acetylglucosaminyltransferase, spectrin, and myotrophin, contribute to hypertrophy and functional sparing of the CS. These data also underscore the muscle-specific responses to dystrophin deficiency and the potential deleterious effects of differential muscle involvement. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. El splicing alternativo del exón 5 de la citocromo p450 aromatasa podría ser un mecanismo de regulación de la producción de estrógenos en humanos Exon 5 alternative splicing of the cytochrome P450 aromatase could be a regulatory mechanism for estrogen production in humans

    Directory of Open Access Journals (Sweden)

    Carolina M. Pepe

    2007-08-01

    adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2 levels suggesting residual P450Aro activity (AA. Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

  14. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T......-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response. Copyright © 2012 Elsevier Inc. All rights reserved. PMID:22698810[PubMed - in process]...

  15. NextSearch: A Search Engine for Mass Spectrometry Data against a Compact Nucleotide Exon Graph.

    Science.gov (United States)

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok

    2015-07-02

    Proteogenomics research has been using six-frame translation of the whole genome or amino acid exon graphs to overcome the limitations of reference protein sequence database; however, six-frame translation is not suitable for annotating genes that span over multiple exons, and amino acid exon graphs are not convenient to represent novel splice variants and exon skipping events between exons of incompatible reading frames. We propose a proteogenomic pipeline NextSearch (Nucleotide EXon-graph Transcriptome Search) that is based on a nucleotide exon graph. This pipeline consists of constructing a compact nucleotide exon graph that systematically incorporates novel splice variations and a search tool that identifies peptides by directly searching the nucleotide exon graph against tandem mass spectra. Because our exon graph stores nucleotide sequences, it can easily represent novel splice variations and exon skipping events between incompatible reading frame exons. Searching for peptide identification is performed against this nucleotide exon graph, without converting it into a protein sequence in FASTA format, achieving an order of magnitude reduction in the size of the sequence database storage. NextSearch outputs the proteome-genome/transcriptome mapping results in a general feature format (GFF) file, which can be visualized by public tools such as the UCSC Genome Browser.

  16. Polymorphism of exon 3 of the HLA-G gene

    DEFF Research Database (Denmark)

    Hviid, T V; Meldgaard, Michael; Sørensen, S

    1997-01-01

    populations have only revealed a limited polymorphism. We investigated the polymorphism of the exon 3 of HLA-G by means of Polymerase Chain Reaction (PCR)-Single Strand Conformation Polymorphism (SSCP)- and DNA sequencing analysis in a Danish population. We detected four single-base substitutions in exon 3...... rate of embryos. HLA-G seems to play an important role in the feto-maternal relationship. The polymorphism of the HLA-G locus is not fully clarified. One study has shown extensive nucleotide sequence variation in the exon 3 (alpha-2 domain) in healthy African Americans. A few studies in other...... compared to the sequence of HLA-6.0 (G*01011); one of these has not been reported before. We also found a deletion of the first base of codon 130 or the third of codon 129 in a heterozygous individual. This study, together with previous results, suggests that the polymorphism of exon 3 of the HLA-G gene...

  17. Translational and Regulatory Challenges for Exon Skipping Therapies

    NARCIS (Netherlands)

    Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M. G.; Wells, Dominic J.; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

    2014-01-01

    Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and int

  18. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene...

  19. Intronic L1 retrotransposons and nested genes cause transcriptional interference by inducing intron retention, exonization and cryptic polyadenylation.

    Directory of Open Access Journals (Sweden)

    Kristel Kaer

    Full Text Available BACKGROUND: Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals. CONCLUSIONS/SIGNIFICANCE: Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression.

  20. The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

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    Nurit Gal-Mark

    2009-11-01

    Full Text Available More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT. In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5' splice site (5'ss, even in the presence of a stronger 5'ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5'ss. This enhancing effect depended on the strength of the downstream 5'ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5'ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within approximately 20 nt downstream of the 5'ss. For most metazoans, the strength of the 5'ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

  1. The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

    Directory of Open Access Journals (Sweden)

    Nurit Gal-Mark

    2009-11-01

    Full Text Available More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT. In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5' splice site (5'ss, even in the presence of a stronger 5'ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5'ss. This enhancing effect depended on the strength of the downstream 5'ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5'ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within approximately 20 nt downstream of the 5'ss. For most metazoans, the strength of the 5'ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

  2. Deep RNA sequencing reveals the smallest known mitochondrial micro exon in animals: The placozoan cox1 single base pair exon.

    Science.gov (United States)

    Osigus, Hans-Jürgen; Eitel, Michael; Schierwater, Bernd

    2017-01-01

    The phylum Placozoa holds a key position for our understanding of the evolution of mitochondrial genomes in Metazoa. Placozoans possess large mitochondrial genomes which harbor several remarkable characteristics such as a fragmented cox1 gene and trans-splicing cox1 introns. A previous study also suggested the existence of cox1 mRNA editing in Trichoplax adhaerens, yet the only formally described species in the phylum Placozoa. We have analyzed RNA-seq data of the undescribed sister species, Placozoa sp. H2 ("Panama" clone), with special focus on the mitochondrial mRNA. While we did not find support for a previously postulated cox1 mRNA editing mechanism, we surprisingly found two independent transcripts representing intermediate cox1 mRNA splicing stages. Both transcripts consist of partial cox1 exon as well as overlapping intron fragments. The data suggest that the cox1 gene harbors a single base pair (cytosine) micro exon. Furthermore, conserved group I intron structures flank this unique micro exon also in other placozoans. We discuss the evolutionary origin of this micro exon in the context of a self-splicing intron gain in the cox1 gene of the last common ancestor of extant placozoans.

  3. Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies.

    Directory of Open Access Journals (Sweden)

    Narinder Janghra

    Full Text Available Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ -sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these

  4. Transactive response DNA-binding protein 43 (TDP-43) regulates alternative splicing of tau exon 10: Implications for the pathogenesis of tauopathies.

    Science.gov (United States)

    Gu, Jianlan; Chen, Feng; Iqbal, Khalid; Gong, Cheng-Xin; Wang, Xinglong; Liu, Fei

    2017-06-23

    Hyperphosphorylation and aggregation of the neuronal protein tau are responsible for neurodegenerative diseases called tauopathies. Dysregulation of the alternative splicing of tau exon 10 results in alterations of the ratio of two tau isoforms, 3R-tau and 4R-tau, which have been seen in several tauopathies. Transactive response DNA-binding protein of 43 kDa (TDP-43) is involved in the regulation of RNA processing, including splicing. Cytoplasmic aggregation of TDP-43 has been observed in the brains of individuals with chronic traumatic encephalopathy or Alzheimer's disease, diseases in which neurofibrillary tangles of hyperphosphorylated tau are hallmarks. Here, we investigated the role of TDP-43 in tau exon 10 splicing. We found that TDP-43 promoted tau exon 10 inclusion, which increased production of the 4R-tau isoform. Moreover, TDP-43 could bind to intron 9 of tau pre-mRNA. Deletion of the TDP-43 N or C terminus promoted its cytoplasmic aggregation and abolished or diminished TDP-43-promoted tau exon 10 inclusion. Several TDP-43 mutations associated with amyotrophic lateral sclerosis or frontotemporal lobar degeneration with ubiquitin inclusions promoted tau exon 10 inclusion more effectively than wild-type TDP-43 but did not affect TDP-43 cytoplasmic aggregation in cultured cells. The ratio of 3R-tau/4R-tau was decreased in transgenic mouse brains expressing human TDP-43 and increased in the brains expressing the disease-causing mutation TDP-43(M337V), in which cytoplasmic TDP-43 was increased. These findings suggest that TDP-43 promotes tau exon 10 inclusion and 4R-tau expression and that disease-related changes of TDP-43, truncations and mutations, affect its function in tau exon 10 splicing, possibly because of TDP-43 mislocalization to the cytoplasm. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

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    Manuel Irimia

    Full Text Available Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans factors that bind to different sequence (cis elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease.

  6. Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome

    NARCIS (Netherlands)

    Albers, C.A.; Paul, D.S.; Schulze, H.; Freson, K.; Stephens, J.C.; Smethurst, P.A.; Jolley, J.D.; Cvejic, A.; Kostadima, M.; Bertone, P.; Breuning, M.H.; Debili, N.; Deloukas, P.; Favier, R.; Fiedler, J.; Hobbs, C.M.; Huang, N.; Hurles, M.E.; Kiddle, G.; Krapels, I.; Nurden, P.; Ruivenkamp, C.A.; Sambrook, J.G.; Smith, K.; Stemple, D.L.; Strauss, G.; Thys, C.; Geet, C. van; Newbury-Ecob, R.; Ouwehand, W.H.; Ghevaert, C.

    2012-01-01

    The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the reg

  7. Evaluating the protein coding potential of exonized transposable element sequences

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2007-11-01

    Full Text Available Abstract Background Transposable element (TE sequences, once thought to be merely selfish or parasitic members of the genomic community, have been shown to contribute a wide variety of functional sequences to their host genomes. Analysis of complete genome sequences have turned up numerous cases where TE sequences have been incorporated as exons into mRNAs, and it is widely assumed that such 'exonized' TEs encode protein sequences. However, the extent to which TE-derived sequences actually encode proteins is unknown and a matter of some controversy. We have tried to address this outstanding issue from two perspectives: i-by evaluating ascertainment biases related to the search methods used to uncover TE-derived protein coding sequences (CDS and ii-through a probabilistic codon-frequency based analysis of the protein coding potential of TE-derived exons. Results We compared the ability of three classes of sequence similarity search methods to detect TE-derived sequences among data sets of experimentally characterized proteins: 1-a profile-based hidden Markov model (HMM approach, 2-BLAST methods and 3-RepeatMasker. Profile based methods are more sensitive and more selective than the other methods evaluated. However, the application of profile-based search methods to the detection of TE-derived sequences among well-curated experimentally characterized protein data sets did not turn up many more cases than had been previously detected and nowhere near as many cases as recent genome-wide searches have. We observed that the different search methods used were complementary in the sense that they yielded largely non-overlapping sets of hits and differed in their ability to recover known cases of TE-derived CDS. The probabilistic analysis of TE-derived exon sequences indicates that these sequences have low protein coding potential on average. In particular, non-autonomous TEs that do not encode protein sequences, such as Alu elements, are frequently

  8. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jianmin, E-mail: jmhuang@partners.org [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Levitsky, Lynne L. [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Rhoads, David B., E-mail: rhoads@helix.mgh.harvard.edu [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States)

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  9. A new chromosome x exon-specific microarray platform for screening of patients with X-linked disorders.

    Science.gov (United States)

    Bashiardes, Stavros; Kousoulidou, Ludmila; van Bokhoven, Hans; Ropers, Hans-Hilger; Chelly, Jamel; Moraine, Claude; de Brouwer, Arjan P M; Van Esch, Hilde; Froyen, Guy; Patsalis, Philippos C

    2009-11-01

    Recent studies and advances in high-density oligonucleotide arrays have shown that microdeletions and microduplications occur at a high frequency in the human genome, causing various genetic conditions including mental retardation. Thus far little is known about the pathways leading to this disease, and implementation of microarrays is hampered by their increasing cost and complexity, underlining the need for new diagnostic tools. The aim of this study was to introduce a new targeted platform called "chromosome X exon-specific array" and to apply this new platform to screening of 20 families (including one blind positive control) with suspected X-linked mental retardation, to identify new causative X-linked mental retardation genes. The new microarray contains of 21,939 oligonucleotides covering 92.9% of all exons of all genes on chromosome X. Patient screening resulted in successful identification of the blind positive control included in the sample of 20 families, and one of the remaining 19 families was found to carry a 1.78-kilobase deletion involving all exons of pseudogene BRAF2. The BRAF2 deletion segregated in the family and was not found in 200 normal male samples, and no copy number variations are reported in this region. Further studies and focused investigation of X-linked disorders have the potential to reveal the molecular basis of human genetic pathological conditions that are caused by copy-number changes in chromosome X genes.

  10. A simple physical model predicts small exon length variations.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available One of the most common splice variations are small exon length variations caused by the use of alternative donor or acceptor splice sites that are in very close proximity on the pre-mRNA. Among these, three-nucleotide variations at so-called NAGNAG tandem acceptor sites have recently attracted considerable attention, and it has been suggested that these variations are regulated and serve to fine-tune protein forms by the addition or removal of a single amino acid. In this paper we first show that in-frame exon length variations are generally overrepresented and that this overrepresentation can be quantitatively explained by the effect of nonsense-mediated decay. Our analysis allows us to estimate that about 50% of frame-shifted coding transcripts are targeted by nonsense-mediated decay. Second, we show that a simple physical model that assumes that the splicing machinery stochastically binds to nearby splice sites in proportion to the affinities of the sites correctly predicts the relative abundances of different small length variations at both boundaries. Finally, using the same simple physical model, we show that for NAGNAG sites, the difference in affinities of the neighboring sites for the splicing machinery accurately predicts whether splicing will occur only at the first site, splicing will occur only at the second site, or three-nucleotide splice variants are likely to occur. Our analysis thus suggests that small exon length variations are the result of stochastic binding of the spliceosome at neighboring splice sites. Small exon length variations occur when there are nearby alternative splice sites that have similar affinity for the splicing machinery.

  11. An Exon-Capture System for the Entire Class Ophiuroidea.

    Science.gov (United States)

    Hugall, Andrew F; O'Hara, Timothy D; Hunjan, Sumitha; Nilsen, Roger; Moussalli, Adnan

    2016-01-01

    Exon-capture studies have typically been restricted to relatively shallow phylogenetic scales due primarily to hybridization constraints. Here, we present an exon-capture system for an entire class of marine invertebrates, the Ophiuroidea, built upon a phylogenetically diverse transcriptome foundation. The system captures approximately 90% of the 1,552 exon target, across all major lineages of the quarter-billion-year-old extant crown group. Key features of our system are 1) basing the target on an alignment of orthologous genes determined from 52 transcriptomes spanning the phylogenetic diversity and trimmed to remove anything difficult to capture, map, or align; 2) use of multiple artificial representatives based on ancestral state reconstructions rather than exemplars to improve capture and mapping of the target; 3) mapping reads to a multi-reference alignment; and 4) using patterns of site polymorphism to distinguish among paralogy, polyploidy, allelic differences, and sample contamination. The resulting data give a well-resolved tree (currently standing at 417 samples, 275,352 sites, 91% data-complete) that will transform our understanding of ophiuroid evolution and biogeography.

  12. SinEx DB: a database for single exon coding sequences in mammalian genomes.

    Science.gov (United States)

    Jorquera, Roddy; Ortiz, Rodrigo; Ossandon, F; Cárdenas, Juan Pablo; Sepúlveda, Rene; González, Carolina; Holmes, David S

    2016-01-01

    Eukaryotic genes are typically interrupted by intragenic, noncoding sequences termed introns. However, some genes lack introns in their coding sequence (CDS) and are generally known as 'single exon genes' (SEGs). In this work, a SEG is defined as a nuclear, protein-coding gene that lacks introns in its CDS. Whereas, many public databases of Eukaryotic multi-exon genes are available, there are only two specialized databases for SEGs. The present work addresses the need for a more extensive and diverse database by creating SinEx DB, a publicly available, searchable database of predicted SEGs from 10 completely sequenced mammalian genomes including human. SinEx DB houses the DNA and protein sequence information of these SEGs and includes their functional predictions (KOG) and the relative distribution of these functions within species. The information is stored in a relational database built with My SQL Server 5.1.33 and the complete dataset of SEG sequences and their functional predictions are available for downloading. SinEx DB can be interrogated by: (i) a browsable phylogenetic schema, (ii) carrying out BLAST searches to the in-house SinEx DB of SEGs and (iii) via an advanced search mode in which the database can be searched by key words and any combination of searches by species and predicted functions. SinEx DB provides a rich source of information for advancing our understanding of the evolution and function of SEGs.Database URL: www.sinex.cl.

  13. Biased exon/intron distribution of cryptic and de novo 3' splice sites.

    Science.gov (United States)

    Královicová, Jana; Christensen, Mikkel B; Vorechovský, Igor

    2005-01-01

    We compiled sequences of previously published aberrant 3' splice sites (3'ss) that were generated by mutations in human disease genes. Cryptic 3'ss, defined here as those resulting from a mutation of the 3'YAG consensus, were more frequent in exons than in introns. They clustered in approximately 20 nt region adjacent to authentic 3'ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3'ss that were induced by mutations outside the 3'YAG consensus (designated 'de novo') were in introns. The activation of intronic de novo 3'ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3'ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro-Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3'ss. Finally, AG-creating mutations in the PPT that produced aberrant 3'ss upstream of the predicted BPS in vivo shared a similar 'BPS-new AG' distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3'ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

  14. Growth hormone receptor exon 3 isoforms and their implication in growth disorders and treatment.

    Science.gov (United States)

    Jorge, Alexander A L; Arnhold, Ivo J P

    2009-04-01

    Human recombinant growth hormone (hGH) has been used to treat short stature in several different conditions, but considerable inter-individual variation in short- and long-term growth response exists. Pharmacogenomics can provide important insights into hGH therapy. The GH receptor (GHR) is the first key molecule mediating GH action. In the past 3 years, a common GHR polymorphism reflecting the presence (GHRfl) or absence (GHRd3) of exon 3 has been under intensive investigation regarding its influence on the response to hGH therapy. Studies that evaluated response to GH treatment determined by these two GHR isoforms in children with GH deficiency, girls with Turner syndrome, children born small for gestational age and patients with acromegaly showed that patients carrying the GHRd3 allele demonstrated a greater GH sensitivity than patients homozygous for the GHRfl allele. Other studies presented contradictory data, however, which may be caused by confounding factors such as small sample sizes and differences in experimental design. This GHR exon 3 genotype is the first identified genetic factor found to modulate the individual response to GH therapy. This article reviews the historical aspects and pharmacogenetic studies published to date in relation to this GHR polymorphism. The analyses of present and future validation studies may define the use of this and other polymorphisms in clinical practice, moving from pharmacogenetics to routine application and allowing individualization of hGH doses to optimize final outcome.

  15. Absence of Dystrophin Related Protein-2 disrupts Cajal bands in a patient with Charcot-Marie-Tooth disease.

    Science.gov (United States)

    Brennan, Kathryn M; Bai, Yunhong; Pisciotta, Chiara; Wang, Suola; Feely, Shawna M E; Hoegger, Mark; Gutmann, Laurie; Moore, Steven A; Gonzalez, Michael; Sherman, Diane L; Brophy, Peter J; Züchner, Stephan; Shy, Michael E

    2015-10-01

    Using exome sequencing in an individual with Charcot-Marie-Tooth disease (CMT) we have identified a mutation in the X-linked dystrophin-related protein 2 (DRP2) gene. A 60-year-old gentleman presented to our clinic and underwent clinical, electrophysiological and skin biopsy studies. The patient had clinical features of a length dependent sensorimotor neuropathy with an age of onset of 50 years. Neurophysiology revealed prolonged latencies with intermediate conduction velocities but no conduction block or temporal dispersion. A panel of 23 disease causing genes was sequenced and ultimately was uninformative. Whole exome sequencing revealed a stop mutation in DRP2, c.805C>T (Q269*). DRP2 interacts with periaxin and dystroglycan to form the periaxin-DRP2-dystroglycan complex which plays a role in the maintenance of the well-characterized Cajal bands of myelinating Schwann cells. Skin biopsies from our patient revealed a lack of DRP2 in myelinated dermal nerves by immunofluorescence. Furthermore electron microscopy failed to identify Cajal bands in the patient's dermal myelinated axons in keeping with ultrastructural pathology seen in the Drp2 knockout mouse. Both the electrophysiologic and dermal nerve twig pathology support the interpretation that this patient's DRP2 mutation causes characteristic morphological abnormalities recapitulating the Drp2 knockout model and potentially represents a novel genetic cause of CMT. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Meghna Pant

    Full Text Available The utrophin-dystrophin deficient (DKO mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD. However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1 and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients.

  17. Identifying alternative hyper-splicing signatures in MG-thymoma by exon arrays.

    Directory of Open Access Journals (Sweden)

    Lilach Soreq

    Full Text Available BACKGROUND: The vast majority of human genes (>70% are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. METHODOLOGY/PRINCIPAL FINDINGS: We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. CONCLUSIONS/SIGNIFICANCE: Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatibility gene HLA-DRB1 and interleukin (IL19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF(2 and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH, Reverse Transcription-Polymerase Chain Reaction (RT-PCR and mass spectrometry (MS followed by peptide sequencing. Our findings

  18. Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

    Science.gov (United States)

    Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

    2016-01-01

    The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

  19. Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

    Science.gov (United States)

    Di Giacomo, Daniela; Gaildrat, Pascaline; Abuli, Anna; Abdat, Julie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2013-11-01

    Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

  20. Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

    Science.gov (United States)

    Betts, Corinne; Saleh, Amer F; Arzumanov, Andrey A; Hammond, Suzan M; Godfrey, Caroline; Coursindel, Thibault; Gait, Michael J; Wood, Matthew Ja

    2012-08-14

    Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

  1. Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment

    Directory of Open Access Journals (Sweden)

    Corinne Betts

    2012-01-01

    Full Text Available Antisense oligonucleotides (AOs are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD. AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO: Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

  2. A founder synonymous COL7A1 mutation in three Danish families with dominant dystrophic epidermolysis bullosa pruriginosa identifies exonic regulatory sequences required for exon 87 splicing

    DEFF Research Database (Denmark)

    Covaciu, C; Grosso, F; Pisaneschi, E

    2011-01-01

    a previously unrecognized translationally silent exonic COL7A1 mutation that results in skipping of exon 87 and is associated with DDEB-Pr phenotypes in several members of three apparently unrelated Danish families. A haplotype segregation study suggested a common ancestor in these kindred. Functional splicing...... shoulders. DEB-Pr is caused by either dominant (DDEB-Pr) or recessive mutations in the COL7A1 gene encoding type VII collagen (COLVII). The full spectrum of COL7A1 mutations in DEB-Pr remains elusive and the genotype-phenotype correlation is largely incomplete. Here, we report and functionally characterize...... analysis of the mutant exon by a COL7A1 minigene construct and computational prediction for splicing regulatory cis-sequences prove that the mutation alters the activity of an exonic splicing enhancer (ESE) critical for exon inclusion. These findings substantiate for the first time the involvement...

  3. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

    Science.gov (United States)

    Quentmeier, H; Martelli, M P; Dirks, W G; Bolli, N; Liso, A; Macleod, R A F; Nicoletti, I; Mannucci, R; Pucciarini, A; Bigerna, B; Martelli, M F; Mecucci, C; Drexler, H G; Falini, B

    2005-10-01

    We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

  4. Common exon 3 polymorphism of the GH receptor (GHR) gene and effect of GH therapy on growth in Korean children with idiopathic short stature (ISS).

    Science.gov (United States)

    Ko, Jung Min; Park, Jung Young; Yoo, Han-Wook

    2009-01-01

    A human GH receptor (GHR) gene exon 3 polymorphism (d3-GHR) has been reported to be associated with responsiveness to GH therapy. We assessed the frequencies of this polymorphism in Korean control and idiopathic short stature (ISS) populations, and analysed short-term growth response to GH therapy according to GHR-exon 3 genotypes in Korean children with ISS. This was a retrospective study in 158 ISS children. Auxological and endocrine parameters were measured, and the GHR-exon 3 genotype was analysed. Allelic frequencies of GHR-exon 3 genotype were compared between the ISS group and a control group. GH had been administered for 62 patients, 52 of whom remained prepubertal after the first follow-up year. Changes in height velocity (HV) and IGF-1 and IGFBP-3 concentrations following GH therapy were compared in patients with these genotypes. There was no difference in GHR-exon 3 genotype frequency between ISS and control groups of Koreans. However, the fl/fl genotype was more frequent in Koreans than in Caucasians. ISS children with d3-GHR showed a significantly higher increment in HV (P = 0.002) and a marginally significant increment in IGF-1 concentration (P = 0.064) at the first year of GH therapy. fl-GHR was more frequently detected in a Korean population than in Caucasians. The growth promotion efficacy of GH therapy differed significantly between ISS patients with and without the d3-GHR allele. These findings indicate that the GHR-exon 3 polymorphism can affect the growth promoting efficacy of short-term GH therapy in Korean children with ISS.

  5. IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

    Science.gov (United States)

    Ke, Xingrao; McKnight, Robert A; Gracey Maniar, Lia E; Sun, Ying; Callaway, Christopher W; Majnik, Amber; Lane, Robert H; Cohen, Susan S

    2015-07-15

    Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats. Copyright © 2015 the American Physiological Society.

  6. Reversible optic neuropathy with OPA1 exon 5b mutation

    DEFF Research Database (Denmark)

    Cornille, K.; Milea, D.; Amati-Bonneau, P.

    2008-01-01

    A new c.740G>A (R247H) mutation in OPA1 alternate spliced exon 5b was found in a patient presenting with bilateral optic neuropathy followed by partial, spontaneous visual recovery. R247H fibroblasts from the patient and his unaffected father presented unusual highly tubular mitochondrial network......, significant increased susceptibility to apoptosis, oxidative phosphorylation uncoupling, and altered OPA1 protein profile, supporting the pathogenicity of this mutation. These results suggest that the clinical spectrum of the OPA1-associated optic neuropathies may be larger than previously described...

  7. Evidence for a novel exon in the coding region of the adenomatous polyposis coli (APC) gene

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Ling; St. Denis, K.A.; Bapat, B. [Univ. of Toronto (Canada)

    1995-08-10

    Germline mutations of the tumor suppressor gene APC cause familial adenomatous polyposis. Somatic APC alterations are involved in several sporadic neoplasma, including colorectal, duodenal, gastric, and esophageal carcinoma. The APC mRNA is encoded by 15 exons. Additional transcripts have been reported, due to alternative splicing of coding as well as noncoding regions. Two mRNA isoforms occur due to a deletion of exon 7 or a partial deletion of exon 9. We have identified a novel exon, flanked by APC exons 10 and 11, which is expressed as an alternatively transcribed product of the gene. Further, we have shown that the novel exon consists of a heptad repeat motif and is conserved across species. 18 refs., 2 figs.

  8. TALE-directed local modulation of H3K9 methylation shapes exon recognition.

    Science.gov (United States)

    Bieberstein, Nicole I; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K; Krchňáková, Zuzana; Krausová, Michaela; Carrillo Oesterreich, Fernando; Staněk, David

    2016-07-21

    In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

  9. Computational analysis of splicing errors and mutations in human transcripts

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2008-01-01

    Full Text Available Abstract Background Most retained introns found in human cDNAs generated by high-throughput sequencing projects seem to result from underspliced transcripts, and thus they capture intermediate steps of pre-mRNA splicing. On the other hand, mutations in splice sites cause exon skipping of the respective exon or activation of pre-existing cryptic sites. Both types of events reflect properties of the splicing mechanism. Results The retained introns were significantly shorter than constitutive ones, and skipped exons are shorter than exons with cryptic sites. Both donor and acceptor splice sites of retained introns were weaker than splice sites of constitutive introns. The authentic acceptor sites affected by mutations were significantly weaker in exons with activated cryptic sites than in skipped exons. The distance from a mutated splice site to the nearest equivalent site is significantly shorter in cases of activated cryptic sites compared to exon skipping events. The prevalence of retained introns within genes monotonically increased in the 5'-to-3' direction (more retained introns close to the 3'-end, consistent with the model of co-transcriptional splicing. The density of exonic splicing enhancers was higher, and the density of exonic splicing silencers lower in retained introns compared to constitutive ones and in exons with cryptic sites compared to skipped exons. Conclusion Thus the analysis of retained introns in human cDNA, exons skipped due to mutations in splice sites and exons with cryptic sites produced results consistent with the intron definition mechanism of splicing of short introns, co-transcriptional splicing, dependence of splicing efficiency on the splice site strength and the density of candidate exonic splicing enhancers and silencers. These results are consistent with other, recently published analyses.

  10. Early right ventricular fibrosis and reduction in biventricular cardiac reserve in the dystrophin-deficient mdx heart.

    Science.gov (United States)

    Meyers, Tatyana A; Townsend, DeWayne

    2015-02-15

    Duchenne muscular dystrophy (DMD) is a progressive disease of striated muscle deterioration. Respiratory and cardiac muscle dysfunction are particularly clinically relevant because they result in the leading causes of death in DMD patients. Despite the clinical and physiological significance of these systems, little has been done to understand the cardiorespiratory interaction in DMD. We show here that prior to the onset of global cardiac dysfunction, dystrophin-deficient mdx mice have increased cardiac fibrosis with the right ventricle being particularly affected. Using a novel biventricular cardiac catheterization technique coupled with cardiac stress testing, we demonstrate that both the right and left ventricles have significant reductions in both systolic and diastolic function in response to dobutamine. Unstimulated cardiac function is relatively normal except for a significant reduction in the ventricular pressure transient duration compared with controls. These biventricular analyses also reveal the absence of a dobutamine-induced increase in isovolumic relaxation in the right ventricle of control hearts. Simultaneous assessment of biventricular pressure demonstrates a dobutamine-dependent enhancement of coupling between the ventricles in control mice, which is absent in mdx mice. Furthermore, studies probing the passive-extension properties of the left ventricle demonstrate that the mdx heart is significantly more compliant compared with age-matched C57BL/10 hearts, which have an age-dependent stiffening that is completely absent from dystrophic hearts. These new results indicate that right ventricular fibrosis is an early indicator of the development of dystrophic cardiomyopathy, suggesting a mechanism by which respiratory insufficiency may accelerate the development of heart failure in DMD.

  11. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion.

    Science.gov (United States)

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-10-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.

  12. Polymorphisms of exon 5, exon 7 and intron 10 of MMP2 gene and their association with wool density in Rex rabbits

    Directory of Open Access Journals (Sweden)

    S.J. Chen

    2017-06-01

    Full Text Available Wool density is an important index that influences Rex rabbit fur quality. In our earlier studies, we found some important differentially expressed genes in different wool density of Rex rabbit by cDNA microarray. Based on the outcome, we conducted an association study to identify single nucleotide polymorphisms (SNPs of exon 1, 5, 7 and 10 of matrix metalloproteinase-2 (MMP2 gene and their ligands associated with wool density. The results showed that exon 1 and exon 10 of MMP2 gene did not occur mutation in 100 Rex rabbits, meanwhile 3 SNPs were identified in exon 5, exon 7 and intron 10 of MMP2 gene sequence respectively, the 3 mutation sites were as follows: MMP2-exon 5-26C/G, MMP2-exon 7-101C/T and MMP2-intron 10-6C/T. The 3 SNPs were all in Hardy-Weinberg equilibrium. Phenotypic correlation analysis results showed the 3 mutations lacked significant associations (P>0.05 with the wool density.

  13. Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy.

    Science.gov (United States)

    Yamashita, Yoshihiro; Matsuura, Tohru; Shinmi, Jun; Amakusa, Yoshinobu; Masuda, Akio; Ito, Mikako; Kinoshita, Masanobu; Furuya, Hirokazu; Abe, Koji; Ibi, Tohru; Sahashi, Ko; Sahashi, Koo; Ohno, Kinji

    2012-06-01

    Myotonic dystrophy type 1 (DM1) is an RNA gain-of-function disorder in which abnormally expanded CTG repeats of DMPK sequestrate a splicing trans-factor MBNL1 and upregulate another splicing trans-factor CUGBP1. To identify a diverse array of aberrantly spliced genes, we performed the exon array analysis of DM1 muscles. We analyzed 72 exons by RT-PCR and found that 27 were aberrantly spliced, whereas 45 were not. Among these, 25 were novel and especially splicing aberrations of LDB3 exon 4 and TTN exon 45 were unique to DM1. Retrospective analysis revealed that four parameters efficiently detect aberrantly spliced exons: (i) the signal intensity is high; (ii) the ratio of probe sets with reliable signal intensities (that is, detection above background P-value=0.000) is high within a gene; (iii) the splice index (SI) is high; and (iv) SI is deviated from SIs of the other exons that can be estimated by calculating the deviation value (DV). Application of the four parameters gave rise to a sensitivity of 77.8% and a specificity of 95.6% in our data set. We propose that calculation of DV, which is unique to our analysis, is of particular importance in analyzing the exon array data.

  14. Lex-SVM: exploring the potential of exon expression profiling for disease classification.

    Science.gov (United States)

    Yuan, Xiongying; Zhao, Yi; Liu, Changning; Bu, Dongbo

    2011-04-01

    Exon expression profiling technologies, including exon arrays and RNA-Seq, measure the abundance of every exon in a gene. Compared with gene expression profiling technologies like 3' array, exon expression profiling technologies could detect alterations in both transcription and alternative splicing, therefore they are expected to be more sensitive in diagnosis. However, exon expression profiling also brings higher dimension, more redundancy, and significant correlation among features. Ignoring the correlation structure among exons of a gene, a popular classification method like L1-SVM selects exons individually from each gene and thus is vulnerable to noise. To overcome this limitation, we present in this paper a new variant of SVM named Lex-SVM to incorporate correlation structure among exons and known splicing patterns to promote classification performance. Specifically, we construct a new norm, ex-norm, including our prior knowledge on exon correlation structure to regularize the coefficients of a linear SVM. Lex-SVM can be solved efficiently using standard linear programming techniques. The advantage of Lex-SVM is that it can select features group-wisely, force features in a subgroup to take equal weihts and exclude the features that contradict the majority in the subgroup. Experimental results suggest that on exon expression profile, Lex-SVM is more accurate than existing methods. Lex-SVM also generates a more compact model and selects genes more consistently in cross-validation. Unlike L1-SVM selecting only one exon in a gene, Lex-SVM assigns equal weights to as many exons in a gene as possible, lending itself easier for further interpretation.

  15. Genomic V exons from whole genome shotgun data in reptiles.

    Science.gov (United States)

    Olivieri, D N; von Haeften, B; Sánchez-Espinel, C; Faro, J; Gambón-Deza, F

    2014-08-01

    Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

  16. Exon Shuffling and Origin of Scorpion Venom Biodiversity

    Directory of Open Access Journals (Sweden)

    Xueli Wang

    2016-12-01

    Full Text Available Scorpion venom is a complex combinatorial library of peptides and proteins with multiple biological functions. A combination of transcriptomic and proteomic techniques has revealed its enormous molecular diversity, as identified by the presence of a large number of ion channel-targeted neurotoxins with different folds, membrane-active antimicrobial peptides, proteases, and protease inhibitors. Although the biodiversity of scorpion venom has long been known, how it arises remains unsolved. In this work, we analyzed the exon-intron structures of an array of scorpion venom protein-encoding genes and unexpectedly found that nearly all of these genes possess a phase-1 intron (one intron located between the first and second nucleotides of a codon near the cleavage site of a signal sequence despite their mature peptides remarkably differ. This observation matches a theory of exon shuffling in the origin of new genes and suggests that recruitment of different folds into scorpion venom might be achieved via shuffling between body protein-coding genes and ancestral venom gland-specific genes that presumably contributed tissue-specific regulatory elements and secretory signal sequences.

  17. Evolutionary characteristics of exons expressed at different abundance levels in mammals

    Institute of Scientific and Technical Information of China (English)

    YU HaiJing; FANG Lin; LI Xin; ZHANG XiaoYan; YE Jia; LI Jie; HU WeiHong; ZHENG BingRong; XIAO ChunJie

    2009-01-01

    With comparative genomics approaches, we evaluated the evolutionary characteristics of conservation of exons which are expressed abundantly, moderately or lowly in mammals. Using non-coding regions and pseudogenes as controls, sequence identity, phastCons and Ka/Ks analyses were carried out and our results showed that as the exons of high abundance are highly conserved, the minor and low exons also showed conservative characteristics in evolution. Our findings suggested that the exons with less abundance which constitute a large proportion of distinct species in transcriptome of organisms are under functional constraint and might play certain roles in enriching biological complexity in the evolution of organisms.

  18. Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE

    Directory of Open Access Journals (Sweden)

    Bjørheim Jens

    2007-08-01

    Full Text Available Abstract Background Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale. Results A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE, has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp was sufficient to design the analyte sequence and predict the expected degree of resolution. Conclusion CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the ~250,000 exons and splice sites of the ~25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S. $500 per person.

  19. Deletion of amelotin exons 3–6 is associated with amelogenesis imperfecta

    Science.gov (United States)

    Smith, Claire E.L.; Murillo, Gina; Brookes, Steven J.; Poulter, James A.; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F.; Mighell, Alan J.

    2016-01-01

    Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. Amelotin (AMTN) is a secreted protein thought to act as a promoter of matrix mineralization in the final stage of enamel development, and is strongly expressed, almost exclusively, in maturation stage ameloblasts. Amtn overexpression and Amtn knockout mouse models have defective enamel with no other associated phenotypes, highlighting AMTN as an excellent candidate gene for human AI. However, no AMTN mutations have yet been associated with human AI. Using whole exome sequencing, we identified an 8,678 bp heterozygous genomic deletion encompassing exons 3-6 of AMTN in a Costa Rican family segregating dominant hypomineralised AI. The deletion corresponds to an in-frame deletion of 92 amino acids, shortening the protein from 209 to 117 residues. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects at least some of which appeared to be associated with organic material as evidenced using elemental analysis. This study demonstrates for the first time that AMTN mutations cause non-syndromic human AI and explores the human phenotype, comparing it with that of mice with disrupted Amtn function. PMID:27412008

  20. An effort to use human-based exome capture methods to analyze chimpanzee and macaque exomes

    DEFF Research Database (Denmark)

    Jin, Xin; He, Mingze; Ferguson, Betsy

    2012-01-01

    Non-human primates have emerged as an important resource for the study of human disease and evolution. The characterization of genomic variation between and within non-human primate species could advance the development of genetically defined non-human primate disease models. However, non-human...... primate specific reagents that would expedite such research, such as exon-capture tools, are lacking. We evaluated the efficiency of using a human exome capture design for the selective enrichment of exonic regions of non-human primates. We compared the exon sequence recovery in nine chimpanzees, two crab...... design exon-capture array can provide efficient enrichment of non-human primate gene regions. Accordingly, use of the human exon-capture methods provides an attractive, cost-effective approach for the comparative analysis of non-human primate genomes, including gene-based DNA variant discovery....

  1. The effects of multiple features of alternatively spliced exons on the KA/KS ratio test

    Directory of Open Access Journals (Sweden)

    Chen Feng-Chi

    2006-05-01

    Full Text Available Abstract Background The evolution of alternatively spliced exons (ASEs is of primary interest because these exons are suggested to be a major source of functional diversity of proteins. Many exon features have been suggested to affect the evolution of ASEs. However, previous studies have relied on the KA/KS ratio test without taking into consideration information sufficiency (i.e., exon length > 75 bp, cross-species divergence > 5% of the studied exons, leading to potentially biased interpretations. Furthermore, which exon feature dominates the results of the KA/KS ratio test and whether multiple exon features have additive effects have remained unexplored. Results In this study, we collect two different datasets for analysis – the ASE dataset (which includes lineage-specific ASEs and conserved ASEs and the ACE dataset (which includes only conserved ASEs. We first show that information sufficiency can significantly affect the interpretation of relationship between exons features and the KA/KS ratio test results. After discarding exons with insufficient information, we use a Boolean method to analyze the relationship between test results and four exon features (namely length, protein domain overlapping, inclusion level, and exonic splicing enhancer (ESE frequency for the ASE dataset. We demonstrate that length and protein domain overlapping are dominant factors, and they have similar impacts on test results of ASEs. In addition, despite the weak impacts of inclusion level and ESE motif frequency when considered individually, combination of these two factors still have minor additive effects on test results. However, the ACE dataset shows a slightly different result in that inclusion level has a marginally significant effect on test results. Lineage-specific ASEs may have contributed to the difference. Overall, in both ASEs and ACEs, protein domain overlapping is the most dominant exon feature while ESE frequency is the weakest one in affecting

  2. The differential roles of Slit2-exon 15 splicing variants in angiogenesis and HUVEC permeability.

    Science.gov (United States)

    Yang, Yun-Chiu; Chen, Pei-Ni; Wang, Siou-Yu; Liao, Chen-Yi; Lin, Yu-Ying; Sun, Shih-Rhong; Chiu, Chun-Ling; Hsieh, Yih-Shou; Shieh, Jia-Ching; Chang, Jinghua Tsai

    2015-07-01

    Slit2, a secreted glycoprotein, is down-regulated in many cancers. Slit2/Robo signaling pathway plays an important, but controversial, role in angiogenesis. We identified splicing variants of Slit2 at exon 15, Slit2-WT and Slit2-ΔE15, with differential effects on proliferation and invasive capability of lung cancer cells. The aim of this study was to elucidate the differential roles of these exon 15 splicing variants in angiogenesis. Our results revealed that both Slit2-WT and Slit2-ΔE15 inhibit motility of human umbilical vein endothelial cells (HUVECs). The conditioned medium (CM) collected from CL1-5/VC or CL1-5/Slit2-WT lung adenocarcinoma cells blocked HUVEC tube formation and angiogenesis on chorioallantoic membrane (CAM) assay when compared with untreated HUVECs and CAM, respectively. However, CM of CL1-5/Slit2-ΔE15 restored the quality of tubes and the size of vessels. Although both Slit2-WT and Slit2-ΔE15 inhibited permeability induced by CM of cancer cells, Slit2-ΔE15 exhibited stronger effect. These results suggested that Slit2-ΔE15 plays important roles in normalization of blood vessels by enhancing tube quality and tightening endothelial cells, while Slit2-WT only enhances tightening of endothelial cells. It appears that Robo4 is responsible for Slit2 isoform-mediated inhibition of permeability, while neither Robo1 nor Robo4 is required for Slit2-ΔE15-enhanced tube quality. The results of this study suggest that Slit2-ΔE15 splicing form is a promising molecule for normalizing blood vessels around a tumor, which, in turn, may increase efficacy of chemotherapy and radiotherapy.

  3. Identification of Nuclear Factor-κB Responsive Element within the Neuronal Nitric Oxide Synthase Exon 1f-specific Promoter

    Institute of Scientific and Technical Information of China (English)

    Yinghui LI; Guangyu LI; Chunyi LI; Yanyan ZHAO

    2007-01-01

    Transcriptional regulation of the neuronal nitric oxide synthase gene (nNOS) is particularly complex as 12 distinct transcripts derived from different first exons are expressed in a tissue- and cellspecific manner. The exon 1f mRNA is relatively highly expressed in nervous system and relies upon exon 1f-specific promoter activity. Using conventional and real-time reverse transcription-polymerase chain reaction,we found exon 1f mRNA was the major transcript of the nNOS gene in human neuroblastoma SK-N-SH cells. We analyzed a 1090 bp fragment of 1f promoter by TRANSFAC-TESS and Match softwares and luciferase assay, and found an important positive transcriptional regulation region that contained a putative nuclear factor (NF)-κB binding site. Subsequently, using electrophoresis mobility shift and chromatin immunoprecipitation assays, we identified this site to be the NF-κB responsive element, a crucial positive regulator in the activation of the nNOS 1f promoter. Taken together, our study identified an NF-κB responsive element within nNOS 1f promoter and showed that it plays an important role in the transactivation of nNOS 1f mRNA, the major transcript of nNOS in SK-N-SH cells.

  4. Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

    Science.gov (United States)

    Linton, Kim; Hey, Yvonne; Dibben, Sian; Miller, Crispin; Freemont, Anthony; Radford, John; Pepper, Stuart

    2009-07-01

    Microarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.

  5. Deletion of Galgt2 (B4Galnt2) Reduces Muscle Growth in Response to Acute Injury and Increases Muscle Inflammation and Pathology in Dystrophin-Deficient Mice

    Science.gov (United States)

    Xu, Rui; Singhal, Neha; Serinagaoglu, Yelda; Chandrasekharan, Kumaran; Joshi, Mandar; Bauer, John A.; Janssen, Paulus M.L.; Martin, Paul T.

    2016-01-01

    Transgenic overexpression of Galgt2 (official name B4Galnt2) in skeletal muscle stimulates the glycosylation of α dystroglycan (αDG) and the up-regulation of laminin α2 and dystrophin surrogates known to inhibit muscle pathology in mouse models of congenital muscular dystrophy 1A and Duchenne muscular dystrophy. Skeletal muscle Galgt2 gene expression is also normally increased in the mdx mouse model of Duchenne muscular dystrophy compared with the wild-type mice. To assess whether this increased endogenous Galgt2 expression could affect disease, we quantified muscular dystrophy measures in mdx mice deleted for Galgt2 (Galgt2−/−mdx). Galgt2−/− mdx mice had increased heart and skeletal muscle pathology and inflammation, and also worsened cardiac function, relative to age-matched mdx mice. Deletion of Galgt2 in wild-type mice also slowed skeletal muscle growth in response to acute muscle injury. In each instance where Galgt2 expression was elevated (developing muscle, regenerating muscle, and dystrophic muscle), Galgt2-dependent glycosylation of αDG was also increased. Overexpression of Galgt2 failed to inhibit skeletal muscle pathology in dystroglycan-deficient muscles, in contrast to previous studies in dystrophin-deficient mdx muscles. This study demonstrates that Galgt2 gene expression and glycosylation of αDG are dynamically regulated in muscle and that endogenous Galgt2 gene expression can ameliorate the extent of muscle pathology, inflammation, and dysfunction in mdx mice. PMID:26435413

  6. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout

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    Marcel Kapahnke

    2016-12-01

    Full Text Available The clustered regularly interspaced short palindromic repeats (CRISPR-associated sequence 9 (CRISPR/Cas9 system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

  7. Computational analysis and prediction for exons of PAC579 genomic sequence

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi(

    2001-01-01

    [1]Milanesi. L.. Kolchanov, N., Rogozin, I. et al.. Sequence functional inference, in Guide to Human Genome Computing (ed.Bishop. M. J.). Cambridge: Academic Press, 1994, 249-312.[2]Solovyev. V. V., Salamov, A. A., Lawrence, C. B., Predicting internal exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames, Nucleic Acids Res., 1994.22(24): 5156-5163.[3]Borodovsky, M., McIninch, J., GeneMark: parallel gene recognition for both DNA stands, Comp, Chem,, 1993, 17:123-133.[4]Guigo. R.. Knudsen, S, Drake, N. et al., Prediction of gene structure, J. Mol. Biol., 1992, 226(1): 141-157.[5]Kulp, D., Haussler, D., Reese, M. G. et al., A generalized Hidden Markov Model for the recognition of human genes in DNA. ISMB-96. St. Louise: AAAI/MIT Press, 1996.[6]Snyder. E. E.. Stormo, G. D., Identification of protein coding regions in genomic DNA, J. Mol. Biol., 1995, 248(1): 1-18.[7]Xu. Y., Einstein, J. R., Mural, R. J. et al., An improved system for exon recognition and gene modeling in human DNA sequences, in Proc. Int. Conf. lntell. Syst. Mol. Biol., Menlo Park, CA: AAAI Press, 1994, 2: 376-384.[8]Burset. M., Guigo, R., Evaluation of gene structure prediction programs, Genomics, 1996, 34(3): 353-367.[9]Burge. C.. Karlin, S., Prediction of complete gene structures in human genomic DNA, J. Mol. Biol., 1997, 268(l): 78-94.[10]Zhang. M. Q., Identification of protein coding regions in the human genome by quadratic discriminant analysis, Proc. Natl.Acad. Sci. USA, 1997,94(2): 565-568.[11]Mount. S. M., A catalogue of splice junction sequences, Nucleic Acids Res., 1982, 10(2): 459-472.[12]Qin Wenxin. Gu Jianren, Loss of heterozygosity on chromosome 17p13.3 in human malignant tumors, Chinese Bulletin of Life Sciences (in Chinese), 1999, 11(2): 75-77.[13]Li, D., Cao, Y., He, L. et al., Aberrations of p53 gene in human hepatocellular carcinoma from China, Carcinogenesis,1993. 14(2): 169-173.[14

  8. Exonic variants associated with development of aspirin exacerbated respiratory diseases.

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    Seung-Woo Shin

    Full Text Available Aspirin-exacerbated respiratory disease (AERD is one phenotype of asthma, often occurring in the form of a severe and sudden attack. Due to the time-consuming nature and difficulty of oral aspirin challenge (OAC for AERD diagnosis, non-invasive biomarkers have been sought. The aim of this study was to identify AERD-associated exonic SNPs and examine the diagnostic potential of a combination of these candidate SNPs to predict AERD. DNA from 165 AERD patients, 397 subjects with aspirin-tolerant asthma (ATA, and 398 normal controls were subjected to an Exome BeadChip assay containing 240K SNPs. 1,023 models (210-1 were generated from combinations of the top 10 SNPs, selected by the p-values in association with AERD. The area under the curve (AUC of the receiver operating characteristic (ROC curves was calculated for each model. SNP Function Portal and PolyPhen-2 were used to validate the functional significance of candidate SNPs. An exonic SNP, exm537513 in HLA-DPB1, showed the lowest p-value (p = 3.40×10-8 in its association with AERD risk. From the top 10 SNPs, a combination model of 7 SNPs (exm537513, exm83523, exm1884673, exm538564, exm2264237, exm396794, and exm791954 showed the best AUC of 0.75 (asymptotic p-value of 7.94×10-21, with 34% sensitivity and 93% specificity to discriminate AERD from ATA. Amino acid changes due to exm83523 in CHIA were predicted to be "probably damaging" to the structure and function of the protein, with a high score of '1'. A combination model of seven SNPs may provide a useful, non-invasive genetic marker combination for predicting AERD.

  9. The role of exon shuffling in shaping protein-protein interaction networks

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    França Gustavo S

    2010-12-01

    Full Text Available Abstract Background Physical protein-protein interaction (PPI is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of PPIs are the result of domain-domain interactions. Exon shuffling, intron-mediated recombination of exons from existing genes, is known to have been a major mechanism of domain shuffling in metazoans. Thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of PPI networks. Results We tested our hypothesis by compiling exon shuffling and PPI data from six eukaryotic species: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Cryptococcus neoformans and Arabidopsis thaliana. For all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree (number of interacting partners in PPI networks. Furthermore, we verified that a set of protein domains that are simultaneously promiscuous (known to interact to multiple types of other domains, self-interacting (able to interact with another copy of themselves and abundant in the genomes presents a stronger signal for exon shuffling. Conclusions Exon shuffling appears to have been a recurrent mechanism for the emergence of new PPIs along metazoan evolution. In metazoan genomes, exon shuffling also promoted the expansion of some protein domains. We speculate that their promiscuous and self-interacting properties may have been decisive for that expansion.

  10. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

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    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

  11. JAK2 exon 12 mutations in patients with Philadelphia(Ph) chromosome-negative myeloproliferative neoplasms

    Institute of Scientific and Technical Information of China (English)

    王婕妤

    2012-01-01

    Objective To investigate JAK2 exon 12 mutations in patients with Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms (MPN) and the clinical characteristics of patients with JAK2 exon 12 mutants. Methods Allele-specific PCR(AS-PCR) was applied to identify JAK2 V617F mutation.

  12. CoNVaDING: Single Exon Variation Detection in Targeted NGS Data.

    Science.gov (United States)

    Johansson, Lennart F; van Dijk, Freerk; de Boer, Eddy N; van Dijk-Bos, Krista K; Jongbloed, Jan D H; van der Hout, Annemieke H; Westers, Helga; Sinke, Richard J; Swertz, Morris A; Sijmons, Rolf H; Sikkema-Raddatz, Birgit

    2016-05-01

    We have developed a tool for detecting single exon copy-number variations (CNVs) in targeted next-generation sequencing data: CoNVaDING (Copy Number Variation Detection In Next-generation sequencing Gene panels). CoNVaDING includes a stringent quality control (QC) metric, that excludes or flags low-quality exons. Since this QC shows exactly which exons can be reliably analyzed and which exons are in need of an alternative analysis method, CoNVaDING is not only useful for CNV detection in a research setting, but also in clinical diagnostics. During the validation phase, CoNVaDING detected all known CNVs in high-quality targets in 320 samples analyzed, giving 100% sensitivity and 99.998% specificity for 308,574 exons. CoNVaDING outperforms existing tools by exhibiting a higher sensitivity and specificity and by precisely identifying low-quality samples and regions.

  13. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice.

    Science.gov (United States)

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith; Dickson, George

    2011-11-01

    Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

  14. On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.

    Science.gov (United States)

    Verhoef, K; Bauer, M; Meyerhans, A; Berkhout, B

    1998-11-20

    Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We introduced a stop codon at the end of the first Tat-coding exon in an expression vector that produces a truncated 71-amino acid Tat protein. This Q72stop mutant displays reduced transcriptional activity of approximately 54% in transient LTR-CAT transfection assays. To test the contribution of the second Tat-coding exon to virus replication, the Q72stop mutation was also introduced in the infectious pLAI molecular clone. The effect on virus replication was analyzed in primary cells and in a transformed T cell line. The fitness of the mutant virus was calculated to be approximately 75% compared with the wild-type control. Thus, a small contribution of the C-terminal Tat domain to viral fitness was measured. It has been proposed that the second Tat-coding exon is involved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was analyzed in T cells infected with the wild-type LAI virus and the replication-competent Q72stop mutant. MHC expression was transiently reduced on infection with either virus, indicating that the second Tat-coding exon is not involved in this downregulation.

  15. ATRX binds to atypical chromatin domains at the 3' exons of zinc finger genes to preserve H3K9me3 enrichment.

    Science.gov (United States)

    Valle-García, David; Qadeer, Zulekha A; McHugh, Domhnall S; Ghiraldini, Flávia G; Chowdhury, Asif H; Hasson, Dan; Dyer, Michael A; Recillas-Targa, Félix; Bernstein, Emily

    2016-06-02

    ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3' exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3' exonic regions encode the zinc finger motifs, which can range from 1-40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3' exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3' exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3' exons of ZNFs to maintain their genomic stability through preservation of H3K9me3.

  16. Immunohistochemical alterations of dystrophin in congenital muscular dystrophy Alterações imuno-hístoquímicas da distrofina na distrofia muscular congênita

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    Lineu Cesar Werneck

    1995-09-01

    Full Text Available The dystrophin distribution in the plasma muscle membrane using immunohystochemistry was studied in 22 children with congenital muscular dystrophy. The dystrophin was detected by immunofluorescence in muscle biopsy through a polyclonal antibody. All the cases had patchy interruptions of the fluorescence in the plasma membrane. A large patchy interruption of the sarcolemma was found in 17 cases, small interruption in 12, and a combination of large and small patchy discontinuity in 7. Small gaps around the fiber like a rosary were found in 15 cases. The frequency of these abnormalities ranged cases from: all fibers in 5 cases, frequent in 8, occasional in 5, and rare in 4. Five cases had total absence of immunofluorescence. These results suggest that the dystrophin expression is abnormal in this group of children and that this type of abnormalities can not be differentiated from early Becker muscular dystrophy nor childhood autosomal recessive muscular dystrophy through immunohystochemistry alone.Foi estudada a distribuição da distrofina na membrana plasmática das fibras musculares em 22 crianças com distrofia muscular congênita, através de técnicas de imuno-histoquímica. A distrofina foi identificada nas biópsias musculares processadas a fresco, por técnicas de imunofluorescência utilizando anticorpos policlonais. Todos os casos tinham interrupções da imunofluorescência na membrana plasmática. Em 17 elas eram grandes, em 12 eram pequenas e em 7 eram de ambos os tipos. Fibras com interrupções pequenas e constantes, como um rosário, foram vistas em 15 casos. Essas anormalidades estavam presentes em todas as fibras em 5 casos, eram frequentes em 8, ocasionais em 5 e raras em 4. Cinco casos mostraram fibras sem distrofina. Esses dados sugerem que a expressão da distrofina é anormal nesse grupo de crianças. Essas anormalidades podem também ser encontradas em casos precoces de distrofia muscular de Becker e distrofia autoss

  17. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    Science.gov (United States)

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons.

  18. Evolutionary constraint helps unmask a splicing regulatory region in BRCA1 exon 11.

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    Michela Raponi

    Full Text Available BACKGROUND: Alternative splicing across exon 11 produces several BRCA1 isoforms. Their proportion varies during the cell cycle, between tissues and in cancer suggesting functional importance of BRCA1 splicing regulation around this exon. Although the regulatory elements driving exon 11 splicing have never been identified, a selective constraint against synonymous substitutions (silent nucleotide variations that do not alter the amino acid residue sequence in a critical region of BRCA1 exon 11 has been reported to be associated with the necessity to maintain regulatory sequences. METHODOLOGY/PRINCIPAL FINDINGS: Here we have designed a specific minigene to investigate the possibility that this bias in synonymous codon usage reflects the need to preserve the BRCA1 alternative splicing program. We report that in-frame deletions and translationally silent nucleotide substitutions in the critical region affect splicing regulation of BRCA1 exon 11. CONCLUSIONS/SIGNIFICANCE: Using a hybrid minigene approach, we have experimentally validated the hypothesis that the need to maintain correct alternative splicing is a selective pressure against translationally silent sequence variations in the critical region of BRCA1 exon 11. Identification of the trans-acting factors involved in regulating exon 11 alternative splicing will be important in understanding BRCA1-associated tumorigenesis.

  19. Genome-wide survey of ds exonization to enrich transcriptomes and proteomes in plants.

    Science.gov (United States)

    Liu, Li-Yu Daisy; Charng, Yuh-Chyang

    2012-01-01

    Insertion of transposable elements (TEs) into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization which can enrich the complexity of transcriptomes and proteomes. Previously, we performed the first experimental assessment of TE exonization by inserting a Ds element into each intron of the rice epsps gene. Exonization of Ds in plants was biased toward providing splice donor sites from the beginning of the inserted Ds sequence. Additionally, Ds inserted in the reverse direction resulted in a continuous splice donor consensus region by offering 4 donor sites in the same intron. The current study involved genome-wide computational analysis of Ds exonization events in the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice). Up to 71% of the exonized transcripts were putative targets for the nonsense-mediated decay (NMD) pathway. The insertion patterns of Ds and the polymorphic splice donor sites increased the transcripts and subsequent protein isoforms. Protein isoforms contain protein sequence due to unspliced intron-TE region and/or a shift of the reading frame. The number of interior protein isoforms would be twice that of C-terminal isoforms, on average. TE exonization provides a promising way for functional expansion of the plant proteome.

  20. Functional analysis of splicing mutations in exon 7 of NF1 gene

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    Calvieri Stefano

    2007-02-01

    Full Text Available Abstract Background Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals. NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to missplicing. Methods In this study we investigated the expression of exon 7 transcripts using bioinformatic identification of splicing regulatory sequences, and functional minigene analysis of four sequence changes [c.910C>T (R304X, c.945G>A/c.946C>A (Q315Q/L316M, c.1005T>C (N335N] identified in exon 7 of three different NF1 patients. Results Our results detected the presence of three exonic splicing enhancers (ESEs and one putative exonic splicing silencer (ESS element. The wild type minigene assay resulted in three alternative isoforms, including a transcript lacking NF1 exon 7 (NF1ΔE7. Both the wild type and the mutated constructs shared NF1ΔE7 in addition to the complete messenger, but displayed a different ratio between the two transcripts. In the presence of R304X and Q315Q/L316M mutations, the relative proportion between the different isoforms is shifted toward the expression of NF1ΔE7, while in the presence of N335N variant, the NF1ΔE7 expression is abolished. Conclusion In conclusion, it appears mandatory to investigate the role of each nucleotide change within the NF1 coding sequence, since a significant proportion of NF1 exon 7 mutations affects pre-mRNA splicing, by disrupting exonic splicing motifs and modifying the delicate balance between aberrantly and correctly spliced transcripts.

  1. Tracking the evolution of alternatively spliced exons within the Dscam family

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    Vision Todd J

    2006-02-01

    Full Text Available Abstract Background The Dscam gene in the fruit fly, Drosophila melanogaster, contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing (4, 6, 9 and 17, potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. We examined the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. Results Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, we iteratively searched sixteen genomes for homologs, and then performed phylogenetic analyses of the resulting sequences to examine their evolutionary history. We found homologs in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. We found no homologs to these exons within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee, we found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248–283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. Conclusion Our results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays

  2. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  3. RNA-guided human genome engineering via Cas9

    National Research Council Canada - National Science Library

    Mali, Prashant; Yang, Luhan; Esvelt, Kevin M; Aach, John; Guell, Marc; DiCarlo, James E; Norville, Julie E; Church, George M

    2013-01-01

    .... We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

  4. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    OpenAIRE

    Dianzani, I; Camaschella, C.; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-01-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results...

  5. Gastrointestinal malignancies harbor actionable MET exon 14 deletions

    Science.gov (United States)

    Hong, Mineui; Kim, Sun Young; Jang, Jiryeon; Ahn, Soomin; Kang, So Young; Lee, Sujin; Kim, Seung Tae; Kim, Bogyou; Choi, Jaehyun; Kim, Kyung-Ah; Lee, Jiyun; Park, Charny; Park, Se Hoon; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Park, Keunchil; Park, Young Suk; Kim, Kyoung-Mee

    2015-01-01

    Recently, MET exon 14 deletion (METex14del) has been postulated to be one potential mechanism for MET protein overexpression. We screened for the presence of METex14del transcript by multiplexed fusion transcript analysis using nCounter assay followed by confirmation with quantitative reverse transcription PCR with correlation to MET protein expression by immunohistochemistry (IHC) and MET amplification by fluorescence in situ hybridization (FISH). We extracted RNAs from 230 patients enrolled onto the prospective molecular profiling clinical trial (NEXT-1) (NCT02141152) between November 2013 and August 2014. Thirteen METex14del cases were identified including 3 gastric cancer, 4 colon cancer, 5 non-small cell lung cancer, and one adenocarcinoma of unknown primary. Of these 13 METex14del cases, 11 were MET IHC 3+ and 2 were 2+. Only one out of the 13 METex14del cases was MET amplified (MET/CEP ratio > 2.0). Growths of two (gastric, colon) METex14del+ patient tumor derived cell lines were profoundly inhibited by both MET tyrosine kinase inhibitors and a monoclonal antibody targeting MET. In conclusion, METex14del is a unique molecular aberration present in gastrointestinal (GI) malignancies corresponding with overexpression of MET protein but rarely with MET amplification. Substantial growth inhibition of METex14del+ patient tumor derived cell lines by several MET targeting drugs strongly suggests METex14del is a potential actionable driver mutation in GI malignancies. PMID:26375439

  6. Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice.

    Science.gov (United States)

    De Stefano, M Egle; Leone, Lucia; Lombardi, Loredana; Paggi, Paola

    2005-12-01

    Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.

  7. Role of mental retardation-associated dystrophin-gene product Dp71 in excitatory synapse organization, synaptic plasticity and behavioral functions.

    Directory of Open Access Journals (Sweden)

    Fatma Daoud

    Full Text Available BACKGROUND: Duchenne muscular dystrophy (DMD is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR, likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. CONCLUSIONS/SIGNIFICANCE: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.

  8. Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy

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    Guttridge Denis C

    2011-05-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain, targeted at blunting Nuclear Factor κB (NF-κB signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient mdx mice. Methods To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice. Results At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice. Conclusions We conclude

  9. Neuronal nitric oxide synthase-rescue of dystrophin/utrophin double knockout mice does not require nNOS localization to the cell membrane.

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    Michelle Wehling-Henricks

    Full Text Available Survival of dystrophin/utrophin double-knockout (dko mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS transgene. Dko mice expressing the transgene (nNOS TG+/dko experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span.

  10. Polymorphisms in exons 1B and 1C of the type I interleukin-1 receptor gene in patients with endometriosis.

    Science.gov (United States)

    D'Amora, Paulo; Sato, Hélio; Girão, Manoel J B C; Silva, Ismael D C G; Schor, Eduardo

    2006-09-01

    To study possible correlation between the prevalence of polymorphisms in the type I interleukin-1 receptor gene and pelvic endometriosis. Genotypes of 223 women were analyzed: 109 women with surgically and histologically confirmed endometriosis and 114 healthy women. Distributions of two single-base polymorphisms of the human interleukin-1 receptor type I (IL-1RI) gene were evaluated: PstI, due to a C-->T transition in exon 1B and BsrBI a C-->A transition at position 52 in exon 1C. Polymorphisms were detected by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP) resolved on 3% agarose gels stained with ethidium bromide. Genotypes for PstI polymorphisms did not differ significantly among control and endometriosis (P = 0.058). However, in relation to BsrBI polymorphism, protective risk was observed for the development of endometriosis [OR 0.39-IC 95% (0.2-0.9)]. BsrBI heterozygote genotype (C/A) showed protective effect against endometriosis development.

  11. Dopamine D4 receptor exon III polymorphism, adverse life events and personality traits in a nonclinical German adult sample.

    Science.gov (United States)

    Reiner, Iris; Spangler, Gottfried

    2011-01-01

    Personality and temperament embrace a wide area of both psychological and behavioral processes which are also based on disposition. A functional polymorphism in exon III of the dopamine D4 receptor gene (DRD4) has been a highly suspect genetic marker for personality in spite of ambiguous results. The present study aimed to further elucidate the relationship between DRD4, negative life events and personality in a representative nonclinical sample. Hundred sixty-seven Germans completed the NEO Five-Factor Inventory, the Tridimensional Personality Questionnaire and the California Adult Q-Sort. A factor analysis revealed 3 factors: emotional stability, social orientation and impulsivity. DNA from buccal cells was genotyped for the DRD4 variable-number tandem-repeat exon III polymorphism with respect to presence versus absence of the DRD4 7-repeat allele. Adverse life events were assessed by means of the Adverse Life Events Scale. Men carrying the DRD4 7-repeat allele were more impulsive than those without. Male 7-repeat carriers were more emotionally instable than others, but only when they experienced a large amount of negative life events. No genotype-personality relationships were found for women. The results indicate gender-specific influences of the DRD4 gene on human behavior and invite researchers to further investigate gene-environment correlations on personality traits.

  12. Functional evaluation of targeted exon deletion reveals prospect for dystrophic epidermolysis bullosa therapy

    NARCIS (Netherlands)

    Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; Van Den Akker, Peter C; Pasmooij, Anna M G; Nyström, Alexander

    2016-01-01

    Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therap

  13. Identification of true EST alignments and exon regions of gene sequences

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yanhong; JING Hui; LI Yanen; LIU Huailan

    2004-01-01

    Expressed sequence tags (ESTs), which have piled up considerably so far, provide a valuable resource for finding new genes, disease-relevant genes, and for recognizing alternative splicing variants, SNP sites, etc. The prerequisite for carrying out these researches is to correctly ascertain the gene-sequence-related ESTs. Based on analysis of the alignment results between some known gene sequences and ESTs in public database, several measures including Identity Check, Gap Check, Inclusion Check and Length Check have been introduced to judge whether an EST alignment is related to a gene sequence or not. A computational program EDSAc1.0 has been developed to identify true EST alignments and exon regions of query gene sequences. When tested with human gene sequences in the standard dataset HMR195 and evaluated with the standard measures of gene prediction performance, EDSAc1.0 can identify protein- coding regions with specificity of 0.997 and sensitivity of 0.88 at the nucleotide level, which outperform that of the counterpart TAP. A web server of EDSAc1.0 is available at http://infosci.hust.edu.cn.

  14. Exon sequencing of PAX3 and T (brachyury) in cases with spina bifida.

    Science.gov (United States)

    Agopian, A J; Bhalla, Angela D; Boerwinkle, Eric; Finnell, Richard H; Grove, Megan L; Hixson, James E; Shimmin, Lawrence C; Sewda, Anshuman; Stuart, Colin; Zhong, Yu; Zhu, Huiping; Mitchell, Laura E

    2013-09-01

    Based on studies in animals and humans, PAX3 and T (brachyury) are candidate genes for spina bifida. However, neither gene has been definitively identified as a risk factor for this condition. Sanger sequencing was used to identify variants in all PAX3 and T exons and promoter regions in 114 spina bifida cases. For known variants, allele frequencies in cases were compared with those from public databases using unadjusted odds ratios. Novel variants were genotyped in parents and assessed for predicted functional impact. We identified common variants in PAX3 (n = 2) and T (n = 3) for which the allele frequencies in cases were significantly different from those reported in at least one public database. We also identified novel variants in both PAX3 (n = 11) and T (n = 1) in spina bifida cases. Several of the novel PAX3 variants are predicted to be highly conserved and/or impact gene function or expression. These studies provide some evidence that common variants of PAX3 and T are associated with spina bifida. Rare and novel variants in these genes were also identified in affected individuals. However, additional studies will be required to determine whether these variants influence the risk of spina bifida. Copyright © 2013 Wiley Periodicals, Inc.

  15. The relationship between gene isoform multiplicity, number of exons and protein divergence.

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    Jordi Morata

    Full Text Available At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly. In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences.

  16. Three types of polymorphisms in exon 14 in porcine Mx1 gene.

    Science.gov (United States)

    Morozumi, T; Sumantri, C; Nakajima, E; Kobayashi, E; Asano, A; Oishi, T; Mitsuhashi, T; Watanabe, T; Hamasima, N

    2001-08-01

    Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1c deletion is associated with variation in resistance to the myxovirus family in the pig.

  17. A genome-wide analysis of putative functional and exonic variation associated with extremely high intelligence.

    Science.gov (United States)

    Spain, S L; Pedroso, I; Kadeva, N; Miller, M B; Iacono, W G; McGue, M; Stergiakouli, E; Smith, G D; Putallaz, M; Lubinski, D; Meaburn, E L; Plomin, R; Simpson, M A

    2016-08-01

    Although individual differences in intelligence (general cognitive ability) are highly heritable, molecular genetic analyses to date have had limited success in identifying specific loci responsible for its heritability. This study is the first to investigate exome variation in individuals of extremely high intelligence. Under the quantitative genetic model, sampling from the high extreme of the distribution should provide increased power to detect associations. We therefore performed a case-control association analysis with 1409 individuals drawn from the top 0.0003 (IQ >170) of the population distribution of intelligence and 3253 unselected population-based controls. Our analysis focused on putative functional exonic variants assayed on the Illumina HumanExome BeadChip. We did not observe any individual protein-altering variants that are reproducibly associated with extremely high intelligence and within the entire distribution of intelligence. Moreover, no significant associations were found for multiple rare alleles within individual genes. However, analyses using genome-wide similarity between unrelated individuals (genome-wide complex trait analysis) indicate that the genotyped functional protein-altering variation yields a heritability estimate of 17.4% (s.e. 1.7%) based on a liability model. In addition, investigation of nominally significant associations revealed fewer rare alleles associated with extremely high intelligence than would be expected under the null hypothesis. This observation is consistent with the hypothesis that rare functional alleles are more frequently detrimental than beneficial to intelligence.

  18. Polymorphism in exon2 of BMP15 gene in Iranian sangsari sheep

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    zana pirkhezranian

    2015-04-01

    Full Text Available Fertility rate is an economically important trait in sheep, which is influenced by genetic and environment. So far, three genes have been identified that affects this trait, one of them would be the BMP family, the most famous one is BMP15. Different mutations in the BMP15 gene, increases reproductive performance and growth rate in sheep. The aim of this study was to investigate the genetic and phylogenetic of BMP15 gene sequence in Iranian Sangsari sheep. For this purpose, the blood samples from 20 animal of Damghan station were collected. After DNA extracting, a segment of 222 bp of exon 2 of BMP15 gene was amplified using polymerase chain reaction. Then, all of the PCR products were sequenced. The results showed existence of four haplotypes and three significant mutations of the gene that which one of them was seen for first. In order to determine the genetic distance of Sansari sheep with other animals especially sheep breeds about 103 sequences were taken from Genebank, Then, phylogenetic trees were drawn. Genetic distances and nucleotide differences were calculated. The results showed that goat, cattle and buffalo have minimum genetic distance and monkey, human and mouse have maximum distance with Sangsari sheep and native Hindi and Kashmiri sheep have not any differences with Iranian Sangsari sheep.

  19. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins

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    Obed Ramírez-Sánchez

    2016-12-01

    Full Text Available Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa, average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81 aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt]. Streptophyta have on average only ∼5.7 exons of medium size (∼230 nt. Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400 nt. Among subcellular compartments, membrane proteins are the largest (∼520 aa, whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240 aa. Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

  20. Intersubtype Genetic Variation of HIV-1 Tat Exon 1.

    Science.gov (United States)

    Roy, Chandra Nath; Khandaker, Irona; Oshitani, Hitoshi

    2015-06-01

    HIV-1 Tat is a regulatory protein that plays a pivotal role in viral transcription and replication. Our study aims to investigate the genetic variation of Tat exon 1 in all subtypes of HIV-1: A, B, C, D, F, G, H, J, and K. We performed phylogenetic, mutation, and selection pressure analyses on a total of 1,179 sequences of different subtypes of HIV-1 Tat obtained from the Los Alamos National Laboratory (LANL). The mean nucleotide divergences (%) among the analyzed sequences of subtypes A, B, C, D, F, G, H, J, and K were 88, 89, 90, 88, 86, 89, 88, 97, and 97, respectively. We revealed that subtype B evolved relatively faster than other subtypes. The second and fifth domains were found comparatively more variable among all subtypes. Site-by-site tests of positive selection revealed that several positions in all subtypes were under significant positive selection. Positively selected sites were found in the acidic domain at positions 3, 4, and 19, in the cysteine-rich domains at positions 24, 29, 32, and 36, in the core domain at position 40, and in the basic domain for the rest of the positions for all subtypes. Positions 58 and 68 in the basic domain were positively selected in subtypes A, B, C and B, C, F, respectively. We also observed high variability within positively selected sites in amino acid positions. Our study findings on HIV-1 Tat genetic variability may contribute to a better understanding of HIV-1 evolution as well as to the development of effective Tat-targeted therapeutics and vaccines.

  1. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

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    Jai Prakash Khichar

    2016-06-01

    Full Text Available Aim: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. Materials and Methods: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan, India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd. as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN gene (Genebank accession no. DQ167575 was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP pattern. Results: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. Conclusion: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan.

  2. Compensatory relationship between splice sites and exonic splicing signals depending on the length of vertebrate introns

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    Rogozin Igor B

    2006-12-01

    Full Text Available Abstract Background The signals that determine the specificity and efficiency of splicing are multiple and complex, and are not fully understood. Among other factors, the relative contributions of different mechanisms appear to depend on intron size inasmuch as long introns might hinder the activity of the spliceosome through interference with the proper positioning of the intron-exon junctions. Indeed, it has been shown that the information content of splice sites positively correlates with intron length in the nematode, Drosophila, and fungi. We explored the connections between the length of vertebrate introns, the strength of splice sites, exonic splicing signals, and evolution of flanking exons. Results A compensatory relationship is shown to exist between different types of signals, namely, the splice sites and the exonic splicing enhancers (ESEs. In the range of relatively short introns (approximately, Conclusion Several weak but statistically significant correlations were observed between vertebrate intron length, splice site strength, and potential exonic splicing signals. Taken together, these findings attest to a compensatory relationship between splice sites and exonic splicing signals, depending on intron length.

  3. Menzerath-Altmann law in mammalian exons reflects the dynamics of gene structure evolution.

    Science.gov (United States)

    Nikolaou, Christoforos

    2014-12-01

    Genomic sequences exhibit self-organization properties at various hierarchical levels. One such is the gene structure of higher eukaryotes with its complex exon/intron arrangement. Exon sizes and exon numbers in genes have been shown to conform to a law derived from statistical linguistics and formulated by Menzerath and Altmann, according to which the mean size of the constituents of an entity is inversely related to the number of these constituents. We herein perform a detailed analysis of this property in the complete exon set of the mouse genome in correlation to the sequence conservation of each exon and the transcriptional complexity of each gene locus. We show that extensive linear fits, representative of accordance to Menzerath-Altmann law are restricted to a particular subset of genes that are formed by exons under low or intermediate sequence constraints and have a small number of alternative transcripts. Based on this observation we propose a hypothesis for the law of Menzerath-Altmann in mammalian genes being predominantly due to genes that are more versatile in function and thus, more prone to undergo changes in their structure. To this end we demonstrate one test case where gene categories of different functionality also show differences in the extent of conformity to Menzerath-Altmann law. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA.

    Science.gov (United States)

    Fu, Yan; Bannach, Oliver; Chen, Hao; Teune, Jan-Hendrik; Schmitz, Axel; Steger, Gerhard; Xiong, Liming; Barbazuk, W Brad

    2009-05-01

    Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

  5. Crystal Structure of the CLOCK Transactivation Domain Exon19 in Complex with a Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Zhiqiang; Su, Lijing; Pei, Jimin; Grishin, Nick V.; Zhang, Hong

    2017-08-01

    In the canonical clock model, CLOCK:BMAL1-mediated transcriptional activation is feedback regulated by its repressors CRY and PER and, in association with other coregulators, ultimately generates oscillatory gene expression patterns. How CLOCK:BMAL1 interacts with coregulator(s) is not well understood. Here we report the crystal structures of the mouse CLOCK transactivating domain Exon19 in complex with CIPC, a potent circadian repressor that functions independently of CRY and PER. The Exon19:CIPC complex adopts a three-helical coiled-coil bundle conformation containing two Exon19 helices and one CIPC. Unique to Exon19:CIPC, three highly conserved polar residues, Asn341 of CIPC and Gln544 of the two Exon19 helices, are located at the mid-section of the coiled-coil bundle interior and form hydrogen bonds with each other. Combining results from protein database search, sequence analysis, and mutagenesis studies, we discovered for the first time that CLOCK Exon19:CIPC interaction is a conserved transcription regulatory mechanism among mammals, fish, flies, and other invertebrates.

  6. NF1 single and multi-exons copy number variations in neurofibromatosis type 1.

    Science.gov (United States)

    Imbard, Apolline; Pasmant, Eric; Sabbagh, Audrey; Luscan, Armelle; Soares, Magali; Goussard, Philippe; Blanché, Hélène; Laurendeau, Ingrid; Ferkal, Salah; Vidaud, Michel; Pinson, Stéphane; Bellanne-Chantelot, Christine; Vidaud, Dominique; Wolkenstein, Pierre; Parfait, Béatrice

    2015-04-01

    Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

  7. Origination of New Immunological Functions in the Costimulatory Molecule B7-H3: The Role of Exon Duplication in Evolution of the Immune System

    OpenAIRE

    Jing Sun; Fengqing Fu; Wenchao Gu; Ruhong Yan; Guangbo Zhang; Zhiyong Shen; Yinghui Zhou; Han Wang; Bairong Shen; Xueguang Zhang

    2011-01-01

    B7-H3, a recently identified B7 family member, has different isoforms in human and mouse. Mouse B7-H3 gene has only one isoform (2IgB7-H3) with two Ig-like domains, whereas human B7-H3 has two isoforms (2IgB7-H3 and 4IgB7-H3). In this study a systematic genomic survey across various species from teleost fishes to mammals revealed that 4IgB7-H3 isoform also appeared in pigs, guinea pigs, cows, dogs, African elephants, pandas, megabats and higher primate animals, which resulted from tandem exon...

  8. Systematic dissection of coding exons at single nucleotide resolution supports an additional role in cell-specific transcriptional regulation.

    Directory of Open Access Journals (Sweden)

    Ramon Y Birnbaum

    2014-10-01

    Full Text Available In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6 at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.

  9. A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

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    Zlatko Radev

    Full Text Available Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD and Bethlem myopathy (BM remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

  10. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  11. Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.

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    Siva Arumugam Saravanaperumal

    Full Text Available Stem cell factor (SCF is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM, SCF is produced either as a membrane-bound (- or soluble (+ forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR. Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp cDNA encodes a precursor protein of 274 amino acids (aa, commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF corresponding to a truncated protein of 181 aa (vs 245 aa with an unique C-terminus lacking the primary proteolytic segment (28 aa right after the D(175G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5-exon 6 (948 bp with a premature termination codon (PTC whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10. We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+ and/or absence (- of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

  12. A Brassica exon array for whole-transcript gene expression profiling.

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    Christopher G Love

    Full Text Available Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3' exons. Plant whole-transcript (WT GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5, with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18, and categorisation by Gene Ontologies (GO based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.